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Subsections

General Bacteriology


General classification of bacteria related to food science

Great efforts were made to classify all living beings.There are three related fields of activities concerning taxonomy:

Nomenclature

Nomenclature provides names to the different groups.

Identification

Identification verifies if an organism belongs to an already described group.

Classification

Classification tries to group bacteria on the basis of similarities or relationships. Bacteria are classified in[1]:
Taxonomic rank intrasubspecific rank Subspecies may be divided into groups with special characters, the intrasubspecific ranks, which are very important in daily practical bacteriology but not part of the official nomenclature.

Intrasubspecific ranks are:


Sections

The Bergey's Manual of Determinative Bacteriology has grouped bacteria in different "sections" based on a few readily determined criteria.
The binary nomenclature was introduced in 1735 by the Swedish scientist Carl von Linné containing usually informations about the genus and the species. It may be completed by the name of the author of the first description followed by the year of the publication.
New discoveries turn out to be necessary to change the position of bacteria in the classification what sometimes means to change their name.
So it may come that in older books one germ is described under a different name as found in new books.

For example the Escherichia coli was described in old publications under the name of Bacterium coli.

The usual classification which is also used in "Our Food" follows the Bergey's Manual of Systematic Bacteriology vol I to IV.

As many bacteria described in Bergey's Manual are not living any more and her description is still incomplete an international community made a general revision of the content of the manual and presented a list denominated as Approved List(Skerman et al.,1980).

Classification of important bacteria found in food

The sections of Bergey's Manual are:


Section 1 Spirochaeta

Includes the genus Spirochaeta, Treponema, Borrelia and Leptospira.

Section 2 Aerobic/microaerophilic, motile, helical, vibrioid, Gram- negative bacteria

Includes the genus Campylobacter and Spirillum.

Section 3 immobile (or rarely motile ), Gram-negative curved bacteria

Section 4 Gram-negative aerobic rods and cocci

Includes the family Pseudomonadaceae with the genus Pseudomonas and Xanthomonas,
the family Azotobacteriaceae with the genus Azotobacter and Azomonas,
the family Rhizobiaceae with the genus Rhizibium and Agrobacterium,
the family Methylococcaceae with the genus Methylococcus and Methylomonas,
the family Halobacteriaceae,with the genus Halobacterium and Halococcus,
the family Acetobacteriaceae with the genus Acetobacter and Gluconobacter,
the family Legionellaceae with the genus Legionella,
the family Neisseriaceae with the genus Neisseria, Moraxella and Acinetobacter.
In section 4 are also included the genus Flavobacterium, Alcaligenes and Brucella.

Section 5 Facultatively anaerobic Gram-negative rods

Includes the family Enterobacteriaceae with the genus Escherichia, Schigella,Salmonella, Citrobacter, Klebsiella, Enterobacter, Erwinia, Serratia, Hafnia, Edwardsiella, Proteus, Providencia, Morganella and Yersinia, the family Vibrionaceae with the genus Vibrio,Photobacterium,Aeromonas and Plesiomonas.

Section 6 Anaerobic Gram-negative straight, curved and helical rods

Section 7 Dissimilatory sulfate- or sulfur-reducing bacteria

Section 8 Anaerobic Gram-negative cocci

Section 9 The Rickettsias and Chlamydias

Section 10 The Mycoplasmas

Section 11 Endosymbionts

Section 12 Gram-positive cocci

Includes the family of Micrococcaceae with the genus Micrococcus, Stomatococcus, Planococcus, Staphylococcus. Section 12 includes also the genus Streptococcus, Enterococcus,Lactococcus, Leuconostoc, Pediococcus, Sarcina.

Section 13 Endospores producing Gram positive rods and cocci

Includes the genus Bacillus, Sporolactobacillus, Clostridium, Desulfotomaculum, Sporosarcina and Oscillospira.
The genus Bacillus has only one aerobic form. This form is Bacillus anthracis which causes skin anthrax or if inhaled the serious form of pulmonary anthraxThe spores are oval

Bacillus anthracis

This organism was seen hundred years ago in the blood of animals ill with anthrax. Robert Koch proved it to be the cause of the disease by inoculating pure cultures into susceptible cattle: Characteristics: Gram-positive rods, tending to form long chains, not motile. The vegetative form are destroyed by chemical and physical agents but the spores can survive for years in dust or soil and on other objects. The spores survive 5 minutes boiling and ordinary disinfectants.

Section 14 Gram-positive regular formed, not sporulated rods

Includes the family Lactobacillaceae with the genus Lactobacillus, Carnobacter,Listeria and Erysopelothrix.

Section 15 Gram-positive, irregular formed, not sporulated rods

Includes the genus Corynebacterium, Clavibacter, Aureobacterium, Arthrobacter, Propionibacterium, Actinomyces and Bifidobacterium.

Section 16 Mycobacteria

Includes the family Mycobacteriaceae with the genus Mycobacterium.

Section 24 Streptomyces and related genus

Includes the genus Streptomyces.
Streptomycetes are the source of an antibioticum Gibco BRL anti PPLO[2]it is Tyclocine and is sold under the name of Tylan. It has a good activity against PPLO from chicken, horses, human and pigs. In cell cultures it has an antiviral activity acting aswell against Meningopneumonitis from mouse and ornithosis. It is not toxic even in high concentrations. In vitro it is more bactericide than bacteriostatic.

Detailed description of some important bacteria

Section 2


Genus Spirillum

The Genus Spirillum has a spiral form, is Gram-negative, aerophylic and microaerophylic. The Genus contains only one species the Spirillum volutans which is the greatest bacteria known. Its length goes up to 60 micrometers.It grows only in culture under microaerobic conditions. Oxidase and phosphatase are positive,catalase is negative.The germ inhabits water and feces of pigs.It produces volutin, a polyphosphate.

Section 4

Family Pseudomonadaceae are straight, curved or ellipsoidal Gram-negative rods, monotrichous or polytrichous. The family is obligatory aerobic,catalase positive, and generally oxidase positive.
The family grows from 4° and below, up to 43°.
Its habit is water, plants, vegetable products and soil. Some species produce diseases on plants.

The genus Pseudomonas can produce yellow-green, blue or red partially fluorescent pigments.These pigments can diffuse in the culture medium.
The genus Pseudomonas is found in soil, water, other substrates and food producing deterioration due to proteases and lipases which decompose albumin and fatty acids with production of bad smell and mucus.

The bacteria prefer a medium without carbohydrates.
There are psychotropic species which are specialized in refrigerated products such as dairy products, meat, fish, poultry and eggs, spoiling these products even under good refrigeration.

With 10.000 germs/g alterations of taste and smell starts. With 100.000 and more there is production of mucus in meat and fish. Pseudomonas grows only at high value of aw (water activity)(0,97 and higher).

Pseudomonas fluorescens and Pseudomonas aeruginosa are frequently found. They may produce alimentary poisoning.


Genus Xanthomonas

The genus Xanthomonas is closely related to the genus Pseudomonas and also belongs to the section 4. Xanthomonas has phytopathological species. It grows on agar plates as yellow colonies. This gave the name from Greek xanthos = yellow.
Nitrates are not reduced.

Some variants of Xanthomonas campestris are used industrially to produce Xanthan.


Family Halobacteriaceae

The family Halobacteriaceae belongs to section 4. It has Gram-negative rods with various shapes or malformed cells.

The family is characterized for necessitating a high concentration of around 15% of salt in the medium, as well as 0,1 to 0,5 mol of MG++ producing carotenoid yellow to strong red pigments. Its name comes from Greek halos=salt.The internal osmotic pressure corresponds to the pressure of the exterior medium turning mechanical supporting of the membrane of the cell unnecessary. These cells die when transfered to water or another medium with low content of salt.

The family grows best at 20% to 30% of NaCl and 40° to 50° . Below 10° there is no growth.
The Halobacteriaceae family lives in salted lakes, in concentrated salt solutions, in meat, in fish, intestines and other salted food


Family Acetobacteraceae

(Aceto=vinegar, bacterion=rods) Recent cultures are Gram-negative,old cultures are Gram-variable.
The germs are rigorously aerobic, generally catalase positive having oxidative activities. They oxidize ethylic acid in acetic acid.

Acetobacter is used in industry to produce vinegar and acetic acid.
The best temperature for growing is 25° to 30° the best ph is 5.4 to 6.3. The genus Acetobacter is undesired in the production of beer and wine because of the resulting acidity.

Acetobacter xilinum causes great damage to non-alcoholic beverages growing in form of a white layer and superficial mucus.
Acetobacter xilinum, Acetobacter aceti- and Acetobacter pasteurianus inhabits fruits and vegetables.

The genus Gluconobacter with the old denomination of Acetomonas also belongs to the family of Acetobacteraceae.
Gluconobacter oxidans is found in flowers, fruits, vegetables, bakery yeast, beer, wine and soil. The germs are ellipsoid or in form of rods. They are Gram-negative weak Gram-positive as they grow old. The germs are isolated, rarely in chain. Some strains produce mucus and a water soluble brown pigment.

Gluconobacter oxydans is obligatory aerophylic, catalase positive. Ethanol is oxidized to acetic acid and glucose to gluconic acid. This has given the name to the genus. Acetate and lactate are not oxidized because of absence of enzymes of the citric acid cycle.
Growth is best at 25° to 30° and ph between 5.5 to 6.0.

Acetic acid bacteria [3]

The acetic acid bacteria are obligate aerobes that oxidise sugars, sugar alcohols, and ethanol with the production of acetic acid as the major end product. They are important in food and beverage production, as well as in the bioproduction of industrial chemicals, but they are also known to spoil food and beverages. The classification of acetic bacteria is being rearranged using 16S rRNA sequence analysis.

Acetic acid bacteria belong to with polar flagellation and no oxidation of acetate, the genus Acetobacter, and the genus Gluconacetobacter comprising G. liquefaciens and G xylinus.

Vinegar is produced by conversion of carbohydrates of ethanol by yeasts, followed by the oxidation of ethanol to acetic acid by acetic acid bacteria, such as Acetobacter, Gluconacetobacter, and Gluconobacter. [4]

Other uses of acetic acid bacteria are important in cocoa production, production of microbial cellulose. Bioproduction uses the enzymes such as 2-keto-L-gulonic acid for the production of vitamin C, the sweetener D-tagatose, and shikimate, an intermediate product for the synthesis of antibiotics.

Macauley and colleagues 2001 evaluated the utility of the genus Gluconobacter in biotechnology and future industrial processes. [5]

A pathogenic acetic acid bacterium was described, representing the tenth genus of acetic acid bacteria:

Granulibacter bethesdensis a new pathogenic acetic acid bacteria [6]

Granulibacter bethesdensis, was isolated from lymph nodes of chronic granulomatous disease patient. The genome of this pathogenic acetic acid bacteria includes the 967 ORFs important for virulence, adherence, DNA uptake, and methanol utilization. G. bethesdensis is a genetically diverse emerging human pathogen that may have recently acquired virulence factors new to this family of organisms.

Grouping acetic acid bacteria by 16S rDNA sequence [7]

De Vero Luciana and Giudici Paolo 2008 developed a method for grouping acetic acid bacteria genera for preliminary screening acetic acid bacteria species used in vinegar production. The authors screened Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, Neoasaia, Saccharibacter, Frateuria and Kozakia acetic acid babcteria strains focusing on 16S rDNA sequences. Using this method it is possible to group the species recovered from vinegar fermentation, being most frequently of the genera Acetobacter, Gluconobacter and Gluconacetobacter say the authors.

Most prominent Gluconobacter genra are:
Gluconobacter frateurii
Gluconobacter thailandicus
Gluconobacter oxydans
Gluconobacter cerinus
Gluconobacter albidus
Gluconobacter kondonii

Gluconobacter japonicus [8]

Malimas and colleagues 2009 describe a cluster of five strains. They were found to differ from the type strains of Gluconobacter frateurii, Gluconobacter thailandicus, Gluconobacter oxydans, Gluconobacter cerinus, Gluconobacter albidus and Gluconobacter kondonii.

The authors propose the name Gluconobacter japonicus sp. Nov for the new cluster. Gluconobacter japonicus produces weakly dihydroxyacetone from glycerol, but not 2,5-diketo-d-gluconate or a water-soluble brown pigment from d-glucose and contained ubiquinone-10.

Intragenic structure of the Genus Gluconobacter using 16S rDNA and ITS sequences [9]

Taqkahashi and colleagues 2006 re-examined the species of the genus gluconobacter analysing The sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). Five cluster were identifien which coorespond to Gluconobacter albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus.

The type strain of G. asaii, NBRC 3276T was included in the G. cerinus cluster. Gluconobacter cerinus, G. frateurii and G. oxydans clusters were heterogeneous. They contained clusters of other species. The authors stress that the species definition must be re-evaluated.

Gluconobacter sphaericus [10]

The strain NBRC 12467T was found by Malimas and colleagues 2008, applying 16S-23S rRNA gene ITS sequences, to form an independent cluster. The strain produced a water-soluble brown pigment and 2,5-diketo-D-gluconate from D-glucose, differing from the type strains of the eight Gluconobacter species. The authors propose the name of Gluconobacter sphaericus (Ameyama 1975) comb. nov.

Gluconobacter thailandicus [11]

Four strains of acetic acid bacteria were isolated from a flower of the Indian cork tree (Millingtonia hortensis) collected in Bangkok, Thailand by Tanasupawat and colleagues 2004. The researchers proposed the name of Gluconobacter thailandicus sp. nov.

Nitrogen fixating bacteria [12]

To avoid or reduce the use of Nitrogen-fertilizers the use of plant growth-promoting bacteria, was proposed by Pedraza 2007. Promising genera include Azospirillum, Azotobacter, Herbaspirillum, Bacillus, Burkholderia, Pseudomonas, Rhizobium, and Gluconacetobacter and others.

They are capable of promoting plant growth through different mechanisms including (in some cases), the biological nitrogen fixation (BNF), the enzymatic reduction of the atmospheric dinitrogen (N(2)) to ammonia, catalyzed by nitrogenase.

Well studied nitrogen-fixing species are Gluconacetobacter diazotrophicus found in sugar plants, Gluconacetobacter johannae and Gluconacetobacter azotocaptans in coffee plants from Mexico, and salt-tolerant bacterium named Swaminathania salitolerans in wild rice plants, which is salt-tolerant. In India Acetobacter peroxydans and Acetobacter nitrogenifigens were found associated with rice plants and Kombucha tea as nitrogen-fixing bacteria.

According to Bhattacharjee and colleagues 2008 nitrogen-fixing bacteria could significantly reduce the use of the nitrogenous fertiliser which contributes to the green house emission (N2O) and underground water leaching. Non-leguminous plants like rice, sugarcane, wheat and maize were also found to be associated with nitrogen-fixing bacteria.

The authors stress the importance of nitrogen-fixing bacteria in non-leguminous plants in face of a higher demand of these crops. [13]

Gluconacetobacter diazotrophicus [14]

Gluconacetobacter diazotrophicus a nitrogen-fixating bactera is important in low nitrogen fertilized sugarcane fields.

Saravanan and colleagues 2007 discusses the survival and transmission of the bacterium. Other nitrogen fixating Acetobacteraceae, such as Gluconacetobacter azotocaptans, Gluconacetobacter johannae and Swaminathania salitolerans, from coffee, corn and rice and other plant-growth-promoting traits of this group of bacteria, such as phytohormone synthesis, P and Zn solubilization and biocontrol, are discussed by the authors.

Munoz-Rojas and Caballero-Mellado 2003 studied the growth effect of Gluconacetobacter diazotrophicus on strains in the different sugarcane varieties. The authors found that the bacterial populations decreased drastically in relation to plant age. The inoculation of Gluconacetobacter diazotrophicus was found beneficial for sugarcane plant growth, but depends on the bacteria genotype and the sugarcane variety. The authors stress the importance of the sugarcane variety for the persistence of the plant-bacteria interaction. [15]

Inoculating sugarcane plants with Gluconacetobacter diazotrophicus and Herbaspirillum sp. was found by Muthukumarasamy and colleagues 2006 to increase nitrogen content in leaves of sugarcane of Co 86032 in South India. The authors found that the number of Herbaspirillum sp. remained stable with the age, but G. diazotrophicus were reduced in old plants.

The authors report that total bio-mass and leaf N were higher in plants inoculated with G. diazotrophicus and Herbaspirillum sp. without fertilization than in plants fertilized with recommended dose of inorganic N (280 kg ha(-1)). This experiment showed that inoculation with these bacteria in sugarcane variety Co 86032 could mitigate fertilizer N application considerably in sugarcane cultivation. [16]

Cocking, Stone and Darwey 2004 recommend the use of Gluconacetobacter diazotrophicus as a substitute of synthetic nitrogen fertilizers in maize, rice and wheat cropping systems for higher yields and environmental protection. The authors perform reseaches witch maize culture under4 zero nitrogen fertiliser input. [17]

Fox and colleagues 2007 stress that the use of synthetic nitrogenous fertilizers, pesticides, and irrigation promoted by the "Green Revolution" doubled the grain production in the past, but crop yields are diminishing. The authors call for a common strategy to reduce dependence on nitrogenous fertilizers by rotating leguminous crops with nonleguminous crops. They point out that organochlorine pesticides, agrichemicals, and environmental contaminants inhibited or delay symbiosis of rhizobia bacteria with host plant roots, reducing overall plant yield. Synthetic chemicals compromise symbiotic nitrogen fixation and increases dependence on synthetic nitrogenous fertilizer and reduces soil fertility. [18]

Biotechnology application of Gluconobacter strains [19]

Gluconobacter strains uses dehydrogenases connected to the respiratory chain located in the periplasmic space. Deppenmeier, Hoffmeister and Prust 2002 explain that transport of substrates and products into, and out of, the cell is, therefore, not necessary. This turns Gluconobacter highly interesting for the production of L-sorbose (vitamin C synthesis), 6-amino- L-sorbose (synthesis of the antidiabetic drug miglitol), dihydroxyacetone, gluconate and ketogluconates.

Adachi and colleagues discusses the oxidative fermentation of Gluconobacter species. The authors highlight two different types of membrane-bound enzymes: The quinoproteins produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. The flavoproteins D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose.

The quinate dehydrogenase is a new quinoprotein which produces 3-dehydroquinate from the oxidation of quinate.

The quinate dehydrogenase can be used to produce shikimate entangled in the production of antibiotics, herbicides, and aromatic amino acids synthesis. [20]


Neisseriaceae

The family Neisseriaceae belongs to the section 4 and bears the genus Neisseria, Moraxella, Acinetobacter and Klingella.
The genus Moraxella has isolated coccoid form or Gram-positive diplococcus. Oxidase and catalase are positive. Moraxella lacunata is psychotropic spoiling meat, fish and shrimp.

The genus Flavobacterium (flavus=yellow) is aerophylic or anaerophillic,Gram- negative rods and produces yellow or red pigments which are insoluble in water. Almost all species are psychophylic and proteolytic. They produce putrefaction and modification of color on fish, poultry, eggs, milk and butter.They are found in in fresh unheated milk, in vegetables, in water and in soil.

Flavobacterium multiplies in the first phase of sauerkraut.One species is pathogen.

The genus Alcaligenes is obligatory aerophylic, Gram-negative rods, rarely coccoid. Its colonies are flat, gray, yellow or brown. Some types are nitrate positive. They do not hydrolyze gelatine and casein. Carbohydrates are not transformed in acids. There is production of alkalinity from amides and other organic salts. This property has given the name to the genus.
There are many species which are generally inhabitants of the intestines from vertebrates as saprophytes.They may act as opportunists on human infections.

Alcaligenes may be present in milk, spoiled eggs and other food.
The most common germ is Alcaligenes faecalis

Achromobacter is an old denomination.Many species from this genus were included in the genus Alcaligenes.

The genus Brucella was denominated in honor to Sir David Bruce. It has Gram-negative short rods or coccoid forms. The germs may present itself isolated or in chain.They are obligatory aerophylic, immobile, growing up to 40°. The best ph is 6.6 to 7.4.Catalase is positive and nitrate is reduced to nitrite.

Culture media for Brucella must contain peptons, liver extract, yeast extract and vitamins such as thiamin, biotin and nicotinic acid. Initial culture needs 5% to 10% CO2.

Brucella causes brucellosis, an infection of animals which can be transmitted to man under the name of bang. The transmission is direct , very seldom the transmission is caused by contaminated milk and milk products.

Brucella abortus and Brucella suis are old denominations which were included under the name of Brucella melitensis.

Section 5


Aeromonas

Aeromonas bacteria can be present in fresh waters, tap waters and food such as fish and other marine animals. It can cause infections in animals and man. That is why controlling water, faeces and food in general is of high importance.

Classification of Aeromonas spp.

Recent genetic studies have cleared some of the confusion in the classification of Aeromonas. The genus Aeromonas has Gram-negative rods is facultative anaerobic,is oxidase positive, catalase positive, resistant to 0/129 vibriostatic agent (2,4-diamino-6,7-diisopropylpteridine).The genus can show very easily two groups:

Aeromonas salmonicida: psychrophilic, non-motile. Some species are pathogenic to frogs, fish and humans. Human disease is usually diarrhoea or bacterimia. Aeromonas hydrophila: motile

The group Aeromonas hydrophila was according to Bergey's Manual of Systematic Bacteriology (1984) divided into three species, being increased today to 12 species. The three species of the classification of Bergey's are:
Aeromonas hydrophila caviae sobria
Esculin hydrolysis + + -
Gas from glucose + - +
Voges-Proskauer + - V
Acid from arabinose + + -

Pathogenic Aeromonas in retail meat [21]

Aeromonas hydrophila was isolated from 6.8% of retail meat in Egypt, other isolate were Aeromonas caviae with 2.7% and Aeromonas sobria with 2.1% from the total meat samples. Three of seventeen meat isolates of Aeromonas hydrophila produced aerolysin toxin gene (aerA). Osman et al 2012 stress the health hazard to humans handling contaminated meat.

Aeromonas hydrophila virulence factor described [22]

According to Suarez et al 2012 Aeromonas hydrophila isolate 7966 possesses a functional repeat in toxin (RtxA) having an actin cross-linking domain (ACD) that contributes to the host cell apoptosis which may represent a virulence factor of Aeromonas hydrophila. However, only the full-length ACD of RtxA from Aeromonas hydrophila catalyses the covalent cross-linking of the host cellular actin and induces host cell apoptosis. The RtxA gene expression required host cell contact to be expressed. The RtxA contained six genes (rtxACHBDE) presenting similarities to the gene organization found for the Rtx of the Vibrio species.

Aeromonas isolates from diseased fish, healthy controls and water environment in China [23]

Hu et al. 2012 report that dead fishes presented Aeromonas veronii and Aeromonas hydrophila as highest frequency of isolation in China. Aeromonas veronii was most the frequent in healthy fish and water samples.

Aeromonas hydrophila isolates were significantly more frequent from diseased fish than from healthy fish and presented the aerolysin (aer), cytotonic enterotoxin. (alt), cytotoxic enterotoxin (act), temperature-sensitive protease (eprCAI) and serine protease (ahp) genes These isolates were more virulent to zebrafish comparing to the other genetic profiles.

The authors point to the fact that Aeromonas species in aquatic environmentsmay present virulence potential. They call for stringent epidemiological studies.

Aeromonas hydrophila and outbreak of abortions and Infertility [24]

Singh et al 2011 report that water-borne A. hydrophila were associated with equine abortions and infertility, and diarrhea in newborn foals. At an equine breeding farm near Hissar (Haryana-India), three mares aborted in their seventh month of pregnancy. The authors found risue samples, swabs and all water samples positive for Aeromonas hydrophila. Water should be included in the control system to avoid this bacteria.

Further classification of aeromonads

The phenotyping of Aeromonas has been for long time confused and is still not yet ready.


Historical classification of aeromonads

Bacillus punctatum, in 1890 classified by Zimmermann: bacillus Gram-negative not sporulated, motile found in tap water.

Bacillus ranicida, classified by Ernst, isolated from frog with red leg disease.


Bacillus stereatothermophilus Donk 1920

The name of this bacterium is presumably intended to mean fat and heat loving. The most distinctive characters are capacity to grow at 65° and a limited tolerance to acid. Bacillus strains capable of growing at temperatures of 65° and above do not belong to a single species, it is however a useful diagnostic character.
Bacillus stereatothermophilus occurs in soil, hot springs, desert sand, arctic waters, ocean sediments, foods and compost.
The biochemical characteristics of bacillus stereatothermophilus are:

Catalase = positive
Voges-Proskauer test = negative
Acid from D-glucose = positive
Acid from L-arabinose = differ
D-xylose = differ
D-mannitol = differ
Gas from glucose = negative
Hydrolysis of casein = differ
Hydrolysis of gelatin = positive
Hydrolysis of starch = positive
Utilization of citrate = differ
Degradation of tyrosine = negative
Desamination of phenylalanine = negative
Nitrate reduced to nitrite = differ
Formation of indole = negative
Dihydroxiacetone = negative
Sodium and potassium chloride required = negative
Alantoin or urate required = negative

Allen (1953) has pointed out that fresh isolates tend to diversity of characteristics. When maintained in culture for some times they are readily classifiable.
The bacterium has a vital importance for canning factories. Bacillus stereatothermophilus, together with Bacillus coagulans as well as other bacteria have high heat resistant spores. The temperature maximum where growth still takes place is 75°. The best growth temperatures for Bacillus stereatothermophilus is 55 to 60°. Bacillus stereatothermophilus is the bacterium whose spores can survive at temperatures higher than other bacteria.
In hot springs bacteria may be found which resist temperatures higher than that.


D-value (Decimal reduction time)

The D-value is the time which is necessary at a specific temperature to reduce the initial population of a bacterium down to 10%. This means it kills 90% of the bacteria. It is measured in minutes. The temperature must always be cited.
For Bacillus stereatothermophilus a D-value of D121,1C= 4 to 5 minutes are given.

D-values for other bacteria in order to draw a comparison

Clostridium botulinum type A and B D121,1C= 0,1 up to 0,2 minutes.
Clostridium sporogenes D121,1C= 4 up to 5 minutes.
Clostridium thermosoccharolyticum D D121,1C= 3 up to 4 minutes.
Desulfotomaculum nigrificans D121,1C 2 up to 3 minutes.

For tropic conserves the sterilization has to be done carefully as Bacillus stereatothermophilus grows at storage temperatures higher than 37°. Below of that there is no growth. To kill its spores F121,1C= 15 to 30 minutes must be used in case of canned food. Another example of high temperature resistant spoilage of canned food is the mould Byssochlamiy nivea which is sometimes found in canned cucumber resisting up to 98degree centigrades in acid medium. Byssochlamis isolated from canned cucumber and cultivated on yeast chloramphenicol dextrose medium:
Image p1010034
Bacillus hydrophillus fuscus from the lymph of a frog with haemorrhagic septicaemia. In 1936 Kluiver and Van Niel created the genus Aeromonas. In 1943 Stanier grouped and rearranged all strains of Aeromonas under the sole species Aeromonas hydrophila. In 1984 Popoff has included the genus Aeromonas under the family of Vibrionaceae under Section 5, facultatively anaerobic Gram-negative rods. He divided the group of A. hydrophila from Stainer in four species:

Some bacteria from underheated food may turn it sour without gas. This is called "flat sour spoilage". Only smell, taste and color may be changed. Deterioration with production of gas such as produced by Clostridium botulinum can easily be detected because of the pressure which is created inside.
Image P7250060
Aeromonas can be present in water with a ph from 5.2 to 9.8, growing by 10° up to 45° being 35° the optimum. They can grow in water with very low organic matter as well in sewage with high content of organic matter. They are not found in sea water an they do not grow in vitro with 4 % of salt. They have been isolated from unchlorinated as well as from chlorinated water. A new family Aeromonadaceae, independent from Vibrionaceae is being proposed.


The culture of aeromonads

Aeromonads grow well on any complex medium such as Nutrient Agar or Trypticase Soy Agar as well as selective media for faecal forms such as McConkey Agar.The most common selective media for aeromonads use carbohydrates as the main carbon source.

Pathogenesis in animals and human

Aeromonas hydrophila is being found in fish, reptiles and turtles. Under certain conditions it may cause infections of the host, such as the haemorrhagic septicemia and the red leg disease. Water is the principal source of Aeromonas infections in man.

Section 2


Campylobacter [25]

History

1886- Theodore Escherich discovered a non-culturable, spiral-shaped bacterium in stools of children and gave him the name of Vibrio felinus.

Other classification followed such as: Vibrio fetus, Vibrio jejuni .
1963- All above micro-organism were united in the new genus of Campylobacter.
1973- Butzler and colleagues in Belgium isolated Campylobacter by filtration techniques from diarrheal stool.
1977- Skirrow in England using blood agar with antibiotics demonstrated that Campylobacter jejuni was responsible for human diarrhoea.
1989- The number of outbreaks caused by Campylobacter surpasses since 1989 the number of outbreaks caused by Salmonella.
1991- Vandamme proposed the following classification:


Family of Campylobacteraceae

Genus

Description

Campylobacter are Gram-negative, slender spiral curved rods. They need a micro-aerobic atmosphere to grow. The pathogenic species grow at 42° and 37°.

Pathogenesis

Campylobacter may cause rare infections in disseminated systemic form. However Campylobacter are the most frequent agents of watery and bloody diarrhoea in the world. Campylobacter-species can cause severe genital or intestinal disease in meat producing lifestocks and poultry.
Genus
Campylobacter has the following species :
C.: is together with C.jejuni ssp. jejuni the most common cause of disease in human and in animals.
Infection takes place during handling raw poultry or eating raw or undercooked poultry meat. [26]
C.conscious
C.curvus
C.fetus ssp.fetus : important animal pathogen associated with abortions in sheep and cattle and human disease such as diarrhoea, meningitis, peritonitis, salpingitis, septic abortion, septicemia in older patients and in immune suppressed persons.
C.fetus ssp.venerealis: causes infertility and embryonic death in cattle and abortion in infected cows. It does not infect man.
C.hyointestinalis
C.jejuni ssp.jejuni : It is together with Campylobacter the most common cause of disease in human and in animals such as diarrhoea in calves and abortion in sheep. In human it may cause septicemia, appendicitis and the Guillain- Barre syndrome (an acute inflammatory polyneuropathy).
C.jejuni ssp.doylei
C.lari: isolated from human , dogs and cat outbreaks.
C.mucosalis
C.rectus
C.sputorum biovar bubulus
C.sputorum biovar fecalis
C.sputorum biovar sputorum
C.upsliensis: isolated from human, dogs and cat outbreaks.
C.helveticus
C.showae
C.hyoillei
C.gracilis
Campylobacter jejuni: It is spiral formed, Gram-negative microaerophil rod.
To cultivate it is necessary to reduce the oxygen between 5% to 7%. Campylobacter jejuni grows at 43° but there is no growth at 25°.

The germ is very sensible to heat, chilling, and acids.
The resistance to heat in skimmed milk = D 55° is 1 to 3 minutes.
Resistent to chilling temperatures: after 5 to 8 days there were no bacteria alive left from a population of 107 in skim milk at -20° .
Resistent to acids: At pH 5,0 all Campylobacter jejuni died after 24 hours.

Campylobacter jejuni can be isolated from feces of children, animals water, beef, in particular poultry (chicken, duck, turkey), on vegetable,fruits and marine animals. An infection with Campylobacter jejuni is characterized with diarrhoea, fever and vomit.

Campylobacter jejuni does not normally multiply outside the host. However the bacteria have the ability to survive a long time in the environment. It is very infectious. If one bird is infected the whole flock will be affected. For the production of Campylobacter free poultry rapid diagnostics are necessary to avoid the contamination to spread out.

If poultry is infected nearly 100% will be bacterial carriers, therefore only a few birds in a flock need to be tested to ensure that the whole flock is Campylobacter free.

Very few cells can produce an infection it is therefore important to enrich suspected material. The infectious level is 103 an 105 germs/g It is being told that even 50 bacteria can cause an infection. [27]

Incubation is two to five days.The onset of symptoms is sudden, often preceded with one or two days with fever and headache with sudden watery and sometimes bloody diarrhoea,abdominal cramps,fever and headache.

After settling in the intestine Campylobacter produces a protein called "adesin" which acts as a glue between the germ and the wall of the mucosa causing the adhesion to the wall of the intestine which makes the invasion of the tissue possible. Campylobacter Jejuni, C. and C. lari produce an enterotoxin, in some cases Zytotoxin is reported.


Complications with Campylobacter infections

A possible complication with Campylobacter infections is an autoimmune disease called Guillain-Barre syndrome which results in weakening and paralysis. It seems that the similar surface of Campylobacter to the surface of the human nervous system can cause the production of antibodies that cross-react with the nervous tissue during an infection. The antibodies will attach to the peripheral nerves causing the disease.

Campylobacter infections decline in response to interventions aimed at poultry [28]

Campylobacter jejuni is the most common cause of acute enteritis in humans, with symptoms such as diarrhoea, fever and abdominal cramps.

Beginning in the 1980s, New Zealand experienced rising annual rates of campylobacteriosis that peaked in 2006. Sears et al 2011 assessed the decline of campylobacteriosis incidence between 2007–2008, when voluntary and regulatory interventions to reduce Campylobacter spp. contamination of poultry were introduced. The authors report a reduction of 74% of cases attributed to poultry, compared with 2002-2006, a period before the introduction these interventions.

Several foodborne pathways of campylobacteriosis (other than poultry) have been identified, including red meat and raw milk consumption. The contribution of these pathways to sporadic campylobacteriosis in New Zealand has been estimated to be notably less than that of poultry.

The authors suggest that the data of their study may help other countries to control foodborne campylobacteriosis linked to specific food sources. The importance of collaboration between industry, food safety regulators, and public health researchers to control food borne diseases.

According to Shaughnessy et al 2011 more than 90% of commercial chickens caecum is colonised asymtomatically by Campylobacter jejuni. The authors used chicken-specific 20K oligonucleotide microarrays to examine global gene expression in Campylobacter jejuni-challenged birds. Increased gene expression of oxidative burst, endothelial cell activation and T cell mediated activity were found. The authors concluded that Campylobacter jejuni alters the global caecal gene expression and a protective intestinal T cell response takes place in the caecum of the chicken. [29]

Conlan et al. 2011 stress that in commercial flocks of chickens are typically found to be Campylobacter free for the first 14-21 days of life However, once detected Campylobacter jejuni spreads rapidly saturating the flock over the course of 2-3 days. Conlan and colleagues suggest that the mechanism of the so-called Campylobacter jejuni-free "lag-phase" is based on the age-dependence of transmissibility between hosts, rather than their susceptibility to colonization. [30]

Identification and quantification of Campylobacter coli and Campylobacter jejuni [31]

Livestock animals are source of foodborne human contamination of Campylobacter jejuni and Campylobacter coli, especially in developing countries. Pigs are often colonized with Campylobacter coli, and their faeces contain high number of the pathogen.

Leblanc-Maridor et al 2011 present a quantitative real-time PCR method for species-specific detection and quantification of Campylobacter coli and Campylobacter jejuni directly in faecal, feed, and environmental samples. The assay can be used for epidemiological studies of Campylobacter coli and C. jejuni by pigs from conventional herds and others.

Campylobacter jejuni survival on packaged beef or pork [32]

According to Balamurugan et al. 2011 the number of Campylobacter jejuni decreased significantly during storage under chilled, vacuum packaged and retail display conditions when inoculated on sterile meat. However, survival of Campylobacter jejuni on commercial vacuum packaged beef and pork was significantly enhanced, decreasing only 1 log cfu cm(-2) reduction at the end of 6 weeks, and could still be detected after seven days of display in a retail case. The authors concluded that natural microflora on vacuum packaged meat foster the survival of Campylobacter jejuni on beef or pork under refrigeration. Strict hygienic practices or the implementation of decontamination technologies are therefore necessary to reduce the risk of campylobacteriosis.

In Denmark, the incidence of human campylobacteriosis cases, as well as the Campylobacter prevalence in broiler flocks, is highest during the summer months of July-August. Boysen et al. 2011 found that the occurrence of Campylobacter in broiler meat at retail increased in domestic chilled mead during summer. The prevalence of Campylobacter in broiler flocks may therefore be used as a predictor of this pathogen in broiler meat at retail, suggest the authors. [33]

Sources of infection

Infected poultry (up to 80% of broiler flocks), untreated water, cattle, pigs, pets, wild animals, birds, Fruits, marine animals.

Continuous air monitoring of Campylobacter for broiler flocks [34]

Campylobacteer is the most common case of diarrhoea in humans, and poultry counts for half of the infections.

Olsen and colleagues 2009 assessed the detection of Campylobacter by PCR in feces, dust, and air samples in poultry houses, and found that the sensitivity of detection of Campylobacter in air is comparable to that in other sample materials.

A low proportions of particles in the 0.5- to 2-µm-diameter range and high proportions in the 2- to 5-µm-diameter range was found. PCR detection of Campylobacter in air samples could also be performed at the hanging stage during the slaughter process but not at other places at the slaughterhouse. The researchers concluded that new detection technologies, allow continuous monitoring of colonization status.

Detailed informations about Culture of Campylobacter

Enrichment broth is spread with a loop on specific agar and typical colonies are inoculated on Columbia Agar. Biochemistry, Latex agglutination and genetic methods like polymerase chain reaction (PCR)

Detection of Campylobacter jejuni

:Detection of Campylobacter is made using enrichment broth according to Wesley et al. 1983 or Blase and Wang (1979)or Campylobacter - selective - broth, under anaerobic atmosphere (5% O2, 10% CO2, 85% N2) at 42° for 24 hours. The atmosphere is easily obtained with the Campylobacter - Gas Generation-Kit from Oxoid.

ingredients amount
Iron sulphate 0,25 g
Sodium metabisulfit 0,25 g
Sodium piruvate 0,25 g
Bicine 10 g
Haematin solution  
Rifampin 25 mg
Cefsulodin 6,25 g
Polymyxin B 20.000 IU


The enrichment broth according to Wesley can demonstrate up to less than 1 germ/g

ingredients amount
Vancomycin 10 mg
Trimethoprim 5 mg
Polymyxin B 2500 IU
Amphotericin 2 mg


Selective Breeding

The enrichment has to be transfered to a selection such as selective medium according to Weslei 1983, Blaser and Wang (1979) or Campy BAP Agar and other. Incubate at 42° for 48 hours under the atmosphere described above.

Biochemical confirm

Suspicious colonies are examined under a phase contrast microscope searching for comma formed bacteria.
As basis for the biochemical reactions of Campylobacter jejuni use Brucella broth with 0,16% agar.
Comparison of the methods of standard microbiological culture versus RFLP-PCR for the identification of Campylobacter was done by V. Atanassova and Ch. Ring. They found that 75.64% of swab samples collected from laying hens were tested positive by RFLP-PCR and only 33.33% by standard microbiological culture[35].

DNA based methods for the Campylobacter diagnostics

DNA diagnostics of Campylobacter is the most specific and sensitive detection technique reducing the diagnostic time of 4 days with normal culture bacteriology to 4 hour with DNA techniques starting from intestinal content and faecal samples. This makes tests of poultry possible prior to slaughter in order to guarantee Campylobacter free products.

The current DNA based tests are not able to distinguish living and dead microorganisms.

The DNA based tests follow the way of separation from matrix, cell lysis, DNA purification and detection. System used are such as Genpoint AS Oslo, Norway.:

The sample is added to a buffer containing magnetic beads which are coated with a surface which absorbs bacteria. The bacteria are immobilized on the beads. The beads are pulled to the side of the tube by a magnet and the sample solution can be removed. Bacterial lysis and DNA immobilization follows so pure DNA is bound to the beads.

This technique allows to isolate and detect several strains and species simultaneously using PCR amplification of the specific DNA labeling it with specific primers and followed by hybridization of the probes to oligonucleotide array giving a signal for the different bacteria in the sample.

Enhanced pathogen bacteria rapid detection systems

In a review in 2004 Stevens and Yaykus wrote that rapid detection technologies of small number of pathogen bacteria should be improved.

Bacterial concentration may perhaps reduce or even eliminate the need for cultural enrichment prior to detection. However, methods such as centrifugation, filtration, and immunomagnetic separation were still not ideal and continued to be a stumbling block in the advancement of molecular methods for the detection of foodborne pathogens. [36]

In this field advances have been achieved, such as immuno-capture magnetic bead systems of Matrix MicroScience that selectively concentrates target microbial pathogens from complex food matrix. It uses paramagnetic beads coated with antibodies facilitates the rapid detection of target bacteria. Viable cultures are produced which enable full and detailed analysis, of any positive result to be carried out. These systems can be used to enhance the performance of other rapid methods such as PCR, lateral flow, ELISA, chromogenic media etc by significantly reducing or eliminating the need for lengthy enrichment and/or selective enrichment steps. [37]

Fukushima and colleagues 2007 developed a density gradient centrifugation method to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. The combined separation and concentration methods and RTi-qPCR may confirme within 3 h the presence of 10 to 100 CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus. The author stresses the feasibility of rapid detection of pathogenic bacteria during outbreaks. [38]

New Zealand produces update on work related to Campylobacter [39]

As concern around New Zealand's high rates of campylobacteriosis continues, the New Zealand Food Safety Authority (NZFSA) has put together a a report "A Background to Campylobacter" [40] which sets out, in easy-to-understand terms, the scientific research that NZFSA has collated on Campylobacter in food and the practical measures currently in place to contain it, as well as an update on what is happening in this regard overseas.

Campylobacter is naturally present throughout the environment, in water, on animals (including birds and pets) as well as being found on meat and food products.

While different interventions may offer reductions in hazard levels at certain points in the farm-to-fork continuum, it is a combination of measures that is more likely to achieve the greatest reduction in risk to consumers.

According Steve Hathaway, Director of NZFSA, the agency is considering some short-term measures that will decrease contamination rates in poultry. However, the aim has always been to focus on not just removing the high levels of the pathogen from the food chain, but to find ways to prevent it getting there in the first place.


Genus Arcobacter

The genus Arcobacter is composed of Campylobacter - like bacteria.
They were found associated with bovine and porcine abortion. They are aero tolerant,gram negative and are strongly motile.
Arcobacter spp. has its habitat in animals like bovines,swine, primates and poultry;human beings are not excluded.


Occurrence of Arcobacter spp. in Food[41]:


Diseases caused by Arcobacter spp.

Acute diarrhea,appedicitis,septicemy and diarrhea in animals.

Culture of Arcobacter spp [42]

The Culture of Arcobacter spp. is made with enrichment broth and isolation medium followed by biochemical and serological identification.


Enrichment in Arcobacter-Selective-Broth (ASB)

Composition of the broth [42]

28 g Brucella broth powder (Difco) in 910 ml A.dest. After sterilization and cooling at about 50-60°.
Add 50 ml of lysated horse blood, 75 mg piperacillin (Sigma) dissolved in 10 ml Aqua dest., sterilized by filtration, 32 mg ceferoperazone (Sigma) dissolved in 10 ml Aqua dest., sterilized by filtration, 20 mg trimethoprim (Sigma)dissolved in 4 ml ethanol (96%) and 6 ml Aqua dest.sterilized by filtration, and 100 mg Cycloheximide (Serva) dissolved in 10 ml Aqua dest., sterilized by filtration. Final pH should be 7,0 +-0,2. Distribution should be made in 10 ml portions in tubes.

Isolation medium for Arcobacter spp.

Isolation is made using Arcobacter selective Medium ASM

Composition

21 g Mueller-Hinton-Broth (Oxoid) and 2,5 g Agar Nr.3 (Oxoid) in 960 ml Aqua dest.
After sterilization and cooling to 50° add all substances cited under ASB medium with exception of blood.Final pH should be 7,4 +- 0,2.
20 g of product to be tested is homogenized in 180 ml physiological Na Cl solution.
1 ml of the suspension is added to 10 ml broth. Incubate 48 hours at 24°.

Identification:Spraying of Arcobacter spp. biochemical and serological identification.
The genus Arcobacter has four species:

Arcobacter cryaerophilus
Arcobacter butzleri: was first describe by Kielbauch et al. Later Vandamme et al. changed this classification to Arcobacter butzleri. These organisms could grow in presence of air and 30° which made the distinction to Campylobacter and were found in diarrhoeas in blood and in peritonial fluids. They were found also in not sufficient cooked poultry, cattle, swine, ovine, equine,primates, sewage and water.The serotyping based on slide agglutination on living bacteria distinguishes between 73 serogroups and biotyping finds out 16 biotypes.

Aerobacter nitrofigilis
Aerobacter skirowii

Helicobacter

The genus contains 16 species. The most important human pathogen are:
Helicobacter pylori
Helicobacter cinaedi
Helicobacter fennelliae
Adult animal pathogen, not found as human pathogen:
Helicobacter mustelae: In 1980 Marshall and Warren cultured in campylobacter media a spiral bacteria from gastric biopsy.
These bacteria were later denominated as Helicobacter pylori being responsible to gastritis and duodenal ulcers and are associated to gastric carcinome and gastric lymphoma.

Claims of an association between Helicobacter pylori and atheroma leading to artheriosclerosis have become less credible, as the organism has not been detected directly from atheromatous lesions and 18 serological studies have failed to support the association [43].
It is being spread from person to person among the family. Cats may harbor the organism.

The genus Helicobacter was created by Goodwin et al. in 1989
Helicobacter grows slowly on brain heart infusion BHI. Growth at 30°. No growths at 25° , optimum at 37°. Colonies: non colored, translucents 1 - 2 mm
mobility: rapid.
Glycine: Growth with 0,5% Glycine and 0,04% triphenyltetrazolium chloride.
NaCl: No growth with 3,5% NaCl.
Catalase: positive
Urea: positive
H2S: negative on TSI and variable on lead acetate paper.
Nitrate: Variable
Hipurate: Variable
Alkaline phosphatase: positive
Gama-glutamyltranspeptidase: positive
Leucine arylamidase: variable
Susceptible to: penicillin, ampicillin, amoxicillin, erythromycine, gentamicin, kanamycin, rifampin, tetracycline.
Resistant to: vancomycin, sulfonamides, and trimethoprim. Variable resistance: nalidixic acid, cephalothin, metronidazole and polymyxin.
Isolation: from the gastric mucosa of primates and ferrets.
Pathogenity: human gastritis and peptic ulcereation (gastric and duodenal)

It includes two species:
  H. mustelae H. pylori
Growth at 42° + -
Growth in 10% CO2 - +
Growth on PSD agar - +
Growth with 1% Glycine d -
Nitrate reduction + -
Susceptible to Cephalothin 30 - +
Causes type B gastritis and    
gastric and duodenal ulcers - +
Causes gastritis and ulcers in    
adult animals + -
Cellular fatty acids 3-OH 18:0 - +


PSD agar: Peptone-starch dextrose agar (Dunkelberg et al Appl.Microbiol. 19: 47-52 , 1970)

Suppression of Helicobacter pylori by green tea extracts

[44]
Keiji Wakabayashi and colleagues 2003 assessed foodstuffs on its capability to suppress the urease enzyme of Helicobacter pylori which is is essential for its colonization.
The authors found some tea such as green tea extracts and rosemary extracts inhibit H. pylori urease in vitro. Catechins, the hydroxyl group of 5'-position were the active components inhibiting urease. H. pylori-infected Monglian gerbils responded to the administration of green tea extract with suppressed gastritis and the absence H.pilori.

The authors concluded that tea and tea catechins may help to control H. pylori-associated gastroduodenal diseases, since H.pylori is getting resistant to antibiotics.

The eradication of Helicobacter pylori after gastric cancer cancer surgery [45]

Nicholas J. Talley wrote that the risk for regression of gastric cancer after gastric surgery was reduced after eradicating the bacteria Helicobacter pylori. Previous studies claimed that there was a relation between Helicobacter pylori infection and gastric cancer. [46]

Despite being classified as carcinogen by the WHO, and the Asian-Pacific consensus conference in 2007 having recommended that population-based screening and antibiotic treatment of Helicobacter pylori in high-risk populations, this is not generally accepted. The author stresses therefore that eradication of this bacterium should be priority in regions with high incidence of gastric cancer.

According to Kazutoshi Fukase and colleagues, 2008 the prophylactic eradication of Helicobacter pylori after endoscopic resection of early gastric cancer should be used to prevent the development of metachronous gastric carcinoma.[47]

The study of Fukase contradicts previous studies which found no relation between the bacterium and stomach cancer. [48] [49]

Transmission and sources of infection of Campylobacter

Enteritis cause by Campylobacter is a zoonosis, resulting from contact to poultry, cattle, raw milk, surface water and pets, following the faecal-oral route.
The consumption of undercooked chicken is one major cause of outbreaks.

Avoid contamination of Campylobacter

To avoid contamination it is necessary to interrupt the chain of cross-contamination in kitchen caused by utensils, chopping-boards, hands and raw vegetables, handling poultry and red meats.

Campylobacter were found in 5% of retail red meats and ground beef in USA and Canada and up to 23% in beef in UK. Important outbreaks were noted in Water and raw milk , especially in school children who drank raw milk.

Pasteurization kills Campylobacter. A contamination after pasteurization however cannot be excluded.
Nonchlorinated water can be contaminated with sewage, birds and animal faecal material, so that there were many outbreaks of Campylobacter caused by municipal waters in USA and Sweden.

In England the findings of Campylobacter in sea water and fresh water from rivers were always associated with Escherichia .
The infection dose is very low: 2 bacteria/ml which turns the contamination through water very likely.

The transmission from person to person is however very unlikely with exclusion in the case of mother/neonate.
More likely is however the transmission from dogs and cats, birds or monkeys.

The infections prevail in the summer, in the equatorial zone during the rain season.
Campylobacter should always be presumed as a traveler risk. Campylobacter jejuni and C. are present in up to 100% in broiler chicken and as normal intestinal flora or domestic animals such as poultry and turkeys.

Prevention of infection with Campylobacter

General hygienic measures will prevent the spread of infection. Hand washing after contact with animal or their products, proper cooking and storage of food, pasteurization of milk and chlorination of water are important to prevent the disease.

Irradiation of food should be allowed to reduce significantly the number of food born pathogens.

Isolation and identification

Phenotyping of Campylobacter includes species identification, serotyping , biotyping, phagetyping.
Molecular genotyping methods includes pulsed field gel electrophoresis (PFGE) random amplified polymorphic DNA (RAPD)

Selective medias such as charcoal media filtration techniques on media without antibiotics are used to grow Campylobacter which do not grow on media with antibiotics.

Enrichment broth

It is used only on food and water.

Presumptive identification

Gram-stain, wet mount for specific motility, oxidase test and hyppurate hydrolysis.

Presumptive confirmation

Commercial latex tests are avaliable: Campyslide (BBL) which identifies the genus and Meritec-Campy (Meridian Diagnostics) to identify C.jejuni, C., and C. upsaliensis.The use of nalidixic acid as antibiotic to select Campylobacter is not reliable because the bacteria has developed resistance to quinolones Non-culturable forms:
Campylobacter as well as Vibrio cholerae, Salmonella enteritides and enteropathogenic E.coli have developed Campylobacteroid forms to resist environmental conditions which do not grow on media. To recover these forms a passage in suckling mice is necessary.

The polymerase chain reaction ( PCR ) has made possible to detect non-culturable forms by amplification of specific DNA sequences.
PCR with a primer for the flagellin gene is used to detect Campylobacter jejuni and Campylobacter coli in stools in chicken and in water.
Indoxyl acetate hydrolysis test: Specification of Campylobacter which can be used as a simple test.
C.lari C.jejuni spp.jejuni C.coli C.lari C.lari C.lari C.lari C.lari C.lari
biotype I II III IV I II I II
Test                
Hippurate hydrolises + + + + - - - -
Rapid H2S test - - + + - - + +
DNA hydrolysis - + - + - + - +
Serogroups 74 46 0          


Culture of Campylobacter spp using filter technique from the Veterinary department of Münster,1998

20 ml or 20 g of the sample are minced with a scissors and tweezers.
Mix without shaking (squeeze by hand) in 90 ml Preston-Broth and incubate at 42°.
Place a filter type DA, 0,65m, Fa Millipore, Kat Nr. DAW PO 4700 avoiding air bubbles. Distribute 300l from the enrichment broth on the filter paper. Incubate the Petry plate for 1 hour at room temperature.Remove the filter and incubated the medium at 42° microaerophile for 48 hours

Escherichia Coli [50]

Escherichia coli is a normal inhabitant of the intestinal human and animal tract.
His presence in food is considered to be an indicator of faecal contamination and causes deterioration.
Some strains of Escherichia coli can however be pathogenic. The number of food born infections are increasing continuously.The outbreaks become greater by high shelf life of raw material and end products, increasing number of communal feeding and worldwide increasing distribution of food and animal food.

In 1982 a great food born infection caused by underheated hamburger lead to the discovering of Enterohaemorrhagic Escherichia coli (EHEC). It was the E.coli O 157:H7 strain.

In 1955 there was a great outbreak of EHEC-bacteria in Bavaria, Germany.
EHEC are intestinal pathogenic bacteria producing watery and bloody diarrhoea with colical intestinal pain, the haemorrhagic colitis, HC.
This infection can develop with life menacing complications in children under six years and in old people.

First there are intestinal symptoms followed after three to twelve days after contamination by an haemolitic uraemic syndrome, (HUS) with damage of the kidneys with 10% of obit, another 10 to 30% develope a permanent kidney damage which makes a lifelong dialysis necessary.
Neurological and artheroschlerotic complications may also occur.
In Germany there are about 8.000 to 16.000 cases of EHEC each year.

Most common places of infection are communal feeding, such as nurseries,kindergarten, old people's home and restaurants, especially fast food.
Most likely contaminated food are ground meat, underheated hamburgers, sausage,turkey sandwiches and underheated milk, all animal food, especially of ruminant origin as most important reservoir of EHEC and contaminated vegetables.

Prophylaxis should be concentrated on proper instalation of toilets and hygienical conditions.
Other important focus of infections are water, salads, vegetables, fresh appel juice and contamination by smear infection caused by diseased persons.

Smear infection seams to have very great importance in infection with EHEC. The diagnostic of Enterohemorrhagic Escherichia coli EHEC is made by isolation of the germ using enterohemolisine-agar and using latex agglutination test.
Escherichia coli strains may produce verotoxine. The strains of Escherichia coli which produce verotoxine are labeled as VTEC-bacteria. EHEC-strains are classified under the group of VTEC- strains.

Cucumber imported from Spain carrying the EHEC bacterium may be implicated in the actual outbreak of EHEC infections [51]

The EHEC infection outbreak of May 2011 in Germany has now spread to Denmark and other countries. Cucumber from Spain were found carrying the pathogen bacteria at an outlet in Hamburg.

Scientists at the National Consulting Laboratory on Hemolytic Uremic Syndrome in Münster compared the genetic code of previous EHEC cases in Germany with the actual strain which was identified HUSEC41, sequence type ST678 of the seroytpe Escherichia coli O104:H4. It is resistant to different antibiotics.

Enterohaemorrhagic Escherichia coli (EHEC) are the pathogenic subgroup of Shiga toxin (Stx)-producing E. coli. EHEC can cause non-bloody and bloody diarrhoea, and the haemolytic uraemic syndrome (HUS) There are different serotypes of EHEC one of which is the E. coli O57:H7 known from other outbreaks. [52]

Microbiologists suggest that the EHEC bacterium developed from the Escherichia coli which lives in the healthy gut of men and animals. Over a certain period genetic material from bacteriphages were introduced in the DNA of the E.coli which mutated to pathogen strains. The excessive use of antibiotics and toxic agrarian chemicals speeded this process. According to veterinary officials the pathogen strains infects six per cent of cattle without producing disease. Manure containing these strains may contaminate directly vegetables and fruits or indirectly through affected irrigation water.

Helge Karch of the Institute of Münster developed a method to speed the identification of the bacteria. Further work is being done to determine if there are any genetic variations which differ from the original Husec 41 strain.

According to Karch, the O104:H4 is not known to have caused an outbreak before. The laboratory is now sequencing the whole genome of HUSEC41. An important finding is that the strain lacks the eae gene which encodes the protein intimin.

Intimin is a virulence factor (adhesin) of EHEC such as E. coli O127:H6 and EHEC such as E. coli O157:H7 Escherichia coli strains. It is an attaching and effacing protein which together with other virulence factors is responsible for enteropathogenic and enterohaemorrhagic diarrhoea. [53]
Intimin is expressed on the bacterial cell surface. Together with Tir (Translocated intimin receptor) intimin is linked to infections of children. Its absence may explain the fact that mainly adult persons are affected from this outbreak.

The source of the Escherichia coli O104:H4 outbreak in Germany is still unkown []

The Escherichia coli O104:H4 outbreak in Germany has spread to Switzerland, Poland, the Netherlands, Sweden, Denmark, and the United Kingdom. All patients reported outside Germany had visited the country and were probably infected there. German officials initially pointed to contaminated cucumbers originating in Spain as the source of the outbreak, but later recognised that Spanish cucumbers were not the source of the pathogenic strain which causes the epidemic.

Meanwhile a great number of patients are hospitalised with Hemolytic-uremic syndrome (HUS) which requires urgent treatment. In Germany, 15 people have died and around 500 have been hospitalised with HUS. Presenting hemolytic anemia, acute renal failure (uremia) and a low platelet count. It predominantly but not exclusively affects children. Most cases are preceded by an episode of diarrhoea caused by an EHEC strain. The disease is acquired as a foodborne illness with a mortality of 5-10%. Health officials recommend not to eat cucumber salad and fresh tomatoes.

Characterisation of the Escherichia coli strain O104:H4 of the 2011 HUS epidemic [54]

The outbreak of the haemolytic uraemic syndrome and bloody diarrhoea caused by a virulent Escherichia coli strain O104:H4 in Germany counts fro 810 cases of the syndrome and 39 deaths since May, 2011.

Karch et al. 2011 found that all isolates were of the HUSEC041 clone (sequence type 678). All shared virulence profiles combining typical Shiga-toxin-producing E coli (stx2, iha, lpfO26, lpfO113) and enteroaggregative E coli (aggA, aggR, set1, pic, aap) loci and expressed phenotypes that define Shiga-toxin-producing E coli and enteroaggregative E coli, including production of Shiga toxing 2 and aggregative adherence to epithelial cells. Isolates additionally displayed an extended-spectrum beta-lactamase phenotype absent in HUSEC041.

The authors suggest that the high adherence of the strain to intestinal epithelium might facilitate the absorption of Shiga toxin and explain the HUS outcomes. The authors concluded that blended virulence profiles in such pathogens may become extreme virulent.

Rapid next-generation technologies for whole genome characterization [55]

Karch et al. compared the whole genome sequence of the outbreak isolate of 2011 and a historic O104:H4 HUS isolate from 2001. The HUS-associated strains of both isolates carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC).

The HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. The outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes.

The serotype O104:H4 had rarely been associated with HUS in the past. The authors suggest, therefore, that EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor. Stepwise gain and loss of chromosomal and plasmid-encoded virulence factors produced the highly pathogenic hybrid of EAEC and EHEC of the current outbreak.

Chinese/German cooperation unveiled the genetic code of the deadly German EHEC outbreak [56]

The Beijing Genomic Institute (BGI) in cooperation with the University Medical Center Hamburg-Eppendorf researchers, unveiled the genome of the EHEC strain. They report that the genome size is about 5.2 Mb. It is a new serotype of the 0104 Escherichia coli strain with 93 % sequence similarity with the EAEC 55989 E.coli strain from Central African Republic. Through horizontal gene transfer it acquired additional specific sequences of hemorrhagic colitis and hemolytic-uremic syndrome. Several antibiotic resistance genes, including resistance to aminoglycoside, macrolides and Beta-lactam antibiotics increase to deadly potential of this strain. The Chinese BGI sequenced the genome within three days using their technology. The BGI developes diagnostic kits to aid to control the European outbreak.

BGI and University of Lübeck cooperation [57]

German Chancellor Merkel and Chinese Prime Minister Wen Jiabao were present at the ceremony of the signing of the cooperation contract between Beijing Genomics Institute and University of Lübeck, strengthening the collaboration of the two institutions in the field of genomic and structural analysis of new viruses. Many of these, such as the bird flu virus H5N1 or the SARS coronavirus, have their origin in Southern China, from where they spread towards Europe (as presently is the case with H5N1) or Northern America (as with the SARS virus in 2003).

Within the collaboration, the Beijing researchers will analyse the genetic material of the new viruses, whereas the Luebeck group will determine the three-dimensional structures of key components of the pathogens and develop antiviral drugs on this basis. Using such an approach, Professor Hilgenfeld had succeeded in presenting an anti-SARS compound in May 2003, only a few weeks after the discovery of the new virus and during the ongoing global SARS outbreak.

Chinese/German cooperation identified the EHEC bacteria, but will the source of the epidemic remain unidentified for ever? [58]

06.06.2011: Shiga toxin-producing E. coli (STEC) is a group of pathogenic Escherichia coli strains capable of producing Shiga toxins, with the potential to cause severe enteric and systemic disease in humans. The full serotype is usually defined by determining both O and H antigens. There are around 200 different E. coli O serotypes producing Shiga toxin, of which over 100 have been associated with human disease. Two major Shiga toxin types (Stx1 and Stx2) have been associated with strains causing human disease. While the serotype O157:H7 is considered as clinically the most important, it is estimated that up to 50% of STEC infections are caused by non-O157 serotypes. STEC is of public health concern because of the potential for outbreaks and the risk of serious complications. Haemolytic uremic syndrome (HUS) is considered as the most common cause of acute renal failure in European children.

Transmission of STEC infection mainly occurs through contaminated food or water and contact with animals. Person-to-person transmission is also possible among close contacts (families, childcare centres, nursing homes, etc). A wide variety of food has previously been implicated in outbreaks as suspected sources, including raw (unpasteurised) raw milk and cheese, undercooked beef, a variety of fresh produce (e.g. sprouts, spinach, lettuce), unpasteurised apple cider, etc. Recently an outbreak of STEC O157 infections in Canada and the USA was linked to walnuts, thus new sources continue to be identified. Various types of animals, in particular cattle and other ruminants, can be healthy carriers of human-pathogenic STEC that can be spread to humans through faecal contamination.

Secondary clusters of cases from person-to-person exposure may occur and thus personal hygiene messages are important.

First diarrhoea cases were reported in early May 2011. On 22 May Germany informed the European Commission's Early Warning and Response System (EWRS) of a significant increase in the number of patients with hemolytic uremic sysndrome (HUS) and bloody diarrhea caused by enterohemorrhagic Escherichia coli (EHEC).

Four samples of cucumber tested positive for EHEC bacteria, but these germs were not the strain which causes the epidemic in Europe. Later on vegetable sprouts were thought to be implicated in the outbreak, but tests on batches from the Gaertnerhof organic sprouts farm in the northern German village of Bienenbuettel were negative.

Escherichia coli O157:H7 vaccine developed in China [59]

Escherichia coli O157 H7 causes bloody diarrhoea with kidney failure and death. The bacteria uses intimin for the adhesion in the first step of interaction with the host and releases the Shiga toxins (Stxs), the poisons responsible for the sickening of the host.

Gao and colleagues 2011 of the State Key Laboratory of Pathogens and Biosecurity, Beijing, China, report the production of a a novel SSI fusion protein that contains the critical toxin-antigens Stx2B and Stx1B, and the critical adhesion-antigen fragment Int281.

The SSI induced Th2-mediated immune protection in a mouse model, triggering the production of antibodies against both Stx1 and Stx2 toxins, and produced a high level of anti-adhesion antibodies. The protein induced complete immune protection, with both anti-toxin and anti-adhesion effects.

Chinese/German cooperation [56]

The strain of STEC causing these illnesses, STEC O104:H4 is very rare. Beijing Genomic Institute (BGI) in cooperation with the University Medical Center Hamburg-Eppendorf researchers, unveiled the genome of the EHEC strain. They report that the genome size is about 5.2 Mb. It is a new serotype of the 0104 Escherichia coli strain with 93 % sequence similarity with the EAEC 55989 E.coli strain from Central African Republic. Through horizontal gene transfer it acquired additional specific sequences of hemorrhagic colitis and hemolytic-uremic syndrome. Several antibiotic resistance genes, including resistance to aminoglycoside, macrolides and Beta-lactam antibiotics increase to deadly potential of this strain. The Chinese BGI sequenced the genome within three days using their technology. The BGI is now developing diagnostic kits to aid to control the European outbreak.

WHO and EHEC alert [60]

The World Health Organization (WHO) confirms that a total of 1823 cases of STEC O104:H4 have been reported, including 520 cases of hemolytic uremic syndrome (HUS), a potentially life-threatening complication of the infection that can cause kidney failure. Twelve HUS cases were fatal, and 6 deaths were reported among non-HUS cases. Most infections have been reported in people in northern Germany (mainly Bremen, Hamburg, Lower Saxony, and Schleswig-Holstein) or in people who have recently travelled to these areas. Cases in travellers to northern Germany have been reported in Denmark, the Netherlands, Spain, Sweden, and the United Kingdom. German health authorities are investigating the outbreak but have not confirmed a source. They suspect that the source is contaminated food, possibly raw vegetables.

The functioning of epidemiology and global public health system is being questioned [61]

Dr. Michael Osterholm, director of the Center for Infectious Disease Research and Policy at the University of Minnesota calls "incompetence" the way the source of the epidemic is being investigated. However, the problem may be worse than just "incompetence". It seems that the food safety systems based on HACCP, Good Manufacturing Practice, ISO 9000 Certification failed to protect the consumer, resulting in over 20 fatalities up to present. The whole series of global food safety systems must be revised to avoid pathogens to enter the food chain without being detected by routine tests at farm, during processing, and at grocery stores.

EHEC bacterium enters water resources and environment [62]

Experts fear that the EHEC O104:h4 (Husec041) may spread to communal waters and drinking water reservoirs. If so, tap water is endangered as Escherichia coli bacteria are sporadically being found in drinking water samples, says Martin Exner, director of the Hygiene Institute of the Uni-Clinic Bonn and head of the drinking water commission of the German Federal Environmental Agency. The deadly bacterium strain has now been identified in water of a small river near Frankfurt, Germany. The possibility that this strain enters the drinking water systems has been underestimated. According to Helge Karch, director of the Institute of Hygiene of the university-clinic Münster, there are many EHEC patients. They all egest high number of pathogen bacteria. It cannot be excluded that the deadly strain has found its place in the local environment and will cause more human infections through water.

Call for stricter control of waterworks in Germany

The drinking water commission of the German Federal Environmental Agency, on its last meeting, called for strict controls of faecal bacteria in water for overhead sprinkling systems and process water in vegetable ans sprouts production as well as water from small waterworks. Waterworks of great cities have their water tested on Escherichia coli often, however, small waterworks test only once a year. Before the EHEC epidemic started, five per cent of tap water samples of small waterworks of Baden-Wuerttenberg tested positive for E.coli bacteria, and almost every second private well of this German region contained gut bacteria, reports the WHO. At this meeting the Drinking Water Commission called for stringent control

The French EHEC outbreak, sprouts and minced meat [63]

According to Professor Patrick Berche, head of the bacteriology department at Necker Hospital in Paris, the French outbreak of E Coli in Begles, a south-western French city of Bordeaux the strain type 0104 enterohaemorrhagic Escherichia coli (EHEC) was confirmed in two of seven diseased patients. Sprouts grown from rocket, fenugreek and mustard seeds that came from a British mail order seed and plant company. Ipswich-based Thomson &amp had been added to a soup which might have been the source of the infection. No EHEC bacteria on sprouts were found and a lot of sprouts and seeds of this company has been sold all over Europe without complaint. The outbreak comes days after an EHEC outbreak in a group of children in the northern city of Lille. That outbreak came from frozen supermarket beef burgers made in France, using minced meat originating from Germany, Belgium and The Netherlands.

European cattle herds are the primary source of the EHEC strain

All epidemiological investigations are failing to find the source of the European EHEC epidemic. While concentrating on sprouts the real source is being neglected. Health officials must concentrate their investigations on cattle, the primary source of the EHEC coli strain. All positive farms must be closed,sanitised and new EHEC breed introduced. All links point to cows manure which contaminates water resources.

Source of the HUS bacterium [64]

EHEC can grow in temperatures ranging from from 7° to 50°, with an optimum temperature of 37°. Some EHEC can grow in acidic foods, down to a pH of 4.4, and in foods with a minimum water activity (Aw) of 0.95. It is destroyed by thorough cooking of foods until all parts reach a temperature of 70° or higher. E. coli O157:H7 is the most important EHEC serotype in relation to public health, however, other serotypes have frequently been involved in sporadic cases and outbreaks.

WHO reports that in addition to Germany, EHEC cases have also been notified from: Austria 2, Czech Republic 1, Denmark 10, France 6, Netherlands 4, Norway 1, Sweden 28, Switzerland 2, United Kingdom 4.

Cows and camels breed the HUS bacterium says WHO [64]

Ruminants, particularly cows host different coli strains, including the EHEC bacterium without experiencing any harm. Their digestive tract is a real coli cloning laboratory which united the African strain with the local EHEC coli, resulting in the deadly HUS strain.

The reservoir of this pathogen appears to be mainly cattle and other ruminants such as camels. It is transmitted to humans primarily through consumption of contaminated foods, such as raw or undercooked ground meat products and raw milk. Faecal contamination of water and other foods, as well as cross-contamination during food preparation (with beef and other meat products, contaminated surfaces and kitchen utensils), will also lead to infection. EHEC has also been isolated from bodies of water (ponds, streams), wells and water troughs, and has been found to survive for months in manure and water-trough sediments. Waterborne transmission has been reported, both from contaminated drinking-water and from recreational waters.

Italian EHEC case confirms hypothesis of oral-faecal infection route instead of a ruminant source [65]

Scavia et al 2011 report that a HUS case in Italy in 2009 by the STEC strain (ED-703) was found to have the same combination of virulence factors as the strain STEC O104 strain of the German and French epidemic in 2011. It presented identical Stx2 production and enteroaggregative adhesion genetic markers. The Italian strain of 2009 was positive for O104 and H4 antigen-associated genes, was agglutinated by an O104 antiserum and presented high degree of genetic similarity with the outbreak strain from Germany, but did not produce extended-spectrum β -lactamases.

This case reported a travel to Tunisia. No unpasteurized milk or dairy products, undercooked meat, or raw sprouts had been consumed and no direct exposure to ruminants or their manure had taken place. Scavia and colleagues suggest, therefore, that the infection was probably acquired through person-to-person transmission, and strains of STEC O104 strictly related to the epidemic strain in Germany had already caused sporadic infections in Europe. Such cases had been reported in 2001 in Germany, in 2004 in France, and in 2010 in Finland in a patient with diarrhoea who had travelled to Egypt from which the sprout seeds associated with both outbreaks could be traced

The authors concluded that ruminants would not have had a specific role in the transmission of STEC O104:H4, as already suggested by the epidemiological features of the recent outbreaks. In fact, STEC O104 cannot be considered true STEC but rather EAEC strains that acquired the Stx2-coding phages by horizontal gene transfer, and EAEC is considered to be a human pathogen usually transmitted by the oral–faecal route

Enteric viruses in European recreational waters

Low concentrations of viruses were found in water of European rivers and at beaches. Their number may increase after heavy rain. Researchers recommend to include viruses in the monitoring of recreational waters

The European Bathing Water Directive [66] establishes maximum levels for bacteria, in particular Escherichia coli and intestinal enterococcus, which must not be exceeded in order to maintain water quality, but does not include enteric viruses to monitore recreational waters. The EU Virobathe project is aimed at the rapid detection of viruses in recreational waters.Viruses which cause gastroenteritis in humans may be present in sewage-polluted bathing waters. Virobathe improves the molecular DNA technology to detect viruses in water and will contribute to the improvements in European bathing water quality. Virobathe

Wyn-Jones et al. 2011 and other groups from the EU Virobathe project, determined the presence of adenoviruses and noroviruses in swimming water, both freshwater and seawater, in different European countries.

The authors selected adenoviruses for their study, because these viruses are shed along the whole year and are resistant to environmental stress. Noroviruses are of importance as indicator of enteric viruses because they are widespread gastroenteritis agents. The authors detected enteric viruses in 39,2% of samples of water, adenoviruses in 36.4%, noroviruses in9.4%. More freshwater samples than marine water were found contaminated with viruses, and one-quarter remained infective. According to the authors the presence of infectious viruses in recreational waters are a threat to public health, and adenoviruses should be used as an indicator of bathing water quality. Virus numbers in bathing waters increase following heavy rains, and adenoviruses may dangerous levels Viruses take longer than bacteria to return to acceptable levels following heavy rains, and survive waste water treatment processes better than bacteria, and are more resistant to seawater. [67]

According to Bofill-Mas et al.2010 human adenoviruses in recreational water present a threat to the population. The authors found no relation between human adenoviruses and E. coli , intestinal enterococci or somatic coliphages concentrations. [68]

Importance for environmental microbiology of the prevalence of enteric viruses in wastewater [69]

La Rosa et al. 2010 point to the potential health risks of the release into the environment of enteric viruses in treated wastewaters,

Using quantitative TaqMan real-time PCR (polymerase chain reaction) the authors determined the presence of adenoviruse,enteroviruses and noroviruses (GI and GII) in influent and effluent wastewaters of treatment plants at Rome, and compared the results to classical bacterial indicator of faecal contamination.

The authors report a removal efficiencies which ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Infectivity assays confirmed potential public health risks through effluent wasters of communal water treatment plants. Bacterial indicators were found inappropriate to monitor wastewater viral removal efficiency.

Control and prevention methods

The prevention of infection requires control measures at all stages of the food chain, from agricultural production on the farm to processing, manufacturing and preparation of foods in both commercial establishments and the domestic environment. Available data are not sufficient to enable the recommendation of specific intervention methods on the farm in order to reduce the incidence of EHEC in cattle. However, risk assessments conducted at national level have predicted that the number of cases of disease might be reduced by various mitigation strategies for ground beef (for example, screening the animals preslaughter to reduce the introduction of large numbers of pathogens in the slaughtering environment)

Since a number of EHEC infections have been caused by contact with recreational water, it is also important to protect such water areas, as well as drinking-water sources, from animal wastes.

Recommendations to reduce the public health risk

To ensure that those who come directly or indirectly into contact with food are not likely to contaminate it with EHEC, food handlers should follow the Recommended International Code of Practice, General Principles of Food Hygiene. [70]

Basic good food hygiene practice, as described in the WHO Five keys to safer food , can prevent the transmission of pathogens responsible for many foodborne diseases, and also protect against foodborne diseases caused by EHEC. Such recommendations should in all cases be implemented, especially "Cook thoroughly" so that at least the centre of the food reaches 70°.

Specific recommendations to sprout producers

In recent years, the popularity of sprouted seeds has increased significantly owing to their nutritional value. However, reports of foodborne outbreaks associated with such raw vegetable sprouts have raised concerns among public health agencies and consumers. Outbreak investigations have indicated that pathogens found on sprouts most likely originate from the seeds. The seed may be contaminated in the field or during harvesting, storage or transportation. During the germination process in sprout production, low levels of pathogens present on seeds may quickly reach levels high enough to cause disease. Therefore specific care is needed. Guidance is available in the Codex Code of Hygienic Practice for Fresh Fruits and Vegetables, Annex for sprout production (document CAC/RCP 53-2003 of the Codex Alimentarius Commission. [71]

Seeds and sprouted seeds, health risks and recommendations of the EFSA [72]

The European Food Safety Authority (EFSA) assessed the risk of pathogenic bacteria that may contaminate seeds intended for sprouting and sprouted seeds (sprouts, shoots and cress) which are generally consumed raw or minimally processed.

Pathogenic bacteria can contaminate seeds and grow during sprouting. Furthermore, preventing initial contamination during production, storage and distribution of seeds is of the foremost importance.

The Germany and France sprout-associated outbreaks in 2011 had been preceded by large outbreaks in the EU and worldwide commonly caused by Salmonella and pathogenic E. coli (including STEC). Very low levels of the bacteria-as little as 4 bacteria/kg-in seeds intended for sprouting have been sufficient to cause outbreaks. Other bacterial pathogens (e.g. Bacillus cereus, Listeria monocytogenes and Yersinia enterocolitica) have also been implicated in sprout-associated outbreaks, but very rarely.

These pathogenic bacteria can contaminate the seeds intended for sprouting during production, storage and distribution through, for example, contaminated irrigation water and soil particles. The high temperature and humidity needed for the germination and sprouting of seeds are also favourable conditions for pathogenic bacteria to further grow and spread.


Panel recommends additional safety measures for the sprouted seed production chain

Panel considers sprouted seeds as ready-to-eat foods and therefore recommends that general EU food safety hygiene rules should be applied across the whole chain from seed production to the final sprouted product. Food safety systems such as Hazard Analysis and Critical Control Point (HACCP) principles, Good Hygiene Practices (GHP), Good Agricultural Practices (GAP) and Good Manufacturing Practices (GMP) should be applied. The Panel concludes that preventing initial contamination of seeds intended for sprouting is of particular importance, as there are currently no methods to ensure elimination of pathogens in all types of seeds used for sprouting.

Suggested mitigation options include but are not limited to: identifying seed crops intended for sprout production before planting; safe use of fertilizers and irrigation water; minimizing contamination of seeds with soil during harvest and preventing mechanical damage of seeds; ensuring that workers harvesting and handling seeds follow hygiene and health requirements; ensuring that seeds are transported, processed and stored under conditions which will minimize the potential for microbial contamination; removing damaged seeds; and improving traceability and minimizing mixing of seed lots.

Operators producing sprouted seeds should strive to implement additional food safety management measures across the whole sprout production chain. Stakeholders at all parts of the production chain and consumers, including also those practising home-sprouting, should be informed of the food safety risk posed by sprouted seeds.

French outbreak: First EHEC death confirms that the outbreak is still going on [73]

A patient at an hospital at Bordeux, Southwest o France die this morning suffering hemolytic uremic syndrome. The deadly strain of Escherichia.coli which infected the women differed from the strain which caused the death of 48 people in Germany and one each in the United States and Sweden. Health experts suppose that contaminated Egyptian fenugreek seeds could be a n infection source, but again the source of the infection remains uncertain.

Seven other patients hospitalised in Bordeaux were infected by the German strain EHEC O104:h4 (Husec041) and one patient has another strain, but also deadly as the German strain. This suggests that the EHEC bacteria is mutating quickly. It is known that deadly strains of Escherichia coli are bread by cattle and camels without doing harm to these animals. From the intensive farming of cattle in Europe the bacteria spread to the environment.

Testing cattle at farms should be started to eliminate animals which shed the bacteria. Experts fear that the EHEC O104:h4 (Husec041) may spread to communal waters and drinking water reservoirs. If so, tap water is endangered as Escherichia coli bacteria are sporadically being found in drinking water samples, says expert. [74]

CDC renews raw sprout warning as outbreaks go on in 2011 [75]

Cook them or stay away to avoid salmonella, E. coli. The sprouts should be thoroughly cooked to kill the bacteria, and some people should stay away altogether, the Centers for Disease Control and Prevention said. Sprouted seed is a perfect vehicle for pathogens which cannot be washed off. Shiga toxin-producing E coli (STEC) have a low infectious dose, sprouts are a great vehicle.

Sprouts have caused many outbreaks of illness. Since 1998, more than 30 outbreaks have been reported to the CDC, due to many different kinds of sprouts of alfalfa, bean, clover, and others.

Sprouts can make even young and healthy people ill and can be particularly severe in vulnerable populations, such as young children, older adults, pregnant women, and people with compromised immunity.

It can be hard for those who become ill to remember having eaten sprouts. People often do not remember having eaten them, because they are often just a garnish or just one of many ingredients in a food dish. It is not necessary to eat large quantities of sprouts to make a person sick.

Raw produce can present a challenge, however, especially when it comes to salads, salsas, guacamoles or similar mixed dishes, and patients can't recall all the ingredients.

Genetic characterisation of Escherichia coli O 104 H4 of the HUS German and French outbreak 2011 [76] [77]

Weill et al 2011 suggest contaminated seeds of one type of sprouts to be the most probable source of the outbreak of bloody diarrhoea and haemolytic-uraemic syndrome caused by Shiga-toxin-producing Escherichia coli O104:H4 in France and Germany. The authors present microbiological data from the outbreak of the bloody diarrhoea and haemolytic-uraemic syndrome, associated with consumption of sprouts that occurred in June-July, 2011 in Southwest of France.

Bielaszewska et al 2011 presented the virulence profile of isolates from the German outbreak, and stress that blended virulence profiles in enteric pathogens, may be fatal for susceptible populations. [78]

The Serotype O104:H4AND has rarely been associated with HUS in the past. The HUS-associated strains carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Mellmann et al 2011 suggest that EAEC 55989 of 2001 and the actual EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, creating the actual highly pathogenic hybrid of EAEC and EHEC. [79]

The German and the French strain belong to the O104:H4 serotype serotype group B1. Extraintestinal virulence genes were found positive for irp2, fuyA and aerobactin genes. The extended-spectrum β -lactamase phenotype was due to the blaCTX-M-15 gene. The Shiga-toxin-producing E coli virulence-factor genes were positive for the stx2 gene (stx2a variant), whereas stx1, eae, and EHEC-hlyA genes were negative, Enteroaggregative E coli virulence factors was positive for aggR and pic, whereas astA was negative.

FDA Guidance for Industry to reduce microbial hazards from sprouted seeds [80]

It is difficult to grow "safe" sprouts. the US Food and Drug Administration developed guidance to help sprout growers reduce the risk for pathogen contamination in sprouts they produce and sell. The Food and Drug Administration recommends that producers soak the seeds in a calcium hypochlorite solution for 15 minutes. Many sprouts growers have implemented practices to decontaminate seeds before sprouting, but no available method has proved completely effective, because the contamination typically starts with the seed.

Idaho sprouts linked to outbreak of Salmonella Enteritidis sickening 25 people [81]

Epidemiological investigations linked an outbreak, which to alfalfa sprouts and spicy sprouts (a mix of alfalfa, clover and radish sprouts) grown by Evergreen Produce in Moyie, Idaho.

Investigating Foodborne Outbreaks [82]

If a larger number of people than expected appear to have the same illness in a given period and area, it’s called a cluster. When an investigation shows that ill persons in a cluster have something in common to explain why they all got the same illness, the group of illnesses is called an outbreak. Informations related to rising illness numbers are gathered by report systems. Laboratory testings include serotyping of markers on the surface of the bacteria, DNA fingerprinting identifies the bacteria’s specific genetic pattern or DNA fingerprint. It can take 2 to 3 weeks from the day the person became ill to the day such data are available. Identify mode of transmission such as food, water, or direct contact with an infected person or animal. Questionaire will help to clear this.

Internalisation of E.coli and Samonella in vegetable products

[83] The number of outbreaks of Salmonella spp. and Escherichia coli O157:H7 involving fresh produce, including leafy greens, fruits, and nuts is increasing. Salmonella spp. and Escherichia coli O157:H7 can internalize within a variety of plant tissues.

Deering et al. 2011describes the various routes of internalization into the plant, survival following internalization, and interactions between internalized bacteria and the plant. There are numerous factors that can influence the extent of internalization, such as greater demand, cross-country shipping and/or importation of fresh produce, increasing the time from harvest to consumption, more intensive use of land, and increasing demand by consumers for ready-to-eat fresh produce items.

Deering and colleagues developed immunocytochemical techniques to localize internalized E. coli O157:H7 expressing green fluorescent protein in germinated mung bean. The bacteria were found found in every plant tissue and cell type, particularly in cortical cells on the outside of the vascular bundles. Mung bean plants were found to support bacterial growth up to 10(7) CFU per plant keeping such high level of bacterial for more than 12 days. The authors remind that E. coli O157:H7 can be internalized in plant tissue following seed contamination. [84]

Deering and colleagues also investigated internalisation of peanut seed with Salmonella. Their tests demonstrated a high internal contamination of the seeds with Salmonella after an external exposure to the bacterium. The researchers concluded that Salmonella Typhimurium can get into de cells of different plants and grow there. Washing or even rinsing with sanitizers will not eliminate the bacteria, while only cooking will be on the safe side. [85] Other pathogenic Escherichia coli strains are:

Escherichia coli enteroinvasiv (EIEC)

This group produces disenteria.It is similar to Schigella and may penetrate the cells of the mucous membrane and cause their death.
The germs are none motile, they are gas negative and do not ferment lactose in 24 hours.
Virulence of EIEC strains are due to the presence of the plasmids 120- until 140-kDA.
These plasmids bear the code for different proteins of the cell membrane, being reponsible for the ability of invasion.

Among these proteins there are filamentous fibrils with which the germ may fixate to the host cells. EIEC has great affinity to the small intestines.

Escherichia coli enterotoxic ETEC

Escherichia coli enterotoxic ETEC characterizes bacteria which produce thermolabile and or thermostable.

The diarrhoeas caused by this group of bacteria are aqueous, accompanied by low fever and nausea. In acute cases they behave cholera accompanied by acute dehydration.

Infection occurs by ingestion of food and water with faecal contamination.
The ETEC group is responsible for most of the diarrhoea during travels (Travellers diarrhoea).
To develop his toxic activities it is necessary to produce fibrils whose code is placed in the plasmids.With theses fibrils the germ can fixate itself to the host cell and start the invasion.
There is a small affinity to the colon.


Enterotoxines thermolabiles (LT)

There are toxins whose code is situated in the plasmids such as the serogroup (LT-I) with the toxins LTh and LTp.

Less frequent is the enterotoxine of the serogroup II (LT-II) whose code is situated in the chromosomes.These toxins are similar to the toxin of cholera. The toxins are composed by part A and part B.

Part A can be separated in the subunits A1 and A2 with trypsin. The subunit A1 is responsible for the toxic activity producing cyclic AMP (cAMP) which produces cholera similar diarrhoea.
unit B is built of 5 identical parts. This subunit is responsible for the union of the toxins with the cell receptor gangliosides of the host as well as it is responsible for the union of the toxins with the enterocites (epitelial cells of the colon).


Enterotoxines thermostable (ST)

The toxins ST from type A stay active even after heating at boiling point for about 15 to 30 minutes (stable up to 120° ) and are stabil against many proteases.
The toxin ST A is believed to activate the guanilateciclase stimulating thus the formation of GMP which produces loss of liquid similar to cAMP.


Sorbitol-fermenting EHEC O157:H- [86]

According to Mellmann and colleagues 2010 Enterohemorrhagic Escherichia coli (EHEC) O157:H7 cause painful bloody diarrhea. Hemolytic uremic syndrome (HUS) develops in about 15% of infected children in around 1 week after the first loose stool. Sorbitol-fermenting (SF) E. coli O157:H- (nonmotile) strains cause about 20% of all cases of HUS. Unlike E. coli O157:H7, organisms within this clone can ferment sorbitol after overnight incubation on sorbitol MacConkey agar.

Variations of the genome of enterohaemorrhagic Escherichia coli O157:H7 affects its adherence and virulence [87]

In strain of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 the Tellurite resistance (TelR) is encoded by duplicate ter cluster in O islands (OI) 43 and 48, which also harbour iha, encoding the adhesin and siderophore receptor Iha.

Bielaszewska et al 2011 described five EHEC O157:H7 strains that differentiate into large (L) colonies and small (S) colonies with high and low Tel minimal inhibitory concentrations (MICs) respectively. S colonies sustained large internal deletions within the TelR OIs via homologous recombination between IS elements and lost ter and iha. According to the authors, iha-negative phenotypes adhered less well to human intestinal epithelial cells and grew slower than did their iha-positive counterparts, and variations of TelR OIs affects EHEC O157 virulence concluded the authors


PulseNet

To identify reservoirs of EHEC O157:H7 infections and of other foodborne pathogens and to elucidate the molecular epidemiology of these pathogens in the United States, PulseNet was established in 1996. This US national molecular subtyping network for foodborne disease surveillance facilitates subtyping of bacterial foodborne pathogens for epidemiologic purposes.

Multilocus variable number tandem repeat analysis (MLVA), used by PulseNet is a subtyping technique for characterizing human pathogenic bacteria such as enterohemorrhagic Escherichia coli (EHEC) O157.

Multilocus variable number tandem repeat (VNTR) analysis (MLVA) is based on the characterization of different VNTR regions throughout the bacterial genome. Repeat regions are amplified by using PCRs, and resulting fragments are sized to determine the number of repeats. The combination of numbers of repeats of different VNTR loci results in an allelic profile known as the typing result.

Mellmann and colleagues determined the phylogeny of epidemiologically unrelated EHEC O157:H7/H- clinical isolates through 8 MLVA loci obtained in Germany during 1987-2008. Specific MLVA profiles with an evolutionary persistence were identified, particularly within sorbitol-fermenting EHEC O157:H-.These pathogens belonged to the same MLVA cluster.


Enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli ( EPEC) causes diarrhoea different from that of Shigella and different from EIEC.They do not produce enterotoxines ETEC.

Enteropathogenic Escherichia coli EPEC produces typical lesions. Especially dangerous are the serotypes O55, O111, O 127, O86 and others being found in babies. Diagnosis of outbreaks in nurseries is only possible through culture and identification of the germ using biochemistry and serology.

Other diagnosis are done using hibridisated DNA sonde with the adherence factor (EAF) plasmid from the EPEC group.

In milk and meat products VTEC- bacteria, but no EHEC strains were recently found in milk,yoghurt, cheese, meat products, fine salads and meals from great kitchens.
The main danger is therefore unheated or underheated ground meat and milk.
Heat treatment makes sausages safe if there is no reinfection after heating.

The Belgian E.coli outbreak report [88]

Eurosurveillance published a case occurred in October 2007, related to an outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145 and E. coli O26 in ice cream in the province of Antwerp (Belgium). Five children, aged between two and 11 years, developed haemolytic uraemic syndrome (HUS).

According to Eurosurveillance three VTEC O145 and one VTEC O26 infections were laboratory confirmed in three children. The infection was traced back ice cream leftovers, detected with PCR and PFGE in faecal samples taken from calves, and in samples of soiled straw from the farm at which the ice cream was produced. Contamination took place after the pasteurisation process.

Verocytotoxin-producing Escherichia coli (VTEC), including E. coli O157:H7, O26, O145 and other E. coli serotypes, are important causes of gastrointestinal illness and haemolytic uraemic syndrome (HUS) in young children. This syndrome is characterised by haemolytic anaemia, thrombocytopenia and acute renal failure, a complication occurring in 5-14% of VTEC infections [89] [90]. HUS is a potential life-threatening disease and can induce hypertension, proteinuria and chronic renal failure in 5% of affected patients. The age group primarily affected are children under five years.

The Eurosurveillance authors stress that the incidence of VTEC in Belgium is probably underestimated, as most of the country's clinical laboratories do not test for these micro-organisms in routine gastroenteritis samples. They point to the need to consider zoonotic transmission and to highlight the prevention measures in facilities where there is easy contact with farm animals and their environment. Moreover, in our case the presence of VTEC in cattle at the farm and the shared activities of food-handling are problematic, as these pathogens can survive for months on surfaces, cross-contamination is a significant risk and there is the need to reinforce hygienic measures for food-handlers working at farms where food products are prepared.


Ways of infection with EHEC

The most important reservoir of the bacteria are ruminants.Unheated ground meat and unheated milk are the main source of infection.Human carrier of the bacteria are an important vector of the disease through smear infection of food and utensils as well as direct contact.Very important is the hygiene of communal toilets in kindergarten,in home and in homes for old people.


Measures to avoid contamination with EHEC

A new, drug-resistant strain of E. coli is causing serious infectious diseases [91]

The ST131 Escherichia coli strain, was identified in serious antimicrobial-resistant infections in USA by James Johnson and colleagues 2010. Isolates of 2007 exhibited fluoroquinolone or extended-spectrum cephalosporin resistance.

The authors stress that the ST 131 strain has a high level of virulence together with an antimicrobial resistance. It is becoming almost untreatable, compared with older high virulent Escherichia coli which were known as susceptible, and other resistant strains which were less virulent.

Antibiotic resistance gene NDM-1 from India and Pakistan [92]

Walsh and colleagues 2010 studied the carbapenem drug-resistant bacterial gene called NDM-1, or New Delhi metallo-beta-lactamase 1, first identified in bacteria in India, Pakistan, and recently in UK. This may become a new global health problem.

Most isolates carried the NDM-1 gene on plasmids. The authors assessed the prevalence of bla(NDM-1) carbapenem resistance gene, in multidrug-resistant Enterobacteriaceae in India, Pakistan, and the UK, using PCR and pulsed-field gel electrophoresis of XbaI-restricted genomic DNA methods. Plasmids were analysed by S1 nuclease digestion and PCR typing.

The authors report that NDM-1 was mostly found among Escherichia coli and Klebsiella pneumoniae, which were highly resistant to all antibiotics except to tigecycline and colistin. The NDM-1 gene produces an enzyme which breaks up the central ringed structure (beta-lactam), common to most of antibiotics. This structure halts bacteria replication. A beta-lactam ring has an heteroatomic ring structure, consisting of three carbon atoms and one nitrogen atom. [93]
Image Antibiotics_3
Carbapenems are a class of beta-lactam antibiotics with a broad spectrum of antibacterial activity. They have a structure that renders them highly resistant to beta-lactamases.
The carbapenems are structurally very similar to the penicillins, but the sulfur atom in position 1 of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenems. [94]

The NDM-1 gene renders bacteria also resistant to carbapenems, antibiotics of last resort, and the gene can be transferred between different bacteria.
Carbapenems resistance-conferring gene is spreading in India because of an irrational use of antibiotics. Due to an increasing tourism to India for cosmetic surgery the infection spreads to USA, Israel, Greece and Turkey.

High-risk clones disseminating antibiotic resistance [95]

Woodford et al. 2011 report that many bacterial species have a clonal structure which may be widespread. Some global clones are multiresistant and have disseminated from single foci, such as the KPC carbapenemase-positive Klebsiella pneumoniae ST258 from Greece to northwest Europe. The global ST131 Escherichia coli clone most often has CTX-M-15 extended-spectrum beta-lactamase (ESBL). However there are also strains which do not depend on ESBLs or have different ESBL types. The authors describe clonal diversity Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa which presents the serotype O12/Burst Group 4 cluster presenting resistances variety from PSE-1 to VIM-1 beta-lactamases. These high-risk clones are entangled in spread of resistance presenting ability to accumulate and switch resistance.

Carbapenem-resistant Enterobacteriaceae include strains of metallo-beta-lactamases (IMP, NDM or VIM) and non-metallo (KPC and OXA-48) enzymes, extended-spectrum beta-lactamase (ESBL) or AmpC enzyme with porin loss. Most strains with NDM-1 carbapenemase also have 16S rRNA methylases, conferring complete aminoglycoside resistance. Livermore et al. 2011 evaluated the activity of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline against carbapenem-resistant Enterobacteriaceae isolates. Acording to the authors the most important genes encoding carbapenem-destroying enzymes are KPCin the US and NDM, which is rapidly spreading internationally from India and Pakistan. [96]

Livermore reports that NLX104 plus ceftazidime is particularly effective against Klebsiella pneumoniae strains carrying K. pneumoniae carbapenem (KPC) resistance genes. Combining NXL104 with the antibiotic aztreonam it becomes effective against a wide range of carbapenemase-producing strains, including those carrying the Verona imipenemase (VIM) and the recently identified New Delhi metallo-beta- lactamase (NDM). [97] [98]

Metallo-beta-lactamases [99]

According to Zhao and Hu 2011 metallo-beta-lactamases (MBLs) hydrolyze most beta-lactams including the carbapenems. VIMs families include 27 variants of metallo-beta lactamases found in Gram-negative bacilli found in more than 40 countries/regions. The authors report further that most of the bla (VIM)s are situated on a class 1 integron, which is a genetic platform able to acquire and express gene cassettes. The fact that integrons are positioned in transposons which are situated on plasmids, making them highly mobile.

Reduction of antibiotic-resistant bacteria in municipal wastewater [100]

Diehl and LaPara studied the reduction of the quantity of antibiotic resistant bacteria in wastewater solids in anaerobic and aerobic digesters. The authors used lab-scale digesters at 22 °, 37 °, 46 °, and 55 °. five genes encoding tetracycline resistance (tet(A), tet(L), tet(O), tet(W), and tet(X)) and the gene encoding the integrase (intI1) of class 1 integrons. A reduction of 99,9% of these genes were obtained with the anaerobic digester at 55 °. Aerobic digesters did not reduce gene quantities. The authors stress that bacteria produce methane gas that can be used to heat the reactor, reducing thus the heating costs. They conclude that high temperature anaerobic digestion of wastewater solids may eliminate various antibiotic resistance genes. This technology together with reduced use of antibiotics in human and veterinary medicine and other uses, such as aquaculture may decrease the risk of antibiotic- resistant strains.

Aztreonam resistance leaded by bla NDM-1 gene [101]

Aztreonam is a synthetic monocyclic beta-lactam antibiotic (a monobactam), with the nucleus based on a simpler monobactam isolated from Chromobacterium violaceum. [102]
The CDC reported that during January-June 2010, three Enterobacteriaceae isolates carrying a newly described resistance mechanism, the New Delhi metallo-beta-lactamase (NDM-1), were identified from three U.S. states. These isolates, which include an Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, were resistant to all beta-lactam agents except aztreonam Enterobacteriaceae containing blaNDM-1 are linked to patients which recived medical care in India and Pakistan.

Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene from India isolates [103]

Young and colleagues 2009 describe a metallo-beta-lactamase gene, bla(NDM-1), and a n erythromycin esterase gene from Klebsiella pneumoniae 05-506 causing an urinary tract infection of a Swedish patient which travelled to Indian. The isolate was shown to possess a metallo-beta-lactamase (MBL) with three resistance-conferring regions: The first contained bla(CMY-4) flanked by ISEcP1 and blc. The second region contained gene cassettes arr-2, a new erythromycin esterase gene. The third region consisted of a new MBL gene, designated bla(NDM-1) which can hydrolyze all beta-lactams except aztreonam.

The authors stress that In addition to K. pneumoniae 05-506, the bla(NDM-1) identified on the Klebsiella pneumoniae , was also found on a plasmid in an Escherichia coli isolated from the patient's feces. Which rises worries that in vivo conjugation takes place.

Antibiotic resistance genes transfer between lactic acid abcteria strains [104]

Toomey and colleagues 2009 describe the ability to transfer antibiotic resistance determinants (plasmid or transposon located) to other lactic acid bacteria. In vitro transfer experiments resulted in high transfer frequencies between all LAB pairs. In a rumen model transconjugants were detected but the frequencies of transfer was low and inconsistent The plant model using alfafa showed higher transfer rates. The authors concluded that lactic acid bacteria can act as a source of genetic elements encoding antibiotic resistance which can be transmitted to other lactic acid bacteria.


Chloramphenicol antibiotic found in German pigs[105]

In Bavaria, Germany 25 farms were closed off in mid January 2012 after urine of pigs tested positive for chloramphenicol, an important antibiotic. Officials found that feed supplied to pig farms by the Bavarian dairy factory Ehrmann contained the antibiotic. Ehrman says that the laboratory of the quality management wrongly discarded the antibiotic. Choramphenicol is used in minimal amounts for selective culturing of moulds. Laboratory waste must be disposed properly or incinerated.

How much?

It is worth to look after details on how much chloramphenicol got into the production water, the so called “white water”. This production water contains a considerable amount of milk products and is used as feed in pig farms. To contaminate animals of 25 farms, over a period which made it possible to be detected by veterinary controls, implies considerable amount of antibiotics. Otherwise, the dilution factor of the “white water” would have turned “laboratory ” residues undetectable.


Antibiotics in pig farms[106]

Antibiotics are commonly used in commercial swine production in the United States and around the world. They are used for disease treatment, disease prevention and control, and growth promotion. When used for growth promoting purposes, antibiotics are given at low concentrations for long periods of time given as feed and water additives which improve daily weight gain and feed efficiency through alterations in digestion and disease suppression.

Residues of Chloramphenicol, no matter how low, are of risk to humans. In humans chloramphenicol is being suspected to cause aplastic anemia, a condition where bone marrow does not produce sufficient new cells to replenish blood cells. Other studies suggest that chloramphenicol is toxic to the embryo/foetus and reproduction system.

Chloranphenicol prohibited in animal breeding

Chloramphenicol is since 1994 prohibited in animal breeding for food, according to the EU regulation 2377/90, Annex IV [107], ammended by the regulation 37/2010 Table 2 (Prohibited substances) [108]. The directive 96/23 lays down measures to monitor the substances and groups of residues of toxicologic importance [109]. The directive 2003/181 setts the minimum required performance limits (MRPLs) of 0,3 microg/kg for Chloramphenicol [110]. Minimum required performance limit(MRPL) means minimum content of an analyte in a sample, which at least has to be detected and confirmed.

Bacteriocins in food industry, veterinary use and importance for human health

Bacteriocins are bacteriocidal toxins released by almost all bacteria. They may inhibit the growth of similar or closely related bacterial strains. They act somehow like yeast and paramecium killing factors. The use of bacteriocins in the food industry may reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods that are more naturally preserved.

The anti-infective potential of bacteriocins for inhibiting pathogens has been shown in various food matrices including cheese, meat, and vegetables, and are promising in treating infections in humans and animals, writes Hammami et al. 2012. [111]


Classification of bacteriocins [112]

Class I bacteriocins
The class I bacteriocins are small peptide inhibitors and include nisin and other lantibiotics. Lantibiotics contain the amino acid Lanthionine as part of their structure.

Class II bacteriocins
The class II bacteriocins are small heat-stable proteins:
The class IIa bacteriocins (pediocin-like bacteriocins) they have a Tyr-Gly-Asn-Gly-Val-Xaa-Cys sequence. This subgroup causes cell-leakage permeabilizing the target cell wall. This group is important in food preservation an medicine, with strong antilisterial activity. Most prominent bacteriocin of this subgroup is pediocin PA-1.
The class IIb bacteriocins (two-peptide bacteriocins) require two different peptides for activity, such as the lactococcin G, which permeabilizes cell membranes for monovalent ions such as Na and K, but not for divalents ones.
Class IIc bacteriocibs are cyclic. Enterocin AS-48 is the prototype of this group.
Class IId are single-peptide bacteriocins, which do not have pediocin-like caracteristics, such as aureocin A53, stable under high acidity, is not affected by proteases and thermoresistant.
Class Iie, are bacteriocins composed by three or four non-pediocin like peptides. Aureocin A70 is a four-peptides bacteriocin, highly active against L. monocytogenes,

Class III bacteriocins
Class III bacteriocins are large, heat-labile.
Subclass IIIa comprises those peptides (bacteriolysins)that kill bacterial cells by cell-wall degradation, such as lysostaphin, hydrolises several Staphylococcus spp. cell walls, principally Staphylococcus aureus.
Subclass IIIb, do not cause cell lysis, but disrupte the membrane potential. This causes ATP efflux.

Class IV bacteriocins
Class IV bacteriocins are composed of an undefined mixture proteins, lipids and carbohydrates. Examples of this class are Sublanicin and Glycocin F (GccF).

Gálvez et al. 2007 names the available commercial preparations of nisin and pediocin PA-1/AcH, together with bacteriocins such as lacticin 3147, enterocin AS-48 or variacin. These bacteriocins provide news strategies for food biopreservation, reduce the use of chemical preservatives, turns it possible to rerduce heat treatments,use less acids and reduce salt content. Broad-spectrum bacteriocins focuses a wide area inhibition. Narrow-spectrum bacteriocins can be used specific pathogens like Listeria monocytogenes without affecting starter cultures. Bacteriocins are available concentrated form to be added to the food, or they can be produced by starter cultures. They may be integrated in the hurdle strategy. [113]


Streptocuccus bovis HC5 cells as oral administration of bacteriocin found as safe

Paiva et al 2012 suggest to add bacteriocin-producing bacteria to animal feeds to ruminant and monogastric animals aiming to reduce the use of antibiotics. The authors report that adding viable or heat-killed Streptococcus bovis HC5 cells to the feed of mice did not affect weight gain and general health condition of the animals. Streptococcus bovis may become a safe way to feed bacteriocin to livestock animals.


Streptococcus macedonicus ACA-DC 198 important producer of bacteriocins [114]

Georgalaki et al. 2013 describe the bacteriocin production of Streptococcus macedonicus ACA-DC 198. The bacterium produces two important lantibiotics: macedovicin and macedocin. Macedovicin was found to be identical to bovicin HJ50 of Streptococcus bovis and thermophilin 1277 produced by Streptococcus thermophilus.

The authors report that macedovicin inhibits a broad spectrum of lactic acid bacteria, several food spoilage species such as Clostridium spp. and oral streptococci. Bovicin HJ50-like lantibiotics were also found to be encoded by Streptococcus suis strains SC84 and D12, Enterococcus columbae PLCH2, Clostridium perfringens JGS1721 and several Bacillus strains. The multiple bacteriocins of Streptococcus macedonicus improve bio-protection during food fermentation, according to the authors.

Lantibiotics of Bacillus subtilis promising bacteriotricins for food production

Members of the genus Bacillus produce bacteriocins, mostly lantibiotics some arer similar to the pediocin-like bacteriocins of the lactic acid bacteria. Bacillus bacteriocins have a broader spectra of inhibition as compared with lactic acid bacteria bacteriocins. Abriouel et al. 2011 discuss their applications in food preservation and in human, animal health as alternatives to conventional antibiotics. [115]

Antimicrobial peptides produced by the genus Bacillus can be classified into two different groups based on whether they are ribosomally (bacteriocins) or nonribosomally (polymyxins and iturins) synthesized. These compounds may become important for applications in the food, agricultural, and pharmaceutical industries to prevent or control spoilage and pathogenic microorganisms. such as the ribosomally synthesized lantibiotic bacteriocins produced by members of Bacillus, where subtilin from Bacillus subtilis is a promising candidate. [116]


Listeria and other foodborne pathogens resistance to nisin [117]

Recently resistance to nisin and Class IIa bacteriocins among the foodborne pathogens were noted. Kaurs et al. 2011 assessed the mechanisms of bacteriocin resistance development among various foodborne pathogens which decrease the efficiency of bacteriocins as biopreservatives, involving physiological activity profile of bacteria, alteration of cell-envelope lipid composition, and alteration of the antibiotic susceptibility/resistance profile of bacteria.


Bacteriocin-like substances of Bifidobacterium [118]

Bifidobacterial strains, isolated from humans have strain specific and not species-dependent antagonistic activity. Bifidobacterium sp. 278 and Bifidobacterium bifidum 174 produced antimicrobial substances of the 4th class of bacteriocins with wide spectrum and have the highest antagonistic activity of all studied strains. The maximal bacteriocin production happens between 8-16 hours of cultivation, write Poltavska and Kovalenko 2012.

Cronobacter in milk and infant formula [119]

Cronobacter species are opportunistic pathogens, and a mortality rate of 40 to 80% is associated with infections resulting in meningitis, septicemia, necrotizing enterocolitis, and brain abscesses. Norberg et al. 2012 stress the risk of Cronobacter infections caused by powdered infant formula, creating immense problem for the powdered baby food industry.

Norberg and colleagues refer to the official World Health Organization guidelines for preparation of powdered infant formula. [120]

There are six genomospecies of Cronobacter spp. (Enterobacter sakazakii) being isolated from a wide variety of foods. According to Healy et al. 2010 Cronobacter spp has a high tolerance to osmotic stress and elevated temperatures improving its survival in dried foods such as powdered infant formula. Cronobacter spp. may survive in macrophage cells and may attach to and invade epithelial cell lines. Cronobacter produce biofilm of exopolysaccharide and active efflux pumps protect the bacteria of antimicrobial agents such as bile salts and disinfectants. [121]


Bacteriocins inhibit Cronobacter sakazakii in milk and infant formula [122]

Oshima et al. 201r report inhibition of Cronobacter sakazakii in milk and infant formula by bacteriocins and organic acids. Combination of these compound may improve the safety of infant milk formula.

The antimicrobial activity were tested in reconstituted whole milk and in reconstituted powdered infant formula under low temperature of reconstitution of 40 and 50°, and long storage or feeding times at ambient temperature.
Propionic acid and monocaprylin (as POEM M-100) in combination inhibted Cronobacter sakazakii at concentrations of 0.1 to 0.2% in milk and formulas.
Lactoperoxidase system (LPOS), combined with bacteriocins nisin or lacticin 3147, inhibited outgrowth of Cronobacter sakazakii at 37° for 8 h, and 12 h when the infant formula was rehydrated at 40 or 50°.

Powdered milk and infant formulas must comply with the EU food safety criteria for ready-to-eat food (EC 1441/2007) which establishes the absence in 25 g of Listeria monocitogenes and Salmonella, and absence of Enterobacter sakazakii in 25 g. [123]


Bacteriocins of Enterococcus faecium [124]

Banwo, Sanni and Tan 2012 studied nono, an African fermented milk. Enterococcus faecium CM4 and Enterococcus faecium 2CM1 were isolated from raw cow's milk. The bacteriocin, entercin A, produced by these strains were active against Bacillus cereus DSM 2301, Bacillus subtilis ATCC 6633, Micrococcus luteus and Listeria monocytogenes. The bacteriocins were stable at pH 4-9, resisted heating of 100° for 10 min and treatment with lipase, catalase, α-amylase and pepsin, but did not resist to other proteases. The authors concluded that Enterococcus faecium has probiotic activity, and may be interesting as starter culture, depending on safety studies.

Nisin is effective against Staphylococcus aureus in dairy products, however, lysozyme is not [125]

Sudagidan and Yemenicioglu report that 25 µg/ml of nisin inhibited the growth of all strains and the biofilm formation of Staphylococcus aureus isolated from raw milk and cheese. Lower concentrations, however, were ineffective. Lysozyme tested at 5.0 mg/ml showed no inhibition. Strains of Staphylococcus containing both biofilm-related protease genes (sspA, sspB, and aur) and active proteases were nisin resistant. The authors concluded that lysozyme do not reduce the risk of staphylococcus aureus growth in dairy products. Nisin can be used to control growth and biofilm formation of foodborne Staphylococcus aureus as biopreservative. Possible formation of resistance must, however, be monitored.

Silver nanoparticles is an effective antibacterial and antifungal agent [126]

Silver is used in wound care products, dental work, catheters, in water disinfecting filters, in textiles, cosmetics, domestic appliances, as preservative cosmetic products, in the form of silica gel microspheres with silver thiosulfate complex mixed into plastics provides long-lasting antibacterial protection.

In this review Lara et al. 2011 state that silver nanoparticles bind with external membrane of lipid enveloped virus to prevent the infection with bacteria viruses and fungus. In particular silver nanoparticles have been studied in the inhibition of HIV infections. Interaction of silver nanoparticles with other viruses was, however, insufficiently studied. Silver nanoparticles interact with disulfide bonds of the glycoprotein/protein contents of microorganisms such as viruses, bacteria and fungi. Nanoparticles and silver ions change the three dimensional structure of proteins by interfering with S-S bonds and block the functional operations of the microorganism. The authors also stress that the size of nanoparticle is important, shape and morphology are important for best performance of the nanoparticles. Smaller silver nanoprticles than 3 nm are more citotoxic than particles with 25 nm.

Mechanism of bacteria swimming speed explained [127]

Boehm and colleagues 2010 stress that Escherichia coli moves by means of rotating flagella that are powered by ion influx through membrane-spanning motor. The bacteria governs its swimming speed with the help of a molecular brake (YcgR) which binds around a messenger nucleotide, a cyclic dimeric GMP (di-GMP). In this bounded form the molecular brake YcgR interacts with the motor protein MotA on the base of the flagella causing it to rotate. The authors found that at least five signaling proteins are engaged in controlling the cellular concentration of cyclic di-GMP determining the swimming speed of the bacteria in response to variations of the environment.

New method to study motility of bacteria [127]

Golding and colleagues 2009 describe a new method for studying the swimming of bacteria such as Escherichia coli bacteria. The researchers used optical traps with laser lights, microfluidic chambers and fluorescence microscopy to track pattern of movement of the organism and the response of a stimulus under a controlled environment. The optical traps confine individual cells without impeding their rotation or the movement of their flagella.

Movement of the bacterial cell alters the light from the laser informing about the direction of the movement, and fluorescent markers improves visualization of the bacteria and their flagella under a microscope. According to Golding Escherichia coli presentes three to six helical flagella. If they all rotate in one direction the bacteria moves corkscrew-like in one direction. However, when one or more flagella rotate in the opposite direction the bacteria seems to tumble.

The authors determined long-term statistics of the run-tumble time series and changes in velocity and reversals of swimming direction of Escherichia coli.

Food spoiling [128]

Bacteria which may spoil mayonnaise, ketchup, sauces and salads are : Yeasts, molds, lactic acid bacteria such as Lactobacillus, Leuconostoc and Pediococcus.

Salads with pH under 4.5: Lactic acid bacteria, yeasts and molds.
Salads with pH over 4.5:Lactic acid bacteria, yeasts and molds, Enterobacteriaceen, Salmonella and coagulase positive Staphylococcus.
Ketchup may be spoiled by:

Bacillus coagulans, Bacillus stearothermophilus, yeast, molds
lactic acid bacteria such as: Lactobacillus, Leuconostoc and
Pediococcus, acetic acid, thermophilic Bacillus
Sauces and dressings are spoiled by:
Acid resistant microorganism such as yeasts, molds and lactic acid bacteria.

Frozen Food:
During the feezing of food water activity is reduced to a point where bacteria cannot divide and her biological activity is significantly reduced.
A part of the bacteria die during freezing. Another part is sublethal damaged so that special revival medias are necessary for further detection. A greater part of the bacteria remain alive and can start the activity as soon as temperatures rises.

Important bacteria in frozen food are: Staphylococcus aureus coagulase positive Escherichia coli,Enterobacteriaceae,
Clostridium perfringens in precooked meat,
Salmonella, especially in poultry, fish, crustaceans, eggs and milk,


Salmonella serotype Enteritides in poultry and in Eggs

In live state, poultry is recognised as a major reservoir of various salmonella serotypes that can cause food poisoning and infection on man. The body temperature of poultry is 38° allowing the germs to settel iheated n the intestinal trackt and breeding stock were eggs may be contaminated with Salmonella.

To reduce the danger of contamination of entire flocks, feed should be heated at 85° for 12 minutes in the presence of 15% moisture.

Salmonella antibiotics-resistant strains should be declared as food adulterants, says CSPI [129]

Meats like chicken, ground beef, ground turkey, and pork are known to cause outbreaks from Salmonella strains Hadar, Heidelberg, Newport, and Typhimurium, presenting growing antibiotic-resistance.

In a letter to USDA Secretary Vilsack, the CSPI urges the agency to declare these strains as adulterants. This could prevent meat contaminated with these pathogens to be placed at market.

The USDA is considering increasing line speeds and reducing microbial testing in poultry facilities, which could make illnesses tied to chicken even more common. Undercooked meat can bear pathogens, improper handling after cooking allows bacteria to grow to dangerous levels. Keeping hot foods hot and cold foods cold can help limit your risk. Don't let cooked meat sit out for more than two hours. [130]

EU Regulation on the control of Salmonella and other zoonosis [131]

Salmonella spp. is one of the major causes of food borne illnesses in humans. According to the Community Summary Report on Trends and Sources of Zoonoses[1] a total of 192 703 cases of human salmonellosis were reported by 25 Member States in 2004.

Pork, after eggs and poultry meat, is a major source of human food borne salmonellosis in the European Union (EU), although the participation of pork-associated salmonellosis in food borne salmonellosis varies between countries or is unclear as, for most Member States, data on the true contribution of pig/pork to human food borne salmonellosis are not available.

Regulation (EC) No 2160/2003 on the control of Salmonella and other specified zoonotic agents[2] provides for the setting of Community targets, for reducing the prevalence of Salmonella serovars with public health significance in pig herds.

The most frequently reported zoonotic diseases in humans are salmonellosis and campylobacteriosis. Listeria monocytogenes accounted for the highest number of reported fatalities (107 deaths) in 2004.
According to EFSA, the report targets the reduction of Salmonella in laying hens in accordance with Article 4 of Regulation No 2160/2003 of the European Parliament and of the Council on the control of salmonella and other specified food-borne zoonotic agents. A final report will be released in October 2006.

Two main options exist for the implementation of monitoring schemes aimed at detecting/evaluating Salmonella prevalence and/or previous exposure to Salmonella in pig production. These options are based on bacteriological and immunological methods. When used appropriately, for specific purposes, each of these approaches is of benefit. However, for monitoring purposes the results of immunological and bacteriological investigations cannot be compared directly, as they give different information. The choice between immunology and bacteriology, or their use in combination, therefore, will depend on the actual situation and the questions that have to be answered.

Bacteriology can be of use when (a) isolation of the strain is necessary for identification, (b) information about all Salmonella infections (all serovars) is required, (c) antimicrobial sensitivity testing is required, (d) the current Salmonella status of individual animals is to be determined, (e) a description of the general diversity of infections with different Salmonellaserovars in a population is the purpose of the investigation, and (f) the evaluation of Salmonella-free status of herds is required. Immunology can be of use for the screening of large numbers of blood and other samples, for example, for monitoring the effectiveness of control programmes in endemic regions or establishing the current immunological status of a population (e.g. herd) and the prevalence of infection.

Risk mitigation options were identified according to three lines of defence formulated by the World Health Organization (WHO): the first line focuses on the control of Salmonella in the food producing animal (Pre-harvest control), the second line deals with improvement of hygiene during slaughter and further processing of meat (Harvest control) and the third line concentrates on measures during the final preparation of the food and the education of the industry and the consumer concerning the application of effective hygienic measures (Post-harvest control).

In general, the control of Salmonella is based upon the implementation of preventive actions throughout the whole production chain.

More specifically, measures should be addressed to (i) the prevention of introduction of Salmonella into the herd, (ii) the prevention of in-herd transmission, and (iii) the increase of the resistance to the infection.

No universal mitigation option capable of eliminating Salmonella entirely from the harvest and post-harvest level was identified. A combination of measures aimed at the prevention of vertical and horizontal transmission is likely to be the most effective approach, as is the case with most other food borne pathogens.

Reduction of the pathogen load in live pigs in each phase of the food chain, including the transportation phase, can be incrementally achieved by separation of batches, the implementation of Good Hygiene Practices (GHP) and hygiene management and optimisation of transport and lairage time.

Slaughter and dressing has to be performed with a high level of hygiene, according to Hazard Analysis and Critical Control Points (HACCP) principles in association with GHP, and focusing on the avoidance of direct or indirect faecal/intestinal contamination of carcasses. Logistic slaughter is a further option for reducing the pathogen load on the carcasses of slaughtered pigs.

Meat/carcass decontamination may be considered in specified situations, under the supervision of the competent health authorities. However decontamination should not be regarded as a substitute for any of the above mentioned recommendations.

Risk mitigation during processing requires maintenance of the cold chain and the application of the so-called "hurdle concept" and the implementation of GHP and the principles of HACCP.

At retail and consumer level mitigation includes hygienic handling and proper cooling or heating of pork and pork products. These options and procedures should be communicated to retailers and consumers.

Monitoring at harvest level is of relevance in regard to both process hygiene evaluation purposes and evaluates the current Salmonella status of the entire food chain. For human exposure assessment, monitoring requires to be conducted at the pre-consumption level.

The EU Zoonose Directive

The Community system for monitoring and collection of information on zoonoses was established by Council Directive 92/117/ECC (The Zoonoses Directive).The new Zoonoses Directive 2003/99/EC was adopted by the Council and the European Parliament on 17 November 2003.

This Directive setting rules for the Member States of European Union (EU) to collect, evaluate and report to the Commission, each year, data on specific zoonoses and zoonotic agents in animals, foodstuffs and feedingstuffs.

Summary Report of Zoonosis 2004 [132]

EFSA's First Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Antimicrobial resistance in the European Union in 2004 was published in December 2005. The zoonoses, meaning infectious diseases transmissible from animals to humans, affected over 380,000 EU citizens in 2004.

Often the human form of the disease is acquired through contaminated food. According to the report, the two most frequently reported zoonotic diseases in humans were Salmonella and Campylobacter infections. These bacteria were also commonly found in food and animals. The report includes information of 11 zoonoses, antimicrobial resistance in zoonotic agents as well as foodborne outbreaks.
The five most frequently isolated Salmonella species in the EU were, in descending order: Salmonella Enteritidis, Salmonella Infantis, Salmonella Typhimurium, Salmonella Mbandaka and Salmonella Livingstone.
Data on Salmonella were reported for a wide range of foodstuffs. The majority of samples were collected from various types of meat and meat products. The lowest levels of contamination in poultry, pig, and bovine meat during the last five-year period have been reported from Finland, Sweden and Norway.

Salmonella was detected at all levels of the poultry meat production, with the highest rates of contamination observed at the slaughterhouse and processing plants. Proportions of positive samples in poultry meat were generally lower than 10%, with the lowest proportions reported in countries with control programmes in the poultry production. At retail Salmonella was reported in fresh poultry meat ranging from 2% to 18.5% positive samples.

A general decreasing trend of Salmonella in table eggs was observed in those countries that had reported consistently. In pig meat, no clear trend was discernable, except for The Netherlands where a clear reduction was observed. Most countries reported Salmonella prevalences in pig meat below 10%. The contamination levels in bovine meat were generally considerably lower.

Some Member States reported contamination of ready-to-eat-meat products at the same level as in fresh meat. Such products constitute a particular risk to human health. In milk and dairy products Salmonella was rarely reported. Several surveys covering spices and herbs revealed relatively high Salmonella contamination.

Salmonellosis along with campylobacteriosis, are by far the most frequently reported food borne diseases in the EU. Both diseases are frequently caught through ingesting poultry and poultry products, such as eggs. On average about one in five large scale commercial egg producers have laying hens infected with the Salmonella spp. pathogen.

The testing did not find the Salmonella spp. species in any large scale commercial egg producers in Luxembourg and Sweden. The maximum level was found in Portugal, where about 80 per cent of the holdings had at least one hen test positive for the pathogen.

Salmonella Enteritidis and Salmonella Typhimurium were not found in Sweden, Ireland, Cyprus, Luxembourg, Latvia. Meanwhile, about 64 % per cent of the egg farms in the Czech Republic tested positive, followed by Poland, where 56 % had one or the other species of the pathogen, and Estonia, with 52 % testing positive.

According to EFSA holdings having Salmonella enteritidis vaccinated flocks were less likely to test positive for the pathogen. However in eight countries with both vaccinated and unvaccinated flocks, there was no different in the proportion of laying hens infected with Salmonella enteritidis. At EU-level the presence of any Salmonella spp. was detected in about 31 per cent of the large-scale laying hen holdings surveyed. The number of positive samples in a holding varied between one and seven, with an important proportion of the holdings found positive on the basis of only one or two of the tested samples.

Food-borne outbreaks were reported by eight new Member States. The Czech Republic reported proportionally more outbreaks than any other Member States in the EU, predominantly caused by Salmonella. The Czech Republic reported also 547 Campylobacter outbreaks (44% of the outbreaks) affecting 1,555 people with 90 hospitalisations. Hungary reported one large waterborne outbreak of campylobacteriosis. Lithuania and Poland recorded together 8 outbreaks of trichinellosis.
With few exceptions, levels of Salmonella contamination in new Member States that reported testing for in food, were similar to that of the old Member States. Some higher prevalences were reported by Malta in fresh pig meat at slaughter (32.8% Salmonella positive) and by Cyprus in fresh broiler meat at processing (36.6% positive). New Member States also reported testing for Campylobacter in food, particularly in poultry meat.

Considerable prevalences (30-40%) were recorded by the Czech Republic, Slovenia and Cyprus. Testing for Listeria monocytogenes in food was performed in all new Member States across a variety of foods. The level of contamination was in general similar to the level in the old Member States, although Estonia found 22.9% samples of fishery products positive for L. monocytogenes.

Seven new Member States reported testing for VTEC in food. Cyprus, Estonia and Slovenia did not detect any VTEC from the samples tested. Poland reported 8.3% of bovine meat samples were positive and Latvia reported 4.9% of pigs tested were positive.

The new Member States reported lower incidence of brucellosis in humans and animals compared to the old Member States. This also applies to tuberculosis in cattle. Many of the new Member States are seeking to receive an officially free status regarding these diseases in accordance with the EU legislation.

The new Member States reported the majority of rabies cases in animals, where wildlife (especially foxes) were frequently infected. Estonia, Hungary, Latvia, Lithuania, Poland and Slovakia reported cases both in farm animals, pets and wildlife.

Some new Member States reported findings of parasites from slaughter animals. Lithuania, Poland and Slovakia found Trichinella in slaughter pigs, and Poland reported remarkable high Echinococcus findings in sheep, goats and pigs.

Data on antimicrobial resistance, primarily in Salmonella from humans, food and animals, was received from eight new Member States: Czech Republic, Estonia, Hungary, Lithuania, Latvia, Poland, Slovakia and Slovenia. In general, the prevalence of antimicrobial resistance reported by new Member States was similar to, or lower than, the prevalence reported by old Member States.

Unsafe EU food safety From Farm to Fork [133] [134]

UK Cadbury Schweppes pleads guilty for selling chocolate contaminated by a leaking waste water pipe with causing Salmonella montevideo strain outbreak

According to the Birmingham City Council the company knew about the contamination but still sold the product, recalled the chocolate bars only after the UK Food Standards Agency. and the Health Protection Agency got envolved on the fact that 37 people were reported being infected by Cadbury chocolate products from January to June 2006.

Cadbury is accused of not reporting positive private tests revealing the presence of salmonella strain in January last year alleging the levels present did not pose a danger.

This is an infringement against article 19 (3) of the EU General Food Law. Food business operator is obliged to immediately inform the competent authorities if it considers or has reason to believe that a food which it has placed on the market may be injurious to human health. Cadbury Sweppes allege that the low level of Salmonella had been considered by them as harmless and will get through the loophole of the Food Law, which leaves up to the producer to decide whether or he responds to the deviation. [135]

Dripping wate water contaminated the chocolate crumb (a mixture of sugar, milk and cocoa) during production at the plant in Marlbrook, Herefordshire.

Poor layout of the factory and inadequate drainage and disinfection equipment. and not having the EU hygiene rules Hazard Analysis and Critical Control Point (HACCP) analysis in place are the cause of this scandal.

Salmonella reduction in poultry production at the primary production level [136]

Vandeplas et al.2010 describe preventive strategies to reduce the incidence of Salmonella colonization in broiler chickens at the farm level and additional preventive hygienic measures, such as feed and drinking water acidification with organic acids and immune strategies based on passive and active immunity, modification of the bird's diet to reduce a bird's susceptibility to Salmonella infection, in ovo feeding to accelerate small intestine development and enhances epithelial cell function. Feed additives such as antibiotics, prebiotics, probiotics, and synbiotics, the use of chlorate products and bacteriophages also being studied to reduce enteropathogens I broiler.

Mulder 1997 stresses that safe poultry meat production begins with the live birds. Mulder calls therefore, for measures focused on the live bird an such critical control point approach of the HACCP concept should be considered. The capability of microorganisms to colonise the gastrointestinal tract is to be included use of vaccines, antimicrobials and competitive exclusion microfloras as well as the implementation of new processing technology should be encouraged. [137]


Nonmotile Salmonella enterica variant bypassed safety procedures in France [138]

Le Hello et al 2012 report a food-related outbreak of salmonellosis in humans caused by a nonmotile variant of Salmonella enterica serotype Typhimurium in France in 2009 circulating in laying hens. This variant was not typed as Typhimurium escaping thus the European poultry flock regulations.

The French Regulation has extended the target for reduction of prevalence of Salmonella spp. in poultry producers to include notification of monophasic (because of the recent emergence in humans) and nonmotile (because of this food poisoning) variants of Typhimurium after January 2010. This food poisoning outbreak also highlighted the need for a second selective enrichment media for Salmonella spp. detection not based on the motility in complement to the modified semisolid Rappaport-Vassiladis medium recommended as a single medium by the European Directive [139].

Comparative risk assessment applied to pig slaughtering in Europe [140]

Fosse J, Seegers H, Magras present a calculation of the incidence and severity of human cases due to pork consumption in Europe together with the ratio of non-control (incidence of human cases divided by the prevalence of hazards on pork).
Using these data highest incidence rates were determined for Salmonella enterica, Yersinia enterocolitica and Campylobacter spp. The highest severity scores were calculated for Listeria monocytogenes, Clostridium botulinum and Mycobacterium spp. The main high risk hazards presenting high non-control ratios were Y. enterocolitica, S. enterica and Campylobacter spp..

The authors stress that these risks cannot be detected by macroscopic examination of carcasses during meat inspection at slaughterhouse, which is based on an ante mortem clinical examination and a macroscopic post mortem examination of the carcass, including incision or palpation of lymph nodes and organs. New means of hazard control, such as presented here may improve the inspection at slaughterhouse.

Awareness of cross-contamination is of greater importance than undercooking [141]

Luber 2009 assessed epidemiological studies of Salmonella and Campylobacter infections from poultry meat and eggs Efforts to reduce the contamination of poultry meat or eggs faces the production. Information of the consumer regarding food preparation behaviour is another risk management tool, using messages such as "cook poultry meat and eggs thoroughly"or "wash your hands" to tackle cross-contamination events and undercooking.

Reviewing available studies the authors concluded that cross-contamination due to the use of the same cutting board for chicken meat and salad without intermediate cleaning or spreading of pathogens via the kitchen environment seem to be of greater importance than the risk associated with undercooking of poultry meat or eggs.

Affected Cadbury products [142]

Cadbury Dairy Milk Turkish 250g; Cadbury Dairy Milk Caramel 250g; Cadbury Dairy Milk Mint 250g; Cadbury Dairy Milk eight chunk; Cadbury Dairy Milk 1kg; Cadbury Dairy Milk Button Easter Egg 105g; Cadbury Freddo 10p.

According to bacteriologist Professor Hugh Pennington of Aberdeen University the fat in chocolate actually preserves the salmonella from the normal intestinal defences. Very few salmonellas cause an infection. The infection dose from chocolate is a thousand times smaller than eating it from traditional sources like meats.

According to the Food Standards Agency the presence of salmonella in ready-to-eat foods such as chocolate is unacceptable at any level.

Serious situation of European food industry ethic

The responsible head of Cadbury Sweppes has overlook serious problems of the layout of the factory. This situation must have gone on for a long time. The head of the company has knowingly failed to report and recall contaminated products. This is a serious disruption commitment to ethic The company producing 97,000 tonnes of milk chocolate crumb every year placed financial costs over food safety, selling chocolate with poisonous bacteria and disgusting waste water.

Serious situation of the official controls

The Food authority in UK not noticing poor layout of the Cadbury factory during normal check ups is an alarming loophole of the European food safety system and private certification auditings.

Stronger surveillance by food authority of factories producing high fat and high sugar items are necessary. Carelessness in cleaning and disinfection is frequent with these produces because they do not present signs of spoilage. UK food authority surveillance is to be blame, at least in part, for the Cadbury Sweppes salmonella scandal.

Vibrio parahaemolyticus in fish and crustaceans

Is a Gram-negative mobile rod. It belongs to the family of Vibrionaceae being found in fish, mussels oysters and shrimps.
It grows between 5° and 44° , it is halotolerant (it can grow at high content of salt), growing between 2% and 3% of salt and pH 4.8.
Food with high contamination with Vibrio parahaemolyticus (1.000.000 /g) cause vomits, diarrohea and haemolysis
positive at Wagatsuma-Agar = Kanagawa positive.
Cytochromoxidase +
Motility +
Katalase +
L- Arginindihydrolase -
L-Lysinedecarboxylase +
Growth at 42° +
Voges Proskauer -
TSI Agar  
inclined surface basic (red)
bottom sour ( yellow )
gas negative
H2S negative

Enriching culture

Demonstrate the presence or absence of Vibrio parahaemolyticus
100 ml or g food are mixed with Broth.
Incubate at 37° C for 18 +- 2 hours Inoculate the broth at TCBS-agar. Incubation and biochemical differentiation.

Total count

On TCBS-agar on surface, incubated at 37° for 18 hours.


Kanagawa reaction

Point inoculum of dried Wagatsuma-agar. Incubate at 37° for 18 hours +- 2 hours. Colonies with positive haemolysis are labeled as Kanagawa positive.

MPN method

MPN-method MPN= most probable number(using 3 tubes. This method is used for total count with very low contamination.

Dilution

Make dilutions with peptone-salt-broth (0,1% Peptone,3% Salt).
1 ml of the dilutions are added to 10 ml SPB-broth.

Incubation

37° for 18 +- 2 hours.

Identification

Inoculate the highest dilution on TCBS-agar. Incubate at 37° for 18 hours.
Typical colonies are 2 to 3 mm in diameter with green-blue center. Differentiated in mediums with 2% to 3% NaCl.
gram per liter  
Peptone from casein 5,0
Peptone from beef 5,0
Yeast extract 5,0
Sodium citrate 10,0
Sodium tiosulphate 10,0
Dried bovine bile 5,0
Sodiumcholate 3,0
Saccharose 20,0
Sodium chloride 10,0
Iron III-citrate 1,0
Thymolblue 0,04
Agar-Agar 14,0

Warning from untreated oysters [143]

A bacteria from the marine and and estuarine environments Vibrio vulnificus is being found in untreated oysters from the Gulf Coast. For people with compromise the immune system the bacteria is deadly for 50% of infections.

The Vibrio vulnificus is a bacterium is present in seawater, sediments, plankton and shellfish (oysters, clams and crabs) in the Gulf of Mexico, the Atlantic Coast and the entire U.S. West Coast. As a member of the Vibrionaceae family, Vibrio vulnificus it is related to Vibrio cholerae and Vibrio parahaemolyticus which produce diarrhea. Vibrio vulnificus causes wound infections, gastroenteritis, or a syndrome known as primary septicemia.

The CSPI says untreated oysters harvested from Gulf Coast waters from April to October should be subject to state bans and, meanwhile, consumers should avoid consuming such oysters.

Raw shellfish northern regions are seen less dangerous as untreated shellfish from the Mexican Gulf because the water temperatures are lower than what is found in the Gulf. However, rising temperatures caused by the climate change will turn northern waters also a potential hazzard.

Action demanded to control deadly Vibrio vulnificus bacteria of raw oysters harvested from the Gulf Coast region [144]

The Center for Science in the Public Interest CSPI points out that eating raw oysters or other shellfish contaminated with deadly Vibrio vulnificus bacteria present a serious health risk. The contaminated oysters are mainly harvested from the Gulf Coast region, especially during the warmer summer months.

The CSPI urges the FDA to set a performance standard for the shellfish industry to reduce this threat to consumers. An infection with Vibrio vulnificus presents symptoms of classic foodborne illness but for some consumers, it may develop to ulcerous skin lesions and septicemia, half of such cases die and those that survive can suffer lifetime infirmities. At risk include consumers with diabetes, hemochromatosis (iron overload), compromised immune systems or liver disease.

While the shellfish industry resisted a 2009 FDA attempt to require mandatory post-harvest processing of contaminated oysters, the new food safety law signed by President Obama in January 2011 requires the FDA to set performance standards for significant foodborne contaminants like Vibrio vulnificus.

The CSPI stresses that the shellfish industry has a number of commercially viable methods of eliminating Vibrio, including four validated methods for pasteurizing oysters, or it could simply divert contaminated oysters to be cooked. Some restaurateurs and retailers, such as Legal Sea Food and Costco, only sell treated Gulf Coast oysters or oysters harvested from colder waters.

Water and mussels were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were identified using biochemical tests and PCR using primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR)


Vibrio bacteria in French mussels [145]

French mussels and marine and estuarine water was found by Hervio-Heath et al. 2002 to be contaminated with Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus. Identified Vibrio cholerae were confirmed to be non-O1/non-O139. All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene. The authors emphasise the health risk of Vibrio bacteria in French mussels.

Growth prediction model of Vibrio vulnificus stored at different temperatures. [146]

Dasilva et al 2012 developed a predictive model for Vibrio vulnificus stored at different temperatures. The Number of Vibrio vulnificus was slowly reduced at 5 and 10°C, but increased at 15, 20, 25, and 30°C. Using this model growth rate estimates of oysters stored at 25 and 30°C were lower than Food and Agricultural Organization (FAO)/WHO model predictions. The model described by the authors may be implemented in food safety plans to reduce Vibrio vulnificus health risk in oysters.

Prevalence in waters and shellfish is not associated to faecal indicator organisms. None of selective-differential media developed for isolation of Vibrio spp., and specifically for Vibrio vulnificus detection, are satisfying in sensitivity to low numbers and specificity to inhibit growth of other organisms. Harwood Gandhi and Wright 2004 stress, therefore, the importance of immunological and molecular protocols to detect and identify Vibrio vulnificus. [147]


PCR assay based on pilF gene to detect patogenic Vibrio vulnificus [148]

According to Baker-Austin et al. 2012 the bacterium Vibrio vulnificus, a leading cause of seafood-related mortality, has a genetically diverse nature, holds a variety of biotypes, and includes pathogenic as well as non-pathogenic variants hampering detection and enumeration by genetic methods. The researchers report the development of a real-time PCR assay for the detection of pathogenic Vibrio vulnificus strains based on a polymorphism in pilF, a gene associated with human pathogenicity, andmay detect biotype 2 and 3 strains.

Vibrio vulnificus phylogroup clade A [149]

According to Broza et al 2012 the biotype 3 group of the human pathogen Vibrio vulnificus emerged in Israel probably as a result of genome hybridization of two bacterial populations. Broza and colleagues studied the genetic relations between the biotypes 1,2 and 3 looking at 12 variable number tandem repeat (VNTR, also termed SSR) loci. They soon detected a new cluster which could not be associated to one of the three biotypes. Multilocus sequence typing (MLST) supports the creation of a new phylogroup called by the authors as clade A. Broza et al. call for caution as fish aquaculture environment, and possibly man-made ecological niches as a whole, may provide as a source of new pathogenic strains.


Sialic acid of mucous membranes can be catabolised by pathogen variants of Vibrio vulnificus [150]

Sialic or nonulosonic acids are sugars that are present in mucous membranes. Pathogen bacteria and commensal species of animals are able to digest sialic acids. Lubin et al. 2012 report that the sialic acid transport (SAT) and catabolism (SAC) region genes can catabolize sialic acid as a sole carbon source, and are found only in pathogenic Vibrio vulnificus. The tripartite ATP-independent periplasmic (TRAP) transporter was found to be essential for sialic acid uptake in this species. The authors concluded that the ability to catabolize and transport sialic acid is predominately lineage specific in Vibrio vulnificus.

DNA array of the ISR regions may identify Vibrio species [151]

Kim et al 2012 note that the DNA sequences of the intergenic spacer regions (ISRs) in the groESL gene of 23 Vibrio spp. are variable in. ISR sequence length (41-85 bp). The authors suggest to use DNA array using ISR-specific probes to distinguish between Vibrio parahaemolyticus and Vibrio vulnificus from other species.

Severe epidemics of Vibrio cholerae in India due to faecal contamination of water [152]

Roy et al.2012 describe an outbreak of acute diarrhea occurred in Belgundi area in South India, in June 2010. The isolated Vibrio cholerae O1 El Tor was multiple drug resistant, corresponding to drugs used in empirical treatments. Water samples were found to be heavily contaminated with fecal coliforms. The authors stress that the complex of shortage of potable water, onset of monsoon rains and breakdown of sanitation systems leaded to the outbreak. Decline of the outbreak was attained changing the empirical treatment, chlorination, improvement in sanitation measures backed by information and education activities.

The epidemic of cholera that occurred in Kashipur and Dasmantpur blocks of Orissa, reported during July-September 2007 were caused by Vibrio cholerae O1 Ogawa biotype El Tor positive for the ctxA and tcpA genes, according to Pal et al. 2010. [153]

Pathogenic bacteria in Brazilian coastal waters [154]

Ristori and colleagues 2007 analized oysters and estuarine water samples from coastal waters of São Paulo, Brazil., The authors found Aeromonas spp., Plesiomonas shigelloides, Vibrio cholerae O1, Vibrio parahaemolyticus, Vibrio vulnificus Salmonella, Shigella, Escherichia coli O157:H7 in oysters and in water samples too. The authors point out the Vibrio vulnificus was present in 10the samples, and Vibrio parahaemolyticus Kanagawa-negative was detected in all oyster samples. The study highlight the true microbial hazard in the aquatic environment and oysters.

Vibrio bacteria in Brazilian oysters [155]

Vieira and colleagues 2010 analysed oyster samples from a coastal aquaculture in Euzebio, Ceará, Brazil. The authors identified Vibrio parahaemolyticus. Vibrio carchariae and Vibrio vulnificus, stressing that oysters should never be eaten raw or undercooked becaause of the risk presented by Vibrio bacteria.

Vibrio vulnificus risk from untreated shellfish throughout the year [156]

Ramirez and colleagues 2009 determined the occurrence of Vibrio vulnificus, in south Texas coastal waters. During this study Vibrio vulnificus was isolated at all sites throughout the year even with water temperature was down to 9.71 degrees C. However there was a correlations between concentrations of V. vulnificus and water temperature and dissolved oxygen, as well as between concentrations of V. vulnificus and enterococci. The authors concluded that the risk of infection of Vibrio vulnificus exists throughout the year. Monitoring of data such as water temperatur, dissolved oxygen and enterococciu count are being suggested to predict rising risk.

Treated and untreated marine products pose a health risk according to a German scientist [157]

Uwe Janssen, in a period from 1994 and 1996 examined various edible fishery- and aquaculture-products from the North Sea and the Atlantic Ocean, and several exotic species of fish from Africa and Southeast Asia. The incidence of Vibrionaceae was 43 % in the untreated products, and 11 % in the ready-to-eat foodstuff.

According to the author the high variety of pathogenic species found such as Plesiomonas shigelloides, Aeromonas hydrophila, Aeromonas caviae, Aeromonas sobria,Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio mimicus, and Vibrio alginolyticus indicate a potential heath risk posed by Vibriuonaceae untreated as well as treated fishery products, such as frozen, cooked, fermented, and cold-smoked products.

Vibrio vulnificus in German coastal waters [158]

Ruppert and colleagues 2004 reported two severe cases of Vibrio vulnificus wound infection with secondary septicemia in 2003 on the German island of Usedom in the southwestern Baltic Sea by wading in contaminated sea water. High concentrations of V. vulnificus in the sea water was found when water temperature exceeded 20°C for more than 2 weeks.

Vibrio vulnificus a pathogen in Gulf Coast oysters [159]

Limiting the Sale of Raw Oysters Harvested from the Gulf of Mexico
The Center for Science in the Public Interest warns the consumers from Vibrio vulnificus, a deadly bacteria found in almost all Gulf Coast oysters harvested in warmer months. The Californian plan to reduce the risk of Vibrio vulnificus has proved to be effective, but it was binding only in this state [160]. The plan banned the sale of untreated Gulf Coast oysters reducing number of deaths from about five a year to zero. This approach will now be adopted nationwide by a new FDA policy. The shellfish industry were opposed to the oyster treatment to kill the pathogen.

The Department of Health Services (DHS) has amended California Code of Regulations (CCR), Title 17, Section 13675, [1ab]to prevent V. vulnificus illnesses and deaths associated with the consumption of raw Gulf oyster, restricting the sale of raw oysters harvested from the Gulf of Mexico during April 1 through October 31, unless the oysters are treated with a scientifically validated process to reduce V. vulnificus to non-detectable levels. Raw Gulf oysters received during April through October that have not been processed to reduce V. vulnificus to non-detectable levels are considered adulterated.[161] Treatments to kill Vibrio vulnificus without affecting taste are cold pasteurization, hydrostatic pressure, cost of the processes are low and increase food safety. [162]

Post-Harvest Processed Oysters [163]

Freezing, heat-cool pasteurization, and high hydrostatic pressure are used commercially on oysters as post-harvest processes to kill spoilage bacteria and reducing Vibrio spp. bacteria to non-detectable levels.The treamtment increases the shelf life of the product. The organisation SafeOyster.org, however, stresses that not all bacteria and viruses may be killed. The organisation recommends high-risk patients not to eat oysters, even when they were post-harvest processed. As a safety measure, these patients should eat cooked oysters.

The Center for Science in the Public Interest (CSPI) says Gulf Cost oysters are not safe [162]

According to David W. Plunkett, a CSPI staff attorney, some Gulf oysters may be "safe" from oil contamination, but are not "safe" to eat. Plunkett contradicts several reassuring statements that seafood from the Gulf on the market is safe. Vibrio vulnificus contaminates Gulf oysters in the spring and summer. While it may cause mild illnesses in healthy individuals, it can kill people who have diabetes, liver disease, hemochromatosis or compromised immune systems, causing the death of 10 people last year.

The Food and Drug Administration FDA affirms on its website that shellfish harvested from areas unaffected by the spill are safe to eat. [164] Plunkett remids that last year Mike Taylor, a Deputy of the FDA called Vibrio vulnificus a significant hazard, and now FDA ignores the risk of the pathogen bacteria.

The CSPI says that the FDA eventually backed down from its plans under pressure from Members of Congress who responded to industry posturing over potential job losses, and only California has implemented an effective control plan to protect its consumers.

Vibrio vulnificus infection [165]

Vibrio vulnificus is a bacteria which is present in marine environments such as estuaries, brackish ponds, or coastal areas, It causes an infection eating seafood, especially raw or undercooked oysters; the bacteria can also enter the body through open wounds when swimming or wading in infected waters, or via puncture wounds from the spines of fish such as tilapia. Symptoms include vomiting, diarrhea, abdominal pain, and a blistering dermatitis that is sometimes mistaken for pemphigus or pemphigoid. In people with compromised immune systems such as in chronic liver disease, a cut infected with Vibrio bacteria can quickly become worse and spread into the bloodstream. Severe symptoms and even death can then occur.

Vibrio illness in Florida [166]

Weis and colleagues 2010 report 834 vibrio infections in 825 individuals in Florida from 1998 to 2007. The incidence was Vibrio vulnificus 33%, V. parahaemolyticus 29%, and V. alginolyticus 16%, causing wound infections 45% and gastroenteritis 42%. Prevention is focused on oyster consumption. The authors call for educational messages focusing wound infections of high-risk populations.

Foodborne pathogens in oysters of South China food markets [167]

Chen and colleagues 2010 looking at the pathogens in shellstock Pacific oysters in the food markets in South China say that Vibrio vulnificus and Vibrio parahaemolyticus (89.3%) but no Listeria monototogenes could be detected in the samples. The authors concluded that Vibrio vulnificus and pathogenic Vibrio parahaemolyticus in oysters are a risk to public health in south China.

Temperature effects on the depuration of Vibrio parahaemolyticus and Vibrio vulnificus from oysters [168]

Chae, Cheney and Su 2009 investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster. Depuration of oysters at 22 degrees C had limited effects. Best reults were obtained with water temperature of 15 degrees C after 96 h of depuration at 15 degrees C. Depurations at 10 and 5 degrees C were less effective than at 15 degrees C.

Elektrolyzed water as sanitizer in food industry [169]

Hricova, Stephan and Zweifel 2008 report that electrolyzed water is obtained by electrolysing a dilute sodium chloride solution dissociating into acidic electrolyzed water (AEW) with a pH of 2 to 3, an oxidation-reduction potential of >1,100 mV, and an active chlorine content of 10 to 90 ppm, and basic electrolyzed water (BEW), which has a pH of 10 to 13 and an oxidation-reduction potential of -800 to -900 mV.

AEW reduced bacteria in suspension > 6.0 log CFU/ml. However, surface type and the presence of organic matter reduce the efficiency of AEW Applying BEW followed by AEW may lead to higher reductions than AEW only. The authors say electrolyzed water technology should be further discussed as industrial sanitization of equipment and decontamination of food products, but must be accompanied by good manufacturing and hygiene practices.

Electrolyzed oxidizing (EO) water treatment on reducing V. parahaemolyticus and V. vulnificus [170]

Holding vibrio contaminated oysters in electrolyzed oxidizing water containing 1% NaCl reduced the number of Vibrio parahaemolyticus and Vibrio vulnificus significantly in 4 to 6 h, but exposure longer than 12 h in electrolyzed oxidizing water and chlorine levels over 30 ppm caused death of oysters. Ren and Su 2006, authors of the study, suggested to use electrolyzed oxidizing water treatment for 4 to 6 h as post-harvest treatment of oysters to reduce Vibrio contamination limited to 4 to 6 h to avoid death of oysters.

Weak acidic electrolyzed oxidizing water post-harvest treatment of oysters [171]

Quan and colleagues 2010 report that Vibrio vulnificus and Vibrio parahaemolyticus were killed with a treatment of 15 s and more of weak acidic electrolyzed oxidizing water (WAEW) containing an available chlorine concentration (ACC) higher than 20mg/L. The effect of the treatment was reduced, when the ACC of WAEW was less than 15mg/L. The authors stress that the bactericidal activity of WAEW was primarily affected by ACC rather than treatment time, and WAEW is more effective than sodium hypochlorite (NaClO).

Vibrio vulnificus resistance to bile and other stresses [172]

Chen, Oliver and Wong 2010 describe the adaptation of Vibrio vulnificus and an rpoS isogenic mutant to bile and other stresses. An in vitro tolerance of the bacterium to 10% bile was attained by the authors, The bile-tolerant strain was more resistant to high pH, heat, high salinity and detergents than the rpoS mutant which had a lower bile-adaption rate.

The authors report further that production of GroEL was not markedly influenced but DnaK was inhibited in the bile-adapted cells, and RpoS plays a significant role in the response of Vibrio vulnificus to bile.

The interaction between low salinity and other common stresses in V. vulnificus [173]

Wong and Liu 2006 studied the cross-protective response of sublethal heat-, acid-, or bile-adapted Vibrio vulnificus against lethal low-salinity stress.

The authors found that Vibrio vulnificus adapted to an acidity of pH 4.4 and 41 degrees C heat-adapted V. vulnificus died in 0.04% NaCl low-salinity environment. The bile-adapted bacteria were resistant to low salinity, however 0.12% NaCl low-salinity adaptation made them sensible to 12% bile stress.

Identification and subtyping of Vibrio parahaemolyticus and V. vulnificus targeting 16S-23S rRNA intergenic spacer regions [174]

Hoffmann and colleagues 2010 developed rapid polymerase chain reaction (PCR)-based intergenic spacer (IGS)-typing system for vibrios based on the IGS regions located between the 16S and 23S rRNA genes of vibrios.

The IGS-typing method demonstrated distinct IGS-typing patterns indicative of subspecies divergence in both populations making this technique equally useful for intraspecies differentiation, as well. The authors concluded that the new method is useful to identify vibrios down to sub-species level, and may be applied in time saving epidemiological investigations.

González-Escalona, Jaykus and DePaola reported in 2006 that amplification of the 16S-23S rDNA spacer region (ISR1) is a simple and rapid procedure for subtyping bacteria, especially those with several ribosomal operons including Vibrio vulnificus. V. vulnificus contains nine ribosomal operons with four or five ISR1 classes that differ in size and sequence. Clinical isolates formed a single cluster containing ISR1 patterns I, V, XI, and XII all carrying the type B 16S rDNA (rrs) sequence associated with human illness. Shellfish isolates presented high variability in the ISR1 patterns. The different classes differed in their tRNA gene composition, allowing subtyping of Vibrio vulnificus. The authors suggest that ISR1 patterns are linked with the virulence of the bacteria. [175]

Two fish-pathogenic serovars of Vibrio vulnificus biotype 2 [176]

According to Fouz and colleagues 2010 Vibrio vulnificus biotype 2 is subdivided into serovar E, infecting fish and humans, and serovar A, infecting only fish, such as eel in brackish water. Serovar A caused infections of freshwater-cultured eels vaccinated against serovar E resulting in haemorrhagic intestine. Both serovars infect healthy eels, tilapia, sea bass and rainbow trout, but not sea bream, serovar A entering mainly by the anus and serovar E by the gills. The authors stress that serovar A form a new antigenic form of Vibrio vulnificus biotype 2 better adapted to fresh water than serovar E.

Vibrio vulnificus found in food and environmental samples of the Mediterranean area [177]

Cañigral and colleagues 2010 found that samples of seawater, oyster and wastewater from a coastal area in Spain near the Mediterranean.were positive by real time PCR, and Vibrio vulnificus could be isolated from these samples. The authors stress that Vibrio vulnificus presents a risk to humans in the Mediterranean area.

Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico [178]

Panicker and colleagues 2004 described the optimization of SYBR Green I-based real-time PCR parameters to detect the presence of vibrios in seafood or environmental samples, using vvh-specific oligonucleotide primers. The method is completed in 8 hours.

Other pathogens from shellfisch [179]

Plesiomonas shigelloides is a Gram-negative, rod-shaped bacterium which has been isolated from freshwater, freshwater fish, and shellfish and from many types of animals.

Infections from this organism cause gastroenteritis, followed by septicemia in immune deficient patients. It is placed among the Enterobacteriaceae. Some Plesiomonas strains share antigens with Shigella sonnei, and cross-reactions with Shigella antisera occur. Plesiomonas can be distinguished from Shigella in diarrheal stools by an oxidase test: Plesiomonas is oxidase positive and Shigella is oxidase negative. Plesiomonas is positive for DNAse; this and other biochemical tests distinguish it from Aeromonas sp.

Other pathogenic microorganisms in Greece seafood [180]

Papadoupolou and colleagues 2007 report in Greece marine fish and shellfish the presence of Aeromonas hydrophilia, Klebsiella ozonae, Escherichia coli, Yersinia enterocolitica, Hafnia alvei, Enterobacter agglomerans, Citrobacter freundii, Proteus vulgaris, Proteus mirabilis, Morganella morganii, Pseudomonas fluorescens, Pseudomonas putida, Plesiomonas shigelloides, Listeria innocua, Vibrio parahaemolyticus, Clostridium perfringens, Staphylococcus aureus and Candida quillermondi, Candida albicans, Penicillium oxalicum and Penicillium italicum.

Chlamydia

The genus Chlamydia contains cocoid nonmotile from 0,2µm to 1,5µm organism which can reproduce only in the vacuoles near the host cell membrane.
The reproduction follows a unique cycle. The development includes the passage through small elementary bodies up to larger reticulate bodies which can divide by fission. At the end the reticulate bodies reorganize into new elementary bodies. These new bodies can survive out of the cells infecting other host cells using a special phagocytosis having no fusion of phagosomes of Chlamydia with the lysosomes.
There is a gradual transition between the elementary and the reticulate form, existing intermediate forms.

The morphology of the genus Chlamydia is very complex:
Characteristics elementary bodies reticulated bodies
Diameter in µm 0,2 - 0,4 0,5 - 1,5
Density in g/cm 1,21 1,18
Infectivity + -
Intracellular Multiplication - +
Intravenous mortality for nice. + -
Immediate toxicity for cells in culture + -


There are 3 types of Chlamydia

:
Chlamydia are Gram negative bacteria. They are nonmotile with reproduction intracellular. They cannot produce ATP so that they need other cells from eucariots as source of ATP.Artropodes are not hosts.
The evolution comprehends two types of cells: After the intracellular reproduction in vacuoles in the cytoplasm of the cells of humans, other mammals or birds the reticular corpuscules which can reproduce itself are liberated and can be assimilated by new host cells [181] [182]. Within 8 to 12 hours the reticular corpuscules divide 10 to 12 times. At the end of this phase the reticular corpuscules are transformed again in elementary corpuscules.The elementary corpuscules can invade new cells.
Chlamydia are susceptible to a series of antibiotic. In cases of infections from Chlamydia there are used tetracyclines, sulfonamides, erythroycine and rifampin.
Chlamydia are resistant to aminoglicosides, bacitracin, vancomycin or ristocetin. Important species of Chlamydia are:


Chlamydia psittaci

This bacterium causes the ornithosis also known as psitacosis. Birds are the normal reservoir of Chlamydia together with other animals like cats, dogs and other mammals. Transmission from person to person is very rare.

Ornithose looks like a pneumonia varying from not serious to mortal. Transmission is made through air.


Chlamydia trachomatis

Transmission from person to person is direct. The species have various serovares which can cause different symptoms.


Chlamydia trachomatis serotype A-K

Produces conjunctivitis by invading epithelial cells. it also invades the epithelial cells of the urinary tract, rectal mucous membrane and feminine genitals.


Chlamydia trachomatis serotype D-K

It is being transmitted through sexual contact. In women it may cause relapsing and chronical diseases resulting in tubal sterility. Infections during pregnancy may cause premature birth and transmission of the germ to the newborn during birth.
Since 1995 the examination of secretions of pregnant to exclude a contamination with Chlamydia is provided by German regulations.


Chlamydia pneumoniae

Taxonomy

Analysis of gene sequence has led to a revision of the taxonomy of the family Chlamydiaceae.
The species Chlamydia pneumoniae was reclassified to Chlamydophila pneumoniae[183].


Cell culture techniques for Chlamydia pneumoniae

Cell culture techniques are used for isolation of Chlamydia pneumoniae from clinical samples. The infection of a monolayer is achieved by centrifugation of the sample onto the monlayer and incubation at 37°. Cells used for culture are HEp-2 cells and Chang cells with cycloheximide as antimetabolite [184]

C. pneumoniae invade the epithelial layer of upper and lower respiratory tract. Many infections occur subclinical or after an incubation of 3 weeks as similar to flue infect.

It is believed that 10 to 15% of treated pneumonia are caused by Chlamydia pneumoniae.
Since 1989 Chlamydia pneumoniae is considered as pathogenic[185][186]. Transmission is made trough droplets. The possibility that amoebae may act as reservoir of Chlamydia is being discussed.

According to seroepidemiological studies 80 percent of adults are infected by Chlamydia.
Chlamydia pneumoniae has been detected in various cases of inflammation of the myocardium and coronary tissue and blood vessels. This started the theory Chlamydia pneumoniae being the main cause of arteriosclerosis as a result of a respiratory infection during childhood[187][188][189][190]. Invasion of the body by Chlamydia is made through the respiratory tract. In vitro studies have shown that infections of macrophages result in elevation of the secretion of zitoquines such as the tumor-necrotic alfa factor (TNF) and interleukines (IL-1 and IL-6)
The inflammatory process is directly linked to the multiplication of the cells of the straight muscles as well as the stimulus of of coagulation.

A high titer of antibodies in plasma shows that there had been a contact with the germ. Studies of Finland in 1988 have demonstrated the link between coronary diseases and the titer of the antibody. This has created the theory that Arteriosclerosis is caused by an old or a chronical infection with Chlamydia pneumoniae or Herpes simplex. It is still being discussed if they are the main cause or if they settle secondary on already existing lesions, accelerating the process. Chlamydia can infect macrophages and survive for long time in its interior. Macrophages play an important part in the etiology of arteriosclerosis. Macrophages are formed by the blood having the property to assimilate oxidized LDL particles and to transform itself in foam-cells.

Macrophage may also be produced by the cells of the straight muscles of the blood vessels.
Foam-cells are the basis of atheromes, which later on will turn out to arteriosclerosis.
It is very likely that Chlamydia pneumoniae causes a local infection with lesions of the blood vessels. These lesions are the points where arteriosclerosis may start. Not always Chlamydia is found in case of arteriosclerosis causing false negative results.

A lage amount of evidence has now accumulated demonstrating a positive association between chronic infection with Chlamydia pneumoniae and atheroma development[191].

Even being confirmed Chlamydia to have a leading role in the etiology of atherosclerosis the participation of nicotine, excess of body weight, hipercholesterinemia and hipertonia will not be denied. These risk factors will have to be treated or even eliminated at the same time.


Chlamydia and arteriosclerosis

There are an increasing number of scientific report about Chlamydia pneumoniae being responsible for arteriosclerosis.
These reports bring up the discussion about reduction of fat, especially saturated fatty acids in the prevention of arteriosclerosis.

The annual edition of "Ernährungs-Umschau" 1998 has printed an interview with Prof.Dr. Wolfram, president of the "Deutsche Gesellschaft für Ernährung[192]. According to Dr. Wolfram there is no reason to modify the prevention of arteriosclerosis.Even in case that Chlamydia pneumoniae is proved to be the main cause of alterations of blood vessels the classic risks will not loose their validity.

Helicobacter pylori is considered as being an agent of infections of the gastric mucous membrane. There are evidence that Chlamydia pneumoniae is responsible for the start of arteriosclerosis. The classic prevention such as reduction of weight, reduction of fat in food and increase of physical activity is now on doubt[193].

Heart infarcts, clinical reports

Studies in Salt Lake ,Utah, USA[194] have demonstrated the presence of Chlamydia pneumoniae in 79% with coronary arteriosclerosis.Only 4% of the patients bearing Chlamydia had no coronary alterations.

This study followed the comments of the studies of Finland and Sweden.The Authors believe that Chlamydia may elevate the level tissue-factors causing thrombosis and adhesion of plattlets causing coronary diseases. They admit however that the results of their studies only satisfy the first postulate of Koch being more research necessary to confirm their theory.

Diagnostic of human infection caused by Chlamydia

Direct diagnostic of Chlamydia is very difficult starting from material of biopsis of blood vessels.The presence of Chlamydia is confirmed by PCR, immunoestequiometry, immunocitochemistry, electronic microscopy and culture of the germ. Indirect diagnostic can be made through a high titer of antibodies IgG and IgA from Chlamydia pneumoniae and specific immunocomplexes in the plasma of the patient.

Chlamydia threatening the population on koala bears (Phascolarctos cinereus) in Australia [195] [196]

Koala bears in Australia are being threatened by Chlamydia pecorum and Chlamydia pneumoniae. The concurrent infection with a retrovirus weakens the immune system of the animals. Fortunately, there is a vaccine available that seems to work on female koalas.

Health professionals warn of getting in contact with koala bears infected with Chlamidia pneumoniae The disease can be transmited to humans. The urine of diseased Koala bears is also infectious. The Australian state of Queensland reports that the number of koals dropped from 60.000 down to 11.000 from 1990 to 2012. The chlamidia strain infecting up to 90% of the koalas is slightly different of the human strain. It is also transmitted through sexual contact and is more severe than the human strain. Symptoms include urinary tract infections and involuntarily bowel excretion, may cause infertility, blindness and death.

Researchers from Queensland University of Technology mapped the koala's genome and are working to develop a vaccine. According to Professor Peter Timms the IFN-g genes were found to control chlamydia and Koala Retrovirus infections in humans and other animals.

Chlamydophila pneumoniae formerly known as Chlamydia pneumoniae

Chlamydia pneumoniae is widespread in humans. It causes acute upper and lower respiratory tract infections in addition to a range of other diseases in humans and animals. It is also infecting animals. Australian koalas are widely infected with Chlamydia, pecorum and Chlamydia pneumoniae. Other Chlamydia, such as Chlamydia psittaci and Chlamydia abortus, are known zoonotic pathogens. [197]

Mathew et al. 2013 provide data on the role of two key cytokines, pro-inflammatory tumour necrosis factor alpha (TNFα) and anti-inflammatory interleukin 10 (IL10), in koala bears infected with Chlamydia pecorum. According to the authors these data may be useful to develop a koala chlamydial vaccine. [198]

Chlamydial vaccine in humans are built on the chlamydial major outer membrane protein (MOMP). Kollipara et al. 2013 report that koalas vaccinated with a MOMP-based Chlamydia pecorum vaccine developed strong antibody and lymphocyte proliferation responses to both homologous as well as heterologous MOMP proteins. The authors concluded that MOMP based Chlamydia pecorum vaccine may protect against a range of strains and vaccine is the best option to protect koalas. [199]

Chlamydia trachomatis cause reproductive damage and infertility in many women. The lack of acute symptoms contributes to the high prevalence of chlamydial infection. [200]

Chlamydophila pneumoniae infection was proposed as an agent of atherosclerosis, however, Joshi et al. 2013 found that negative clinical trials did not find benefits of anti-Chlamydophila drug therapy. The authors call for new paradigms in Chlamydophila-atherosclerosis research. [201]

Treatment of arteriosclerosis

Treating infarcts with azitomicin and roxitromicin could reduce the level of infection indicators.
There were significant reduction of the number of relapses.

In patients with high levels of antibodies IgG azitomicin and roxitromicin reduced the signs of infection.
There are however only a small number of cases under surveillance what does not justify an uncontrolled medication or prophylaxis of arteriosclerosis with these antibiotics.

Even being promising, the use of azitomicin or roxitromicin in the treatment of arteriosclerosis may induce resistance to antibiotic in bacteria and loss of effect in the treatment of other infectious diseases.

Acetylsalicylic acid

Studies over 10 years have demonstrated that a group of patients with high level of C-reactive protein using acetylsalicylic acid had less infarcts as a group with no protection with AAS.
It is being suggested to continue these studies.

Serum salicylic acid is related to fruit and vegetable consumption [202]

Spadafranca and colleagues 2007 studied the relationship between fruit and vegetable consume and and the amount of circulating salicylic acid, which is a natural phenolic compound present in fruits and vegetable, and is the active principle of aspirin. The authors found that the median salicylic acid serum concentration was 0.124 mumol/l, varying between 0.188 mumol/l for those who had more than 4.75 serving of fruits and vegetables the day before, and 0.112 for those who had only 2.3 servings. The authors stress that there is a low chronic salicylic acid exposure from vegetal food which may be responsible, at least in part, for the beneficial effects of fruits and vegetable consume.

Vitamin C and arteriosclerosis

A study of Finland has established a link between a hypovitaminosis C and arteriosclerosis.
It is believed that vitamin C protects against infectious diseases, especially of virotic origin and therefore also against Chlamydia. This theory is yet not confirmed.

Classical advises of arteriosclerosis prevention

Clostridium perfringens


Clostridium perfringens:Merck Fluorocult TSC-Supplement

Clostridium perfringens is an anaerobic sporulating bacterium which is known in medicine causing gaseous gangrene, as well in food microbiology associated with dangerous toxins [203].

A rapid technique to detect Clostridium perfringens in food samples is done by using TSC-Agar (Tryptose-Sulfit-Cycloserin Merck Nr. 1.11972) adding Fluorocult TSC-Supplement (Merck Nr. 1.04032).
The supplement contains D-Cycloserin used to inhibit accompanying bacteria and a fluorogen substrate (MUP) which acts as indicator of the specific acid phosphatase enzyme.

Composition of Fluorocult TSC-Supplement

250 mg D-Cycloserin and 50 mg Methylumbelliferylphosphate disodium salt to be used in 500 ml TSC-Agar The supplement is diluted in water and added to the TSC-Agar. The sample is inoculated in pour plate and incubated under strict anaerobic conditions at 44° for 18-24 hours. All under UV light fluorescent colonies are Clostridium perfringens. Further biochemical reactions such as lactose, gelatine, nitrate, motility are unnecessary.


Refrigeration and shelf life

Refrigeration of food is used to increase shelf life. Food freezing made the transportation of perishable food over long distances possible. cooling and freezing food works against the activity of bacteria such as: The speed of enzymatic and biochemical reactions are highly temperature bounded. It is therefore obvious that low temperatures result in low biochemical activity.

Even under low temperatures there may be enzymatic activity without increase of total number. Streptococcus lactis may produce lactic acid at 0° but it does not multiplicate itself. As it grows older, the cells may die. As there are no new generations the total number of bacteria may decrease. Very often bacteriological counts from one laboratory do not compare with the counts of another laboratory when the samples are taken on different days.

Proteolytical activity can take place even at -18° . Lipolytical activity of Pseudomonas in butter is present at -10° .

Diseases transmitted by water

Almost every pathogen agent can be transmitted through water.
Normal safe supply of drinking water for the population of overcrowded areas is very important In disaster areas the supply of uncontaminated fresh water is crucial to avoid epidemics.

In hospitals and nurseries sterilized water and milk is necessary to feed newborn, old, sick and weakened persons. Strictest asepsis is necessary to prevent infections among these groups of patients.

Bacteria found in water

Water specific bacteria: Water bacteria with contamination origin from sewage and other sources:

Legionella pneumophila

In 1976 Vietnam-veterans came together in a dilapidated hotel in Philadelphia. They contracted a lung disease of which 29 died. The disease was called "Veterans disease" and the isolated germ was labeled as Legionella.
Legionella pneumonia is found in old water pipelines carrying warm water with often stand still.

Moisture and water droplets from shower and air conditioning system with poor maintenance can bear the germs and make a lung infection possible.
Old people and persons with reduced immunity are endangered.

Symptoms of Legionella infection

The symptoms of an infection caused by Legionella pneumophila are:
High fever,
Dry cough,
Chest pain
Headache and
Diarrhoea.
As these symptoms are unspecific the disease is often not diagnosed.Antibiotic medication is necessary.
Sterilization of the warm water system can be done flushing the system with water at a temperature of at least 70°. This temperature kills Legionella bacteria.
Even the Buckingham-Palace in London was contaminated by Legionella pneumophila The water system of the palace was heated up to 70° in December 1998 [204].

Test of Legionella pneumophila in water

The method to test the presence of Legionella pneumophila in water was published by the German Health Department:
An inspection concerning the hygienic status of the system and his environment is important.
Transportation to the laboratory should be made as soon as possible.If the sample has to be stored over night it should not be refrigerated, but left in dark and by room temperature.

Method for volumes from 100 ml to 1.000 ml

Membrane filtration with polycarbonate filter with pores from 0,4 to 0,45 milli micra should be used. Resuspend in 0,5 to 1 ml of the water of the sample with ultrasound using low power in order not to damage the cells of Legionella.
Add 0,5 ml of 0,2 ml-KCL/HCL buffer mix and wait for 5 minutes.
Inoculate the whole volume on the surface of several BCYE- Agar-Plate
This culture media contains alfa-ketoglutarat,glycin, L-cysteine-hydrochloride and iron-III-pyrophosphate as well a antibiotic supplement.
Incubate for seven days at 35 to 37° in damp atmosphere.

Differentiation

Inoculate about three typical colonies on a cysteine free culture media such as blood agar. Incubate two to three days at 35 to 37°. If there is no growth, test identical colonies with Immunofluorescent test using mono or polyvalent sera of Legionella pneumophyla.

Clear water samples expecting low number of Legionella

Membrane filtration should be made with "black" celulosenitrate filters.
After filtration of the sample the filter should be covered with 10 ml 0,2-mol-KCl/HCl-buffer.
After 5 minutes the buffer should be filtered off and the filter should be washed with about 10 ml PBS (phosphate buffer, pH 7,6). The filter should now be placed on a plate of blood-agar incubated and differentiated as described above.

Method for small volumes of water sample

The water samples are inoculated directly using not more than 0,5 ml for each plate.
Incubation and differentiation as described above.


Legionella longbeachae, living in composted waste, causes lung infections and endocarditis [205]

Leggieri et al 2012 report a case of infectious endocarditis attributable to Legionella longbeachae. L. longbeachae is usually associated with lung infections. It is a facultative intracellular gram-negative bacillus, which is commonly found in composted waste wood products used in potting mixes. Legionella longbeachae should be regarded as an agent of infectious endocarditis, notably in the context of gardening involving handling of potting soils.

The patient recently used only Australian potting mixes, which may contain Listeria longbeachae, although we were not able to cultivate Legionella longbeachae or other Legionella spp. from the potting mix.

Enterobacteriaceae, culture methods

Culture methods for Enterobacteriaceae used in food bacteriology are:
Official collection of methods according to the 35 of the Food Law in Germany (Amtliche Sammlung von Untersuchungsverfahren nach 35 LMBG) indicate under Methode L 05.00.5 the use of Violet Neutral Red bile Dextrose Agar (VRBD-Agar ) for the culture of Enterobacteriaceae in fine foods such as Mayonnaise emulsified sauces, could sauces ready to eat, fine salads, prime materials and quality control of the production.

This medium is being used by commercial laboratories, by official laboratories of food control laboratories in Germany and with some small modifications by a large number of quality control laboratories of the industry.

This medium is also described by ISO/DIS 552 (1977), DIN 10164 and DIN 10172. Overlaying the plates with the same medium assures anaerobic conditions.Not fermenting gram negative bacteria will so be suppressed and fermentation of dextrose is increased.
Enterobacteriaceae grow as 1-2 mm violet colonies having a precipitation around the colony.Not Enterobacteriaceae grow as colorless colonies.
The Coli-Aerogenes - Group as possible indicator of feces contamination as great importance in the control of water but is of smaller significance on food where the search for exact defined groups of bacteria.

Enterobacteriaceae are considered to be a good indicator of failures by the manufacturing of foods using low heat. The VRBD-agar plate, Violet Red Bile Dextrose Agar gives positive colonies after 24 hours of incubation at 30 ° caused by the following bacteria:


Enterobacteriaceae

: Coliform bacteria
Shigella which do not ferment lactose
Escherichia coli
Escherichia coli which can produce Enterotoxin.They are more resistant to heat than the apathogenic E.coli.
Salmonella
Citrobacter
Klebsiella

Other bacteria which grow on VRBD, not included in the group of Enterobacteriaceae

Aeromonas
Yersinia
Pseudomonas, inhibited by overlay technique
Bacterium anitratum
Achromobacter anitratus.

The selectivity of VRBD reduces after 24 hours incubation.Some other not specified as Enterobacteriaceae bacteria can then grow.
Ingredient g/l
Yeast extract 3,0
Pepton 7,0
Natrium chlorid 5,0
Bile salt Nr.3 1,5
Glucose 10,0
Neutral red 0,03
Violet red 0,002
Agar 12,0

pH 7,4+-0,2
VRBD- Agar is told to have a better recovery rate of Salmonella , Arizona and Shigella as with the lactose bearing VRB-Agar. Unfortunately it does not differentiate between Coli and Salmonella/shigella.

Other media for Enterobacteriaceae

In food control laboratories using VRBD-agar there are often positive findings with low number of Enterobacteriaceae. In order to make a decision about discarding or using the food further media could be used beside VRBD- agar examining food known to have problems , such as potatoes salad.In production of food with short shelf life it is impossible to wait for results of traditional bacteriology.
To speed up final results one or more of the following media could additionaly be used:


Detection of Coli-Aerogenes group in water,foods and dairy products with VRB-Agar; Violet Red Bile Agar

This medium is cited by DIN 10160, APHA 1985, FIL-IDF, Euroglace and the official methode according 35 LMBG L 00.00-21.

It is not exclusive for Enterobacteriaceae, other bacteria such as Aeromonas and Yersinia spp. can produce similar reactions. Violet red and bile salt inhibit the growth of gram positive bacteria. The fermentation of lactose produces acid which makes the pH- indicator neutral red change its color to red and is responsible for the precipitation of bile salts. in the near surroundings of positive colony.

Lactose positive bacteria grow as 0,5-2 mm (24 hours) purple colonies which may be surrounded by a purple zone.(Coliform bacteria grow as 1-2 mm colonies.The Coli-Aerogenes group Enterococcus and Klebsiella) grow as 0,5 mm small colonies after 24 hours at 30° . Escherichia coli should be incubated at 44+-1°
Lactose negative bacteria grow as pale colonies which can be surrounded by a greenish zone.
Ingredient g/l
Yeast extract 3,0
Pepton 7,0
Natrium chlorid 5,0
Bile salt Nr.3 1,5
Lactose 10,0
Neutral red 0,03
Violet red 0,002
Agar 12,0

pH 7,4+-0,2 VRB-Agar is available under Art.-Nr. CM 107 from Oxoid and Ar.Nr. 1406 from Merck.


COLI ID, medium for the detection of coliforms and identification of E.coli

The medium COLI ID contains two chromogenic substrates which allows the direct recognition of coliforms and identification of Escherichia coli, without use of additional reagents.
Colony color rose blue colorless
beta-glucuronidase + - -
beta-galactosidase + + -
  Escherichia coli other coliforms other Gram(-)

Coliforms on COLI ID, Citrobacter,Enterobacter or Klebsiella have blue color. Detection, enumeration and identification of E. coli and coliforms are made after 24-48 hours incubation at 37° on pour-plate.E.coli colonies on COLI ID are of rose color with a rose zone around the colonies.Other Gram negative bacteria a on COLI ID are bight rose , small and have no surrounding zone. Gram positives and yeasts are inhibited.COLI ID is available under Ref 42017 from bioMérieux


Enrichment broth for total coliforms and Escherichia coli

Enrichment of total coliforms together with Escherichia coli can be made with Fluorocult LMX-broth according to Manafi and Ossmer

After 24 - 48 hours the broth will change its color to blue-green.
Fluorocult LMX-broth is available under the number 1.10620.
Positive cultures can be plated on Chromocult coliforms-agar.


Chromocult, coliforms agar

Chromocult, coliforms agar is a combination of two chromogene substrates Salmon-GAL and X-GLUC for the recognition of Escherichia coli, coliforms and other Enterobacteriaceae.
The growth of Coliforms even sublethal damaged cells is granted due to use of peptone, pyruvate, sorbitol and a buffer of phosphate. Gram(+) and some Gram (-) bacteria are inhibited by Tergitol 7. Coliform bacteria are beta-D-galactosidasepositive and ferment the substrate Salmon GAL turning pink/red.

Escherichia coli is beta-D-glucuronidase positive and ferment the substrate Salmon GAL turning blue.
The used substrate is X-Glucuronid.Escherichia coli ferments both Salmon-GAL and also X-Glucuronid turning blue-violet being easy to identify E. coli among other coliforms which turn pink-red.
Tryptophane improves the indol reaction to confirm E. coli adding safety of the detection of the germ with Salmon-GAL and x glucuronid-reaction. To suppress Pseudomonas spp. and Aeromonas spp.add 5 mg Cefsulodin(such as Sigma)in 1 ml demineralised. water sterile filtered to 1liter of medium.

Inoculation

As pour plate, as surface culture or as filter culture.
Read the plate as follows:

Escherichia coli

Deep blue-violet (Salmon-GAL reaction and x-glucuronid-reaction)
To confirm E. coli the colonies may covered with one droplet of Kovac's indol solution. Red color of the solution after few seconds is positive for E.coli.

Coliforms

:Pink-red colonies (Salmon-GAL reaction)(Citrobacter,Enterobacter, Klebsiella and some E.coli and deep blue-violet (E.coli).
Some enterohaemorrhagic Escherichia coli ferment X-Gluc (Mug-) and behave like coliforms. To find these bacteria use Fluorocult E.coli 0157:H7-agar .

Other Enterobacteriaceae

Pale colonies, with exception of some bacteria which have beta-glucuronidase activity. These Bacteria have bright blue turquoise colonies.

Ingredient g/l
Pepton 3,0
Natrium chlorid 5,0
Sodium dihydrogen phosphate 2,2
di-sodium hydrogenphosphate 2,7
Sodium pyrovate 1,0
Sorbitol 1,0
Tryptophane 1,0
Agar-agar 10,0
Tergitol 7 0,15
Chromogen mixture 0,2

pH 7,4+-0,2 Chromocult Coliforms Agar is available under Number 1.10426 Merck.

EHEC, Enterohaemorrhagic Escherichia coli 0157:H7, culture

Verotoxin producing E. coli (VETC) and Escherichia coli 0157:H7 (EHEC) produce diarrhea, kidney failures in children and old patients and are also important in veterinary medicine.
They can be detected on Fluorocult E coli 0157:H7-agar detected.

e.coli 0157:H7 is sorbitol negative and has no beta-D-glucuronidase.This is used to distinguish him from not pathogen E.coli.
The medium also distinguishes between Proteus and Shigella.
Fluorescens of MUG is an important differentiation between the different bacteria.
Inhibition of Gram (+) bacteria is made by sodiumdesoxycholate.

Bacteria colorprecipitationMUG Sorbitol      
E.coli 0157:H7 greenish - - -
E.coli yellow +- + +
Proteus mirabilis black-brown - - -
Enterobacter aerogenes yellow +- - +
Streptococcus faecalis no growth      

Fluorocult E.coli 0157:H7-agar is available under the number 1.04036


Fluorescence methods for detection of Escherichia coli

: E. Meck, Darmstadt, Germany has various culture media to detect by means of fluorescence, production of indol from tryptophan.

The fluorescence methode is done by adding MUG (4-Methylumbelliferyl-beta-D-glucuronid.)
Best results on reading the cultures are at pH 9 to 10 under UV light of 366 nm.


Advantages of MUG (glucuronidase methode )

Image MUG_methode

To avoid false results please observe:

Fluorescence optical methods are cited in the following German DIN methods: DIN 10 110 : Determination on E.coli in meat and meat derivates.
DIN 10 183 , part 3 : Determination of E.coli in milk, milk derivates, icecream, baby food based on milk.

MUG can be added to almost any standard culture medium, however the usual media having MUG already added are available. Some examples are:


Merck Fluorocult Brila-broth (1.12587)

It is used as selective enrichment and enumeration from E. coli and other coliforms in titer test of water from swimming pools.

Bile and brilliant green inhibit Gram positive bacteria. Reading of the cultures is done under 366 nm light. A pale blue fluorescence of the tube stands for the presence of E.coli.
To confirm the result the culture should be covered with approximately 5 mm with KOVACS-indol reagent A cherry-red color of the reagent stands for the presence of E.coli and /or coliforms.


Merck Fluorocult DEV-lactose-peptone-broth (1.04037)

It is used as enrichment and titer of coliform bacteria in the examination of water.
The presence of E. coli is demonstrated with fluorescence under UV light, and the a positive indol reaction.

Merck Fluorocult ECD-Agar (1.04038)

The ECD agar (E.coli Direct Agar) is suitable to examine feces and food samples as well. It is cited under DIN 10 110 for the examination of meat and related material. The bile salt inhibits the not obligatory intestinal flora. The presence of E. coli is demonstrated with fluorescence under UV light, and the a positive indol reaction.


Merck Fluorocult E.coli 0157:H7 Agar(1.04036)

It is a selective agar for the isolation and differentiation of enterohaemorrhagic EHEC Escherichia coli 0157:H7 from food samples and from clinic material.
This bacteria is sorbitol negative, glucuronidase negative, no fluorescence.
Sodium desoxicholat inhibits the concomitant Gram positive flora.

Faecis are inoculated directly on plates and incubated for 14 to 24 hours at 37°. Sorbitol negative colonies presenting no modification of the color of the medium have to be identified under UV light.
E.coli o157:H7 grows as greenish and Proteus mirabilis as black/brown colonies.


Fluorocult Lauryl-Sulfate-Broth (1.12588)

This culture medium is determined to be a reference methode for E.coli in the examination of milk and derivates in DIN 10183, part 3. Lauryl-sulfate is added to inhibit the concomitant organism.


Merck Fluorocult LMX- Broth (Laurylsulfate-MUG-X-Gal)

This medium is used in the simultaneously detection or coliforms and E.coli.
A rapid growth of coliform bacteria is granted due to the high nourishing properties of the medium and the presence of a phosphate buffer.

Lauryl-sulfate is added to inhibit the concomitant organism. The chromogen substrate X-Gal is being hydrolysed causing the change of color to blue-green. The booster substance IPTG intensifying the enzymatic activity of coliforms increasing the sensibility of the test. E. coli is confirmed with fluorescence.


Merck Fluorocult MacConkey-Agar [1.04029]

This culture medium is used to isolate Salmonelleae Shigelleae and coliforms, especially Escherichia coli. Bile salts and violet red inhibit the Gram positive Flora.
Lactose fermentation is demonstrated by the pH indicator neutral red.

Escherichia coli colonies are fluorescent under UV light. Lactose negative colonies are colorless,lactose positive red often with a turbid zone of precipitated bile.


Merck Fluorocult VRB-Agar(Fluorocult Violet Red Bile Agar 1.04030)

This selective culture medium is used to detect and to enumerate coliform bacteria, especially Escherichia coli.Violet red and bile salts inhibit the Gram positive Flora.
Lactose positive bacteria turn red among them E.coli is fluorescent under UV light. Lactose negative Enterobacteriaceae are colorless.


Merck Bactident E.coli, rapid test to identify Escherichia coli isolated on non selective media (1.13303)

[203]
The result is available in about 30 minutes. There is no asepsis needed because the initial amount of bacteria is very high and growth of E. coli more rapid as concomitant bacteria.
A colony on test is suspended in 200 microliter distilled water. A test stick is added and the tub is incubated by 37° for 30 to 120 minutes. under ultra violet light of 366 nm fluorescence is read (glucuronidase with MUG).Indol reaction is done by adding one droplet of KOVACS reagent. Red color of the suspension after some seconds is a positive reaction and confirms E.coli.

Thin layer chromatography with immunologic analysis to detect E.coli 0157:H7

Vip EHEC Thin layer chromatography with immunologic analysis to detect E.coli 0157:H7 is a rapid detection system for E.coli (EHEC) in food from BioControl Systems Inc.USA.

This system is admitted by the AOAC. It makes EHEC specific antigens visible as a blue reaction line reacting with antibody linked to blue latex particle.
To perform the test VIP EHEC Biotest Nr. 928 110 and further utensils are needed:
- Sterile Pipette with 100 µl volume
- Stopwatch
- 225 modified Casein peptone-Soy peptone-Broth with Novobiocin
ingredient quantity
Casein peptone-Soy peptone-broth 30,0 g/l
bile salt Nor.3 1,5 g/l
K2HPO4 1,5 g/l
Deionizated water 1000 ml
Autoclavated and cool  
   
Novobiocin solution:  
   
Novobiocin (Na salt] 100 mg
Deionized water 1,0 ml

The novobiocine solution is to be sterile filtered an kept in dark a glass. The shelf life of the solution is several month at 4°.

Enrichment

25 g from the sample are incubated at 35-37° during 18 hours in 225 ml modified Casein-peptone-Soy peptone-Broth.
0,1 ml of the enrichment culture are place in the start field. The test kit is then incubated for 10 minutes at room temperature. The reading of the test should be made after 10 minutes, after that false positive reactions can take place.
Positive results should be confirmed inoculating the enrichment culture one a selective medium such as: Sorbitol-macConkey -Agar (Heipha Nr. 125e) , Bile-Chrysoidin -Sorbitol-Agar with MUG, (Heipha Nr 1052). After 24 hours of incubation the culture plates are read and suspected colonies are biochemicaly differentiated and identified with specific serotypes.


Polymerase chain reaction (PCR) identifying microorganism

Tougher regulatory standards and the increased attention of the news media in relation to food contamination in restaurants, supermarkets and processing plants trigger higher priority on safety, shelf life and cleanliness. More stringent and rapid testing along the whole supply chain are being developed.

PCR became one of the top laboratory methods for microbacterial detection in the food industry..It can detect small samples of contamination by amplifying the amount of DNA of the contaminant.

Scientist of Campden and Chorleywood Food Researche have automated ribotyping for characterising microorganisms below the species level. The technique can be used for identification. To complement the technology the development of an identification system based on the capture of PCR-amplified DNA sequences onto DNA microarrays is being developed.


Microarray

A microarray is a solid surface as a microscope slide, onto which the amplified DNA is bond. The microarrays can be used to probe an unknown organism to see which of the DNA sequences on the array are also present in the organism.

Microarray probes were designed for selected groups of bacteria, based on regions of the 16S ribosomal RNA. Campden and Chorleywood Food Researche (CCFRA) developed and has validated PCR protocols for Enterobacteriaceae, Pseudomonas, Bacillus and Clostridium species.

Impedance technologies for rapid detection and enumeration of bacteria

The impedance technology is being increasingly used in industrial microbiology.

Principle

Proteins and carbohydrates from the culture medium are electrically neutral or weakly ionized. Microorganisms transform these molecules into several smaller parts like amino acids, lactate etc which have greater charge and electrical motility.
These modifications can be measured between paired electrodes in the culture medium. Even very weak electrical variations of the medium can be measured and the presence of very few bacteria can be detected long before a colony is visible in culture.

Making a curve of the percentage of electrical variation in relation to time there is an unexpected inflection in the curve depending on the number and the metabolic activity of the bacteria being present.

Image Impedance_technology

Detection time [206]: is the point of the inflection of the curve, depending on the number and the metabolic activity of the bacteria being present.


Impedance

is the total resistance measured in a conductive medium.


Conductance

measures the bulk ionic strength in the growth medium.


Capacitance

is measured as the accumulation of electrical charges by increasing the ionic concentration around the electrodes.
Measurement of capacitance is particularly adapted to detection of bacteria which release slightly ionized metabolites (yeasts, moulds and non-fermenting Gram negative organisms).


Sterility testing

a detection time at anytime during the test period signifies a non sterile sample.

Screening or enumeration

the detection time enables samples to be accepted or rejected according to the specification level in a short period of time.

Impedance technology provides results within hours. The more contaminated the sample, the quicker impedance technology detects it saving time.
Automated impedance technology available as BACTOMETER from bioMérieux.This company also sells dehydrated culture media specially adapted to impedance technology:

Total count BHI: Enumeration of total aerobic flora in food.
Total count MPCA: Enumeration of total aerobic flora in food.
Total count GPM: Sterility control and enumeration of total aerobic flora in milk, food, cosmetics, and pharmaceutical products.
Total count GPM Plus: Sterility control and enumeration of total aerobic flora in milk, food, cosmetics and pharmaceutical products. The formula of the medium is enriched vitamins, amino acids etc to favor the growth of fastidious and/or stressed bacteria during the manufacturing process.
Coliforms CM: Enumeration of coliforms in food.
Enterobacteriaceae EM: Enumeration of Enterobacteriaceae in food.
Yeasts/Moulds YMM: Detection of yeasts and moulds in fruit juice, milk, food, cosmetics and pharmaceutical products.

Lactic acid bacteria LM

Detection and enumeration of lactic acid bacteria in fruit juice, dairy products and food. The BACTOMETER impedance technology used for total count,sterility tests, coliforms Enterobacteriaceae, Yeasts and moulds as well as lactic acid bacteria can also be complemented with two other systems:


VITEK

Automated identification of Gram negative bacilli, Grampositive cocci, Bacillus, anaerobes, yeasts.


VIDAS

Automated detection using immunoanalysis of pathogenic bacteria, Listeria, Salmonella, Staphylococcal enterotoxins, E.coli O157, Campylobacter.
The principle of VIDAS is based on a specific reaction between an antibody and an antigen. An antibody coated device captures the antigens of the organism being detected. After several washing procedures a second antibody will fix the antigen using the sandwich technique.

This second antibody is then conjugated with an enzyme, which will produce a fluorescent reaction with the substrate. The intensity of this reaction is measured and interpreted by the system.


The RABIT (Rapid Automated Bacterial Impedance Technique)

is a system developed by Don Whitley Scientific Limited,Shipley, West Yorkshire. Using the principles of the impedance technology RABIT detects and enumerates bacteria. High initial costs of the device still are a disadvantage of the impedance technique.


MALTHUS from Malthus Instruments

, Bury, UK measures electrical conductivity of culture media.It detects and enumerates specific bacteria such as Campylobacter, coliforms, Columbia, Enterobacteriaceae, Listeria monocytogenes, Salmonellae, TMA ( total count), Staphylococcus aureus.

Bioship to detect Listeria monocitogenes [207]

Bhunia and colleagues 2009 developed a biosensor to detect Listeria monocitogenes. Whioch could not easily be detected using antibodies. The researchers used heat shock protein 60 (Hsp60), a eukaryotic mitochondrial chaperon protein which is a receptor for Listeria adhesion protein (LAP) during Listeria monocytogenes infection.

The authors concluded that Hsp60, immobilized on the surface of streptavidin-coated silicon dioxide biochip sensor platform may be used for the detection of Listeria monocytogenes. They report that the capture efficiency of Listeria monocytogenes was 83 times greater than another Listeria receptor, the monoclonal antibody, mAb-C11E9.


BacTrac, impedance analytic

BacTrac is a system using Impedance technology to detect and to enumerate spoilage bacteria in beer, coliform, Salmonellae and Staphylococcus aureus and total bacterial count in milk powder.
BcTrac system can scan three analytical methods:
The Impedance of the medium (M-value)
The Impedance at the Electrode (E-value)
The indirect Measuring of the Gas Production (G-value)

Measuring the three parameters at the same time a more rapid result is possible.
The Impedance at the Electrode (E-Value) often reacts earlier as other values , permitting thus a rapid result. The E-value is relatively stable against variations of salt content. The Impedance of the Medium (M-value) is a confirmation of the E-value and increases the safety of the result.

The biological activity of bacteria decompose big molecules (carbohydrates, proteins, peptides etc) of the medium in which they are growing. Smaller chemical compounds result which are dissociated and and increase conductivity. The electrical resistance is diminished. [208]
There are 5 possible interaction of the bacteria with the substrate: The Impedance of the medium (E-value)
  1. Molecules without charge and polar macromolecules are digested in smaller ions, increasing the conductivity of the medium.
  2. Small molecules are digested in very small electrical charged parts which can move more easily increasing the conductivity.
  3. Ions already existing in solution are united in great ions. The conductivity is diminished.
  4. Small ions are united in electrical charged macromolecules. The conductivity is diminished.
  5. Autolysis of cells: Due to autolytic activities of the cell often observed in yeasts, ions of the interior of the cell are liberated an increase the conductivity . This is not caused by growing number of alien bacteria.
To measure cultural media a metallic electrode of high-grade steel, platinum or gold is dipped into the solution. A layer of ions covers the surface of the electrode. Measuring the electrical resistance of the electrode the variations of this layer is recorded.

The impedance of the electrode ZE acts as a capacitor with a parallel resistor. Cultural media or bacterial suspensions have ions and particles without charges which can also adhere to the electrode. A separate measure of the impedance of the electrode and the impedance of the medium is very useful to get earlier results:

Image Impedance

The growth of Saccharomyces cerevisiae [208]is seen only after 15 hours measuring the impedance of the medium. Measuring the impedance of the electrode the result is already seen after 4 hours.

When high amount of ions from the medium are already present new ions from the activity of microorganisms present no significant modification of conductivity, conductance or resistance which is measured as Impedance of the Medium , M-value. Measuring the impedance of the Electrode E-value, there is a good result after a couple of hours. This is shown in the example of Zygosaccharomyces rouxii.

Other toxigenic bacteria can be detected with the BacTrac system using the impedance technology. Such bacteria and other applications are: Listeria and Clostridium, total count, sterility test, shelf life, activity test of preservatives and antibiotic, activity of starter cultures.

The impedance technology has been validated in DIN 10115 of the German standardization.

Clostridium acetobutylicum

Clostridium acetobutylicum can digest whey, sugar, starch, lignin, cellulose fiber, and other biomass directly into butanol, propionic acid, ether and glycerin. The bacterium is useful in the fuel production using organic waste.

Clostridium difficile Infection

[209] Clostridium difficile infections are related to food infections and 94% are healthcare contamination. The current epidemic strain of Clostridium difficile is the BI/NAP1/027, toxinotype III strain. This strain is more virulent than foregoing strains. It has an increased toxin A and B production, polymorphisms in binding domain of toxin B, and presents increased sporulation.

The Centers for Disease Control and Prevention (CDC) outlined the 6 strategies that have the best chance of eradicating Clostridium difficile from healthcare. These strategies involve elements of antibiotic stewardship, testing for Clostridium difficile, isolation and infection control procedures, environmental cleaning, and communication.
1. Prescribe and use antibiotics carefully. About 50% of antibiotics that are given are not needed.
2. Test for C difficile when patients have diarrhea while taking antibiotics or within several months of taking them.
3. Isolate patients with C difficile immediately.
4. Wear gloves and gown when treating patients with C difficile, even during short visits. Hand sanitizer does not kill C difficile, and handwashing may not be sufficient.
5. Clean room surfaces with bleach or another Environmental Protection Agency (EPA)-approved spore-killing disinfectant after a patient with C difficile has been treated there.
6. When a patient transfers to another facility, notify the new facility if the patient has CDI.

Hand hygiene [210]

The alcohol-based (hand sanitizer) gels don't kill C difficile spores. Essentially, hand gels don't do anything to these spores, says Gerding et al. 2008. Handwashing, with soap, water, and friction, is better than alcohol-based hand sanitizers for Clostridium difficile, because of dilution and physical removal-getting the spores off of the hands. However, alcohol-based hand sanitizers are more effective than handwashing for a huge range of usual pathogens that do not form spores.

Clostridium difficile is probably transmitted primarily between patients on the hands of healthcare personnel who are transiently contaminated after contact with symptomatic patients or their surrounding environment, therefore gloves are the first and best line of defence.

The data suggest that patients with Clostridium difficile infection can continue to shed organisms for up to 7 days after the cessation of diarrhoea. We know these patients will continue to shed; it's the degree of shedding that is important. The important factor is diarrhoea. Most shedding occurs while the patient has diarrhoea. When diarrhoea resolves, shedding diminishes.

Good physical cleaning should be accomplished, because this can lead to the physical removal of spores. This should be augmented with an EPA-approved spore-killing disinfectant, such as chlorine bleach. Standard EPA-registered hospital disinfectants are not effective against Clostridium difficile spores. CDC recommends telling patients to use a separate bathroom at home or make sure it has been cleaned before someone else uses it.

SHEA/IDSA Practice Recommendation 2008: The organisations highlight practical recommendations designed to assist acute care hospitals in implementing and prioritizing their Clostridium difficile infection (CDI) prevention efforts. Hand hygiene and environmental disinfection is elucidated. [211]

SHEA-IDSA Guidelines 2010: Clinical Practice Guidelines for Clostridium difficile Infection in Adults (2010). Update by the Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). The revised guidelines of 2010 stress the need to advise visitors and Healthcare workers to wash hands with soap (or antimicrobial soap) and water after caring for or contacting patients with Clostridium difficile. [212]

Glove use is the only Clostridium difficile infection prevention recommendation with the highest strength of recommendation. If gloves are removed properly to prevent hand contamination in the removal process, any potential benefit of using soap and water over alcohol-based hand hygiene products is likely negated.

Dubberke and Gerding in an update in 2011 conclude that although soap and water is superior to removing Clostridium difficile spores from hands of volunteers compared to alcohol-based hand hygiene products, there have been no studies in acute care settings that have demonstrated an increase in Clostridium difficile infections with alcohol-based hand hygiene products or a decrease in Clostridium difficile infections with soap and water. This is why preferential use of soap and water for hand hygiene after caring for a patient with Clostridium difficile infections is not recommended in non-outbreak settings. The recommendation to use soap and water preferentially in outbreak settings after caring for a patient with Clostridium difficile infections is based on expert opinion as there are no data that demonstrate preferential use of soap and water for hand hygiene after caring for a patient with Clostridium difficile infections in an outbreak setting is effective at preventing Clostridium difficile infections. [213]

Dubberke 2012 stresses the importance of appropriate contact precautions, strict hand hygiene, effective environmental cleaning, identification and removal of environmental sources of Clostridium difficile, and antibiotic stewardship in the prevention of Clostridium difficile infections. [214]

Yoghurt or probiotics to reduce CDI risk

Although not a core CDC recommendation at this time, the idea of using yoghurt or probiotics to prevent CDI-associated diarrhoea makes a lot of sense. It has been studied, but the data don't fully say that the probiotics that are currently available are effective in preventing Clostridium difficile infection.

Over-the-counter drugs used to treat frequent heartburn increase risk of Clostridium difficile infection. []

The U.S. Food and Drug Administration (FDA) is informing the public that the use of stomach acid drugs known as proton pump inhibitors (PPIs) may be associated with an increased risk of Clostridium difficile-associated diarrhoea.

Chlostridium difficile prevention [215]

According to Badger et al 2012, Clostridium difficile may be found in the normal flora in some healthy individuals. Some persons, however, have selective risk factors which turns them susceptible to the morbidity and mortality of the diarrhoea caused by this bacteria.

Toxigenic culture testing from anaerobic culture remains the gold standard, and together with real-time polymerase chain reaction (PCR) has a high specificity, however, both are time consuming. Enzyme immunoassay and common antigen (glutamate dehydrogenase) testing, are performed in a short time. A strategy for prevention and eradication of Clostridium difficile is being suggested by the authors. This strategy includes early disease recognition through appropriate surveillance, implementation of effective contact isolation strategies, adherence to environmental controls, judicious hand hygiene, evidence-based treatment, and management that includes antibiotic stewardship, continuous education of healthcare workers, and administrative support.

Clostridium difficile contamination of food []

Clostridium difficile infection is typically associated with exposure to health care settings, especially among patients who have taken antibiotics, however, it is increasingly recognized as a cause of diarrhoea in persons with no apparent health care contacts.

Recent studies have isolated Clostridium difficile from retail beef, pork, turkey products and ground beef intended for human consumption in the United States, Canada, and Europe and from meat products intended for consumption by pets.

Several studies found overlap among bovine, equine, porcine, canine, and human isolates. Toxinotype (TOX) V/PCR ribotype 078/PFGE type NAP7 or NAP8/REA type BK strains are the predominant strains in cattle and pigs in the United States and Europe and also a common pathogens in humans. The epidemic strain, NAP1/027/BI, affected crowded areas in the Netherlands, and human infections with ribotype 078 was concentrated in more rural areas where pigs are raised. Isolates from food animals were found to be very closely related to or indistinguishable from human isolates, however, it is unclear whether and how zoonotic transmission occurs.

Clostridium difficile on contaminated meat originates from food animal hides, intestinal contents and human handling. Spore contamination may not be preventable during processing of meat. Unlike other bacterial contaminants of meat, cooking might not kill Clostridium difficile. Spores can survive in ground beef despite proper handling and adherence to recommended cooking temperatures.


Laboratory tests used to diagnose Clostridium difficile infection [216]

Clostridium difficile is a spore-forming, Gram-positive anaerobic bacillus that produces two exotoxins: toxin A and toxin B. It accounts for 15-25% of all episodes of antibiotic-associated diarrhoea. Clostridium difficile infections cause nausea, fever, diarrhoea, and abdominal pain.

Often, Clostridium difficile infections cause nausea, fever, diarrhea, and abdominal pain. The infections produce inflammation of the colon. This bacterium is associated with the use of antibiotic therapies and long hospital stays. It can be life-threatenig among elderly and persons with weak immune systems or on prolonged antibiotic regimens. Clostridium difficile is the cause of 14,000 deaths in the US annually.

The preferred test is the nucleic acid amplification test (NAAT), because it has the highest sensitivity. Many hospitals are still using enzyme immunoassay (EIA), which detects stool toxins A and B. These tests provide rapid results but have low sensitivity, which can lead to repeated testing, with the possibility of a false-positive result. Stools should be transported to the laboratory within 2 hours or refrigerated, because the toxins degrade rapidly at room temperature, resulting in a false-negative test result.

Stool culture for Clostridium difficile: While this is the most sensitive test available, it is the one most often associated with false-positive results due to presence nontoxigenic Clostridium difficile strains. Isolates must be tested for toxin production (i.e. so called "toxigenic culture").

Molecular tests: FDA-approved PCR assays, which test for the gene encoding toxin B, are highly sensitive and specific for the presence of a toxin-producing Clostridium difficile organism.

Antigen detection for Clostridium difficile: These are rapid tests (<1 hr) that detect the presence of Clostridium difficile antigen by latex agglutination or immunochromatographic assays. Antigen assays have been employed in combination with tests for toxin detection, PCR, or toxigenic culture in two-step testing algorithms.

Toxin testing for Clostridium difficile: Tissue culture cytotoxicity assay detects toxin B only. It is recognized as less sensitive than PCR or toxigenic culture.

Enzyme immunoassay detects toxin A, toxin B, or both A and B: There are increasing concerns about their relative insensitivity (less than tissue culture cytotoxicity and much less than PCR or toxigenic culture).

AmpliVue(R) Clostridium difficile test: The US Food and Drug Administration approved the AmpliVue C. difficile test. The Clostridium difficile Assay is a time saving molecular test which is more sensitive than current traditional detection methods. According to the U.S. Company Quidel, the C. difficile test combines isothermal helicase dependent amplification with a molecular method that requires no nucleic acids extraction step and no expensive equipment. [217]

Helicases: AmpliVue Clostridium test is based on helicases, which are often utilized to separate strands of a DNA double helix or a self-annealed RNA molecule by breaking of hydrogen bonds between annealed nucleotide bases. The helicase function is required for efficient and accurate replication, repair, and recombination of the genome. They also function to remove nucleic acid-associated proteins and catalyze homologous DNA recombination. Metabolic processes of RNA such as translation, transcription, ribosome biogenesis, RNA splicing, RNA transport, RNA editing, and RNA degradation are all facilitated by helicases. [218]

Corrosion caused by bacteria

Bilge water and standing water in ships,cooling water systems and water tanks may be contaminated by Desulfovibrio desulfuricans. This bacterium reduces sulfates to corrosive sulfides producing mud which can make holes through iron plates of ten millimeters in a period of one year.
A group of scientists of Palo Alto makes experiments experiments to change the bacterial layer (bio film) of water systems to a population of oxygen consuming bacteria which due to genetic modification can produce antimicrobial substances which act against sulfate reducing bacteria. [219]
In the meantime maintenance of water systems is the best way to avoid damages caused by Desulfovibrio.

European standards for plastic pipelines for water and gas supply [220]

HD-PE pipes are increasingly used for water and also for gas distribution. These pipes are easy to install. They have high stability against corrosion, high resistance to mechanical damage, and are resistant to chemicals.

The continuous improvement of the material originated three generations of HD-PE: PE 63, PE 80 and PE 100. The PE 100 has increased resistance.

Water pipes are regulated under Directive 89/106/EEC [221]

EU regulations classify water pipes as "construction products". These regulations define technical specifications and describe how to proceed if special specifications applying for the product are unknown. The regulation 89/106/EEC comprises water pipes as well as gas pipes.

EN 12201 high density polyethylene (HDPE) for drinking water and gas pipes. 05.12.2003. [220]

This standard regulates the requirements be fulfilled by polyethylene pipelines (main and distribution for drinking water. The standard also specifies testing conditions for valves and other components, their welding among themselves or in combined with other material, These specifications must be met under the following requirements: Maximal operational pressure not exceeding 25 bar. Reference temperature is 20°. The colouring of tubes and additives are specified.

EN 1555-1 Plastics piping systems for the supply of gaseous fuels Polyethylene (PE). 2009. [222]

Usually pipe producers meet EN 12201 and EN 1555. The same material may thus be applied for both uses.

US Regulations for water pipes

Food grade pipe must comply with the provisions of Title 21 of the United States FDA Code of Federal Regulations as being safe for use in food contact applications. [223]

Pipe systems should also comply with the 3-A Sanitary Standard. [224]

Disinfection of drinking water [225]

Supply of drinking water may come from surface water or ground-water, from a river, a lake or a spring.

As this water may be contaminated by bacteria or parasites it should be disinfected as follows:


Filtration

Filtration should be made using Berkefeld filters, Chamberlain-filters and others.
Continuous detection of bacterial count is necessary to avoid overgrowing of the filter.

Disinfection of water using heat

Small amount of water can be boiled killing all bacteria. The taste of boiled water is not good enough because of the elimination of carbon acid and alkaline earth carbonates.

Ultraviolet radiation

Ultraviolet radiation is used in small quantities of water. It is to expensive for central communal distribution.
Mercury low pressure lamps are used. Short waves producing ozone are often filtered out.

Chemical disinfection of drinking water


Chlorine

is the most used chemical disinfectant of water. 0,2 to 1,0 g in 1 m³ kills all germs in water.
Chlorine can combine with components such as lipoproteins and cell plasma of the cell of the bacteria.Chlorine can react with the unsaturated molecules. It can also act dehydrating.

With water chlorine forms underchloric acid

H2O + Cl2 = HClO + HCl
Underchloric acid decays in chloric acid and oxygen:
2 HCLO = 2 HCl + O2

The oxygen in molecular form is an active part during disinfection killing bacteria. The resulting chloric acid is neutralized by earth alkaline carbonate generally present in complex media (E. Thofern and collaborators, 1958)
According to drinking water regulations (German regulation) disinfection of water can be made with Chlorine, sodium-, calcium- and magnesium hypochloride, chlorine chalk and chlorine dioxide.

Chlorine dioxide in 20% solution is widely used presenting low taste and low smell compared with chlorine.It has strong disinfectant activity and is used to brake down phenols in drinking water and waste water streams. [226]
Drinking water should not have more than 0,3 mg/l active chlorine.If necessary, in case of highly contaminated water 0,6 mg/l are allowed.

The disinfection of chlorinated water takes place in 30 minutes. the taste level of free chlorine is around 0,5 mg/l.
The smell level of free chlorine is far below taste level.

In case of resistant bacteria strong chlorination may be used.Excess of chlorine can be neutralized with natriumthiosulphate or filtering the water through granulate of calciumsulfide (Katarsit) or charcoal.

Charcoal filters out compounds of chlorphenol of chlorine reacted with natural phenoles of river water.
Chlorination of drinking water was widely introduced in USA beginning with 1908.

Ozonization of drinking water

Ozone is created discharging two electrodes through a dried air stream.
W.Siemens had built in 1857 an ozone glass tube where ozone could be produced. The modern equipment to generate ozone for the disinfection of drinking water have great capacity.

Chlorine dioxide is used primarily for bleaching of wood pulp, flour and disinfection of water. Its most common use in water treatment is as a pre-oxidant prior to chlorination of drinking water to reduce trihalomethanes which are a carcinogenic disinfection by-product associated with chlorination of naturally occurring organics in the raw water. [226]

Chlorine dioxide is also used in conjunction with ozone disinfection of water to reduce the formation of bromates which are regulated carcinogens. Chlorine dioxide is also superior to chlorine when operating above neutral pH, when ammonia is present and for the control of biofilms. [226]

Chlorine dioxide is less corrosive than chlorine and superior for the control of legionella bacteria, viruses, bacteria and protozoa, including cysts of Giardia and the oocysts of Cryptosporidium. [226]

It can also be used for air disinfection, and was the principal agent used in the decontamination of buildings in the United States after the 2001 anthrax attacs. After the disaster of 2007 of Hurricane Katrina in New Orleans, Louisiana and the surrounding Gulf Coast, chlorine dioxide has been used to eradicate dangerous mold from houses inundated by water from massive flooding. [226]

Chorine dioxide as disinfectant on produce, fresh fruits and vegetables [227]

An U.S. epidemic caused by spinach, contaminated with pathogenic Escherichia coli in September 2006 boosted the researches at the Purdue University of Indiana, concerning the use of chlorine dioxide gas as disinfectant on produce, fresh fruits and vegetables. The use of chlorine dioxide to sterilize processing equipment speeding up sterilisation and eliminating the heat energy needed for conventional sterilization is included in this study.

According to Linton, leading author of the project, chlorine dioxide is highly effective at killing microbial pathogens but too much of it can cause a decrease of quality in the product, such as browning of leafy greens. To help to prevent future outbreaks like the spinach contamination in September 2006, Linton stresses the need to follow more stringent sanitary policies, as well as practicing better manure and water management.

High contamination of salad leaves found in United Arab Emirates [228]

According to Dr Dennis Russell mustard salad greens, known as arugula salad leaves, or jargeer greens (Eruca sativa) were found to be highly contaminated with faecal coliform cells and Escherichia coli bacteria after washing the leaves three times. These bacteria were found even after rinsing with diluted chlorine bleach. Some strains of these bacteria can be deadly. Dr Tibor Pal stressed that high levels indicated faecal contamination and risk of other serious diseases, such as viruses and parasites.

The United Kingdom and Germany have limits of 100 E. coli bacteria per gram, and Switzerland has a limit of 10 per gram. Brazil has a limit of 100 viable faecal coliform per gram.

Jargeer greens grown on United Arab Emirates (UAE) produce farms, are a favourite and popular part of the traditional Arab meal. The authors stress, however, that contamination on the farm, with raw or poorly composted manure, used as fertilizer, and untreated liquid manure for irrigation or as a foliage spray. Pose a risk of enteric disease outbreaks.

High efforts to exclude disease organisms from farms growing irrigated lettuce and leafy vegetables at California were started following a deadly outbreak of Escherichia coli O157:H7 on August 2006 regarding fresh spinach, followed by several cases of hemolytic uremic syndrome (HUS) in September 2006. Contaminated surface water and vectoring by wildlife, most likely feral pigs are the most important sources of contamination. [229]

How Salmonella attach to salad leaves [230]

According to Professor Gadi Frankel, Dr. Rob Shaw and colleagues, Salmonella and E. coli O157 - a strain of E. coli can spread to salads and vegetables if they are fertilised with contaminated manure, irrigated with contaminated water, or if they come into contact with contaminated products during cutting, washing, packing and preparation processes. Some Salmonella bacteria use the long stringy appendages (flagella) to attach themselves to salad leaves and other vegetables. The researchers found that Salmonella which were deprived of their flagella could not attach themselves to the leaves, and the salad remained uncontaminated.

Professor Frankel stresses that the flagella play a key role in Salmonella's ability to contaminate salad leaves. He says that different types of salad leaves are affected and focuses his work on how to use the plant protection strategy to increase food safety.

The authors fears that the number of infections will increase as people are eating more bagged salads, choosing organic produces ready to eat.

Chronic health effects of chlorine dioxide

Careful handling and use of chlorine dioxide is imperative because of possible chronic health effects. In the fact sheet about this gas, the National Pollutant Inventory (NPI) of the Australian Department of Environment states that the following chronic (long-term) health effects can occur at some time after exposure to chlorine dioxide and can last for months or years: irritate the lungs; repeated exposure may cause bronchitis to develop with cough, phlegm, and/or shortness of breath. Permanent lung damage may occur, especially with repeated exposure to the vapours. There is limited evidence that chlorine dioxide may damage the developing foetus. [231]

Biofilms


Interspecies bacterial amyloid biofilms

Amyloid fibre formation is responsible for several human diseases including Alzheimer's, Huntington's, and prion diseases. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding.

Amyloid fibrilization is a multistep process characterized by an energetically unfavorable formation of nuclei (lag phase) followed by cooperative amyloid elongation. [232]


Curli, bacterial amyloid fibres [233]

Curli are proteinaceous component of a complex extracellular matrix produced by many Enterobacteriaceae. Curli belong to a class of fibers known as amyloids. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation, invasion and inflammations.

Seeding accelerates polymerization of amyloidogenic proteins [234]

Zou et al 2012 describe the cross-seeding of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development.

Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. The authors found that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2 and Citrobacter koseri were able to cross-seed in vitro. The polymerization of E. coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Interspecies ross-seeding of curli proteins was also observed in mixed colony biofilms with other Enterobacteriaceae enhanced bacterial attachment to agar surfaces formed bacterial biofilm formation.

The ten most dangerous diseases of the world

The WHO has listed the ten most dangerous diseases of the world.


Disease caused affects Million trans
  by   death/Y mission
Influenza Haemophilus influenza Respiratory system 3,7  
Tuberculosis Mycobacterium tuberculosis Lung 2,9 Food (e.g. milk)
Cholera Vibrio cholerae Digestive tract 2,5 food (e.g. water)
Aids Aids virus Immune system 2,3  
Malaria Plasmodium falciparum Blood 1,5  
Measles Measles virus lung and meninges 0,96  
Hepatitis-B Hepatitis-B virus Liver 0,605 Food (e.g. water)
Whooping cough Bortadella pertussis Respiratory system 0,41  
Tetanus Clostridium tetani Infections 0,275  
Dengue fever Flavivirus Fever 0,14  


Three among ten of the most dangerous diseases are transmitted by food. This shows that food born diseases are of great importance. Other, more typical food born diseases like Campylobacter or Salmonella may have a local high incidence, but cause worldwide less casualties. Engineering and food hygiene must be improved to reduce this hazard.

Peanut butter Salmonella outbreak 2009 [235]

The outbreak strain of Salmonella typhimurium in late 2008 and January 2009 may have contributed to six deaths, according to the CDC. The FDA confirmed that the source of the outbreak is peanut butter and peanut paste made by the Peanut Corporation of America (PCA) at its Blakely, Ga., processing plant.

The PCA peanut butter and the peanut paste is sold to institutions throughout the state to long-term care facilities, hospitals, schools, restaurants, delis, universities, cafeterias and bakeries. It is also sold to food manufacturers of products such as crackers, cookies, cakes, cereal, candy, and ice cream.

The FDA advises consumers not to eat commercially prepared products containing peanut butter or peanut paste, or peanut butter served at institutions. Watch the list of ingredients at the label.


Hygiene and infections in flood catastrophe

Flood catastrophes cause enormous destructions, endange human life and cause a high risk of infections because of contaminated drinking water and food resources.
Special care to avoid infections should be taken not only during but also long after the flood period.


The role of Water during flood catastrophes

During and long after flood catatrophes water of rivers,lakes, wells, ground-water and public water suply systems are contaminated by a high count of pathogen bacteria, virus and egs of parasitic worms due to faecal contamination and man to man infections. This is often caused by a breakdown of the sewage treatment systen of the region or even the destruction of the sewerage
Another source of infection are deteriorating corpses of drowened animals.
The contamination of the water due to urine of rat and mouses with Leptospirae should always be considered. Most frequent intestinal infections are typhoid and paratyphoid fever Coli-enteritis, Shigella infections and hepatitis A infections.

Vacination of rescue and aid program groups

All members of rescue or aid program groups should be vaccinated against typhoid fever and hepatitis A. Due to increased possibility of injuries tetanus vaccination should be included or if necessary old vaccinations should be freshed up.

Water quality for recreational waters [236]

According to the WHO guidelines for recreational waters contamination from agriculture and sewage treatment, fish and aquatic mammals and from animals grazing near rivers such as sheep, goats and deer produce inputs into rivers with very high concentrations of bacteria and viruses including a wide range of pathogenic organisms such as liver fluke. In rivers used for swimming, safe levels of bacteria and viruses can be established based on risk assessment.

Under certain conditions bacteria can colonise freshwaters occasionally making large rafts of filamentous mats known as sewage fungus, usually Sphaerotilus natans. The presence of such organisms is almost always an indicator of extreme organic pollution and would be expected to be matched with low dissolved oxygen concentrations and high BOD values. Escherichia coli is used to indicate the presence of recent human or animal faecal contamination.

Free-living microorganisms

Natural inhabitants of marine aquatic environments are also potential pathogens. Vibrio cholerae and other Vibrio species are natural inhabitants of marine aquatic environments. They cause gastrointestinal infections, wound and ear infections. The occurrence of vibrios does not correlate with the occurrence of the traditionally used bacterial faecal index organisms, except in case of disease outbreaks.

Aeromonas spp. are present in surface fresh and marine waters with densities ranging from less than 1 to 1000 cells per ml. Sewage can also contain elevated numbers (106–108 cells per ml) of aeromonads. Aeromonas causes gastroenteritis. wound infections pneumonia.

Free-living amoebae of the members of the genus Acanthamoeba, Naegleria fowleri and Balamuthia mandrillaris infect humans. Some Acanthamoeba species are pathogenic to humans and cause two clinically distinct diseases affecting the central nervous system: granulomatous amoebic encephalitis (GAE) and inflammation of the cornea (keratitis).

Naegleria fowleri, which is found in thermal freshwater habitats worldwide, causes primary amoebic meningoencephalitis in humans, which is usually fatal, with death occurring in 3–10 days after exposure. Balamuthia mandrillaris encephalitis is largely a disease of the immunocompromised host.

No differentiation between human and non-human faecal sources of pathogens [237]

Because faecal matter can be a major source of pathogens in ambient water, and because it is not practical or feasible to monitor for the full spectrum of all pathogens that may occur in water, water quality criteria are specified throughout the world in terms of faecal indicator organism densities. For decades, these faecal indicator organisms have served as surrogates for potential pathogens and subsequent health risks in both recreational and drinking waters.

The EPA recommended recreational water quality criteria do not differentiate between faecal sources of pathogens. Thus, EPA's regulatory premise concerning recreational water quality has been that nonhuman-derived human pathogens in faecally contaminated waters are as hazardous as their human-derived counterparts.

Monitoring pathogens at recreation beaches [238]

Beaches are monitored for faecal indicator bacteria (typically Escherichia coli) in order to protect the public from potential sewage contamination. The authors criticize that there is no universal standard for sample collection and analysis or results interpretation. Monitoring policies are developed by individual beach management jurisdictions, and applications are highly variable across and within lakes, states, and provinces.

Indicator microbes may predict pathogens and help to improve human protection at beaches [239]

Shah et al 2011 report an inverse correlation between moisture content of beach sand and most faecal indicator microbes. Some indicator microbes were found to be associated with pathogens such as nematode larvae, Candida yeasts and methicillin resistant Staphylococcus aureus. The authors suggest, therefore, that indicator microbes may be useful for monitoring sand of beaches to predict the presence of these pathogens and water quality to improve human health protection at recreational beaches.

Marine recreational water quality [240]

Shibata et al. 2004 tested the microbial status at two US beaches using enterococci, Escherichia coli, faecal coliform, total coliform and Clostridium perfringens as indicator microbes. The authors found that the results did not vary between one indicator microbe and another, and did not change between seasons with different rainfall, temperature, pH, and salinity.

The shoreline points presented highest microbiological contamination which decreased at offshore points. Beach sands within the wash zone tested positive for all indicator microbes, suggesting that samples should be taken at this point. The authors stress further that concentrations of indicator microbes do not necessarily correlate with one another. The authors concluded that beach advisories, based on exceedance of water quality guidelines varies according the chosen indicator.

Cladophora algae mats in the Great Lakes are a shelter for pathogens [241]

According to Verhougstraete and colleagues 2010 the algae Cladophora form free-floating mats which may strand on recreational beaches of the Great Lakes. Pathogens such as Escherichia coli, enterococci, Shigella, Campylobacter, and Salmonella find nutrients and shelter in Cladophora mats, affecting the water quality. The authors considers the traditional faecal indicators at beaches with Cladophora presence asinadequate to predicting the presence of faecal contamination.

Bacterial indicators and EPA water quality guidelines for recreational waters [242]

Wade et al. 2003 support the use of enterococci in marine water at U.S. Environmental Protection Agency guideline levels. They found, however, that in fresh water, Escherichia coli was better suited to predict gastrointestinal illness than are enterococci or other bacterial indicators. Indicators of viral contamination were considered by the authors as strong predictors of gastrointestinal illness in both fresh and marine water.

Faecal indicator bacteria monitoring and effect of tide [243]

Boehm et al. 2005 report that marine beach water quality is monitored in early morning once a week without respect to tidal condition. The authors assess the effect of tide on this monitoring system. They found that enterococci concentrations during spring tides were higher than data of neap tides. Spring-ebb tides yielded the highest enterococci concentrations. The authors concluded that tide should be considered in the design of beach monitoring programs.


Guide and warning standards for Foods

It is of great importance for industry, health service and trade agreements to have standards for limits of number of bacteria in foods.Some limits are already set by food regulations such as limits for milk or water. With growing importance of the global standards are becoming more important.
The Deutsche Gesellschaft für Hygiene und Mikrobiologie DGHM has compiled limits which are oficial regulations. They should be used as a help to define Good Manufacturing Practice. The Standards can be downloaded under: http://www.unibonn.de/em-mibi
The standards are divided in two parts:

1 - Guide limits: If the bacterial count is under or is equal to the guide limit changes are not necessary.
If growth of bacteria exceeds guide limits but is under the warning limit weak points in storage, production transport and retail should be analyzed. Veterinary supervision should advice the companies which engaged in the production.
2 - Warning limits: If bacterial surpasses the warning limits error at hygiene during production must be eliminated. Veterinary control system may act against these foods.

The European Regulation on Microbiological Criteria for Foodstuffs [244]

The regulation 2073/2005 [244], amended by the Regulation 1441/2007 in December 2007 [123], lays down limits for specific bacteria in food. It was based on the following EU regulations:

Regulation (EC) No 178/2002 [245]which lays down general food safety requirements, according to which food must not be placed on the market if it is unsafe. Food business operators have an obligation to withdraw unsafe food from the market. In order to contribute to the protection of public health and to prevent differing interpretations, it is appropriate to establish harmonised safety criteria on the acceptability of food, in particular as regards the presence of certain pathogenic micro-organisms.

Microbiological criteria also give guidance on the acceptability of foodstuffs and their manufacturing, handling and distribution processes. The use of microbiological criteria should form an integral part of the implementation of HACCP-based procedures and other hygiene control measures.

According to Regulation (EC) No 852/2004 [246], food business operators are to comply with microbiological criteria. This should include testing against the values set for the criteria through the taking of samples, the conduct of analyses and the implementation of corrective actions, in accordance with food law and the instructions given by the competent authority.

Conflict and Emerging Infectious Diseases [247]

Instability of regions in Far East, Asia and Africa promote infectious diseases. Dr Michelle Gayerfrom the WHO, writing on detection, containment, and control of emerging infectious diseases in conflict situations says that they are major challenges because of multiple risk factors that promote disease transmission and hinder control even more than those in many resource-poor settings. She stresses the moral imperative to alleviate the effects of these diseases on already vulnerable conflict-affected populations.

Risk Factors Enhancing Disease Emergence and Transmission in Conflict Situations, according to Gayer, are:

Population Displacement and Environmental Conditions

Malaria epidemic due to Plasmodium falciparung was caused by massive population displacement to Afghanistan in 1992-1993.
Emergence of Lassa fever in camps in non-disease-endemic areas has been documented and is probably related to the poor condition of dwellings and storage of grain rations in nonsecure canvas sacks, which attracts rodents.
Unsanitary environmental conditions led to the proliferation of rats in postwar Kosovo and resulted in a tularemia outbreak among the displaced population from August 1999 through April 2000.

Breakdown in Infection Control

Poor infection control practices in healthcare facilities have enabled amplification of outbreaks of viral hemorrhagic fevers, several outbreaks of Ebola hemorrhagic fever (EHF) in Yambuku, in 1976, in Sudan in 1976 and 1979, in Kikwit, in 1995, and in Gulu, Uganda, in 2000. The outbreak of Marburg hemorrhagic fever in Angola from October 2004 through July 2005 was booted by healthcare centers reusing needles and syringes and using multidose vials in healthcare centers due to poor training in safe injection practice. An outbreak of Lassa fever occurred in Kenema District Hospital from January through April 2004 and was also caused by reuse of needles and syringes.

Disruption of Disease Control Programs and Collapse of Health Systems

Recurrence of the sleeping sickness (human African trypanosomiasis) in the 1990s, predominantly in conflict-affected Angola, DRC, and Southern Sudan due to interruption of the control measures.

Inadequate Surveillance and Early Warning and Response Systems

Surveillance systems are often weak in conflict situations, which results in delays in detection and reporting of epidemics. Limited laboratory facilities and lack of expertise in specimen collection may delay confirmation of the causative organism. Outbreak investigation and implementation of control measures may be hampered by fighting, impeded access to populations, destroyed infrastructure, limited coverage of healthcare services, poorly trained health staff, and difficult logistics that prevent delivery of drugs. This resulted in the outbreak of Marburg hemorrhagic fever in Durba in northeastern DRC from October 1998 through September 2000.

Impeded Access to Populations

Ongoing conflict can hamper access to populations for timely delivery of supplies and implementation of control measures during an outbreak. Access to populations to conduct vaccination campaigns may also be interrupted for months to years during protracted conflict due to long-term inadequacies in cold chain and logistics or ongoing insecurity.
Several outbreaks of pneumonic plague have been documented in Oriental Province in northeastern DRC, where war has hampered control efforts.

Development of Drug Resistance

Pathogen resistance to drugs can contribute to disease emergence. Resistance may develop more rapidly in conflict situations because of inappropriate diagnoses or inappropriate drug regimens and outdated drugs.
An outbreak of Shigella dysenteriae type 1 infection in a Rwandan camp for Burundian refugees fleeing civil war in 1993, less than 50% of patients complied with their 5-day antimicrobial drug treatment.
Refugee populations had higher anti-tuberculosis (TB) drug resistance rates than nonrefugee populations in northeastern Kenya.

Antibiotic resistance of strains of poultry [248]

Antimicrobials used for therapy, prophylaxis, and growth promotion in broiler chicken production have been associated with antimicrobial-resistant enteric bacteria.

In February 2002 two U.S. poultry companies stopped the use of fluoroquinolones for flock-wide treatment. In 2003 Lance, Silbergeld and colleagues 2005 surveyed Campylobacter isolates on chicken products from these two companies together with two producers claiming total abstention from antibiotic use.

The authors found that antibiotic-free brands were not more likely to be contaminated with Campylobacter. A high percentage of products from the two conventional brands were contaminated with FQ-resistant Campylobacter and these conventional brands had significantly higher odds of carrying resistant strains compared with antibiotic-free products.

The authors concluded that fluoroquinolone resistance may persist in the commercial poultry environment in the absence of fluoroquinolones-selective pressure and that these strains contaminate a larger proportion of foods than reported previously.

Lance, Silbergeld and colleagues 2007 estimated the relative risk for carrying antimicrobial-resistant E. coli among poultry workers compared with community referents. The authors found that poultry workers had 32 times the odds of carrying gentamicin-resistant E. coli compared with community referents. The poultry workers were also at significantly increased risk of carrying multidrug-resistant E. coli.

The researchers concluded that occupational exposure to antimicrobial-resistant E. coli from live-animal contact in the broiler chicken industry may be an important route of entry for antimicrobial-resistant E. coli into the community. [249]

Probiotics to phase out antibiotics in poultry breeding [250]

Antibiotics in poultry feed reduces Salmonella contamination and acts as a growth enhancer. Billy Hargis, director of the Poultry Health Research Laboratory at the University of Arkansas System's Division of Agriculture develops probiotic bacteria to be added to feed or water to reduce Salmonella in either meat-type chicken houses or turkey houses before being transported to the processing plant and reduce the risk of cross contamination.

The researchers are focused on Bacillus, a defined aerobic lactic acid bacterial culture which develops spores resistant to heat treatment of feed during pelleting. These probiotics may phase out the use of antibiotics in feed for poultry. However, the researcher notes that the lack of antibiotics means producers will have to give more feed to the birds to get the raise the same amount of meat. According to Hargis the price of grain is already going up to meet demand for biofuels, so the price of meats produced from small grains will also rise. With rising prices of grain due to high demand for biofuel it will be hard to phase out antibiotics in commercial poultry breeding.

Bacteria are increasing their resistance to antibiotics [251]

Common bacteria have acquired resistance to multiple antibiotics. Most of these strains were confined to hospitals, however, some strains are now being found in free environment, such as methicillin-resistant Staphylococcus aureus and gram negative bacteria. To curb the use of antibiotics Gary Taubes advocates a switch from broad-to narrow-spectrum antibiotics and the reduction of the standard 7 to 10 days drug treatment, adopting a shorter use of antibiotics. Taubes cites the resistance of Clostridium difficile. [252]

Eliot Marshall cites the resistance of Mycobacterium tuberculosis strains in Tomsk, Siberia, where Partners in Health (PIH) made some progress collaborating with local authorities which report that deaths have declined, but resistance to drugs remains high. [253]

The authors claim that changes in natural ecosystems, including the release of large amounts of antimicrobials, might alter the population dynamics of microorganisms, including selection of resistance. Antibiotics in feed used to improve productivity of flocks are a one important door to high releases in the environment, affecting food safety.

Martínez, Monk and Goffeau. describe the evolution of antibiotic resistance genes in bacteria in natural environments and considers possible intervention in the expression of these genes. The authors strategies are based on improved diagnosis and indirect intervention via inhibition of the energy supply for drug efflux as a way to develop of broad-spectrum fungicides. [254] [255]

Bacteria, such as Klesiella pneumoniae develop resistance to carbapenems [256]

Carbapenems, useful to treat infections caused by extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria, is now getting ineffective by bacteria developing enzymes called Klebsiella pneumoniae carbapenemases (KPCs), which inactivate them. KPC-producing organisms can spread inside hospitals as well as in the community setting.

Treatment of KPCs often requires the use of tigecycline and the polymyxins. This epidemic of KPCs Gram-negative resistance reside on transferable plasmids and can hydrolyse all penicillins, cephalosporins, and carbapenems. Increased awareness and intensified infection control practices are the keys to curtail the spread of this terrifying antimicrobial resistance.

The first KPC-isolate was detected in 1995in isolates of Pseudomonas aeruginosa mutants [257]. KPC-resistace is most frequent in Klebsiella pneumoniae, and seems to have spread to the Enterobacteriaceae family, such as Proteus, Serratia, Salmonella, and Citrobacter.

Antibiotic resistance of bacteria [258]

Spízek and colleagues 2010 write that resistance to antibiotics and other antimicrobial compounds continues to increase and suggest protection strategies against pathogenic microorganisms. Such strategies should focus on new vaccines, use of specific bacteriophages, search for new antibiotics, sequencing microbial genomes to identify related genes and analysing DNA from the environment (metagenomics).

Study explains antibiotic resistance mechanism of bacteria [259]

Desai and Miller 2010 assessed the mechanisms used by bacteria to fight antibiotics. The authors used bromoacetate as an environmental threat to simulate antibiotics. In this study Escherichia coli produced protective proteins allowing its survival even at lethal concentration of bromoacetate.

The authors identified nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight genes encode putative transporters or transmembrane proteins, while one encodes the enzyme UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA), which is irreversibly inactivated by bromoacetate via alkylation of a critical active-site cysteine. Also 63 strains were found by the authors to be susceptible to bromoacetate.

The techniques described allows to predict the resistance to specific antibiotics, identify the resistance mechanisms and may enable researchers to reverse the resistance to antibiotics.

Assessing the hygromycyn bacterial resistance [260]

Stogios and colleagues 2010 write that the aminoglycoside phosphotransferase APH(4)-Ia is one of two enzymes responsible for bacterial resistance to hygromycin B (hygB). The authors studied the crystal structure of APH(4)-Ia enzyme. The enzyme was found to form an hydrogen bond network with hygB primarily through polar and acidic side chain groups, and binding affinity is spread across a distributed network. The APH(4)-Ia enzyme contains a cluster of hydrophobic residues which interact with hygromycin, and similar to APH(2") utilize either ATP or GTP for phosphoryl transfer, this pathway may be used to tailor new aminoglycoside antibiotics or inhibitors of this enzyme suggest the authors.

CmeG gene is linked to resistance to antimicrobials and oxidative defence [261]

Campylobacter jejuni presets rising resistance to antimicrobials. Jeon and colleagues studied the cmeG (Cj1375) gene in Campylobacter jejuni which encodes a putative efflux transporter. The authors report that overexpression of the cmeGH operon in Campylobacter jejuni significantly increased its resistance to fluoroquinolones. The authors concluded that CmeG gene is a multidrug efflux transporter involved in antibiotic resistance and oxidative defence of Campylobacter.

Peptide antibiotic bacterial resistance of Bacillus subtilis [262]

Staron, Finkeisen Mascher 2010 describe three peptide antibiotic sensing and detoxification modules in Bacillus subtilis consist of two-component system which induces the expression of an ABC transporter which removes the antibiotic from the bacterial cell wall resulting in resistance of the cell:

BceRS-AB responds to bacitracin, plectasin, mersacidin and actagardine. YxdJK-LM is induced by a cationic antimicrobial peptide, LL-37. The PsdRS-AB system primarily responds to lipid II-binding lantibiotics, such as nisin and gallidermin.

The authors suggest the PbceA and PpsdA promoters as novel whole cell biosensors for high-throughput screening.

Pan-resistant NDM1 bacteria [263]

NDM1 is an enzyme that confers resistance to one of the most potent classes of antibiotics, known as carbapenems. It is found in many different types of bacteria and one in 10 of these strains is resistant to all known antibiotics. For the moment there is no new antimicrobial being developed. The NDM enzyme genes that The NDM1 gene set has been found in Escherichia coli and can move easily from other bacteria.

The WHO in its 2000 report "Overcoming antimicrobial resistance" called the rise of antimicrobial resistance a global crisis. In 2001 the organization released a global strategy and campaign which should be updated and rejuvenated. Of concern is selling for human use antibiotics over the counter in Asia,Africa Central and South America. Also of high concern is the use of antibiotics as growth promoters in animal feed and bee-hives.

WHO calls on the responsibility of governments to regulate these isues, such as happened in the European Union where the use of antimicrobial growth promotion in livestock was banned.

Postgenomic strategies profit from foregoing activities of antibacterial drug development [264]

Brötz-Oesterhelt and Sasss 2010 stress that any use of antibiotics triggers bacterial resistance to the drug due to their short reproduction time leading to mutations and the exchange of genetic material with other strains. The authors call, therefore, for proper hygiene, skilful and restricted use of antibiotic treatment.

The classical strategy

discovered all currently used antibiotic classes by screening natural product extracts or synthetic compounds for their antibacterial activity.

The genomics strategy

New genetic discoveries initiated a "genomic" strategy replaced the classical strategy. However, research focused on synthetic quinolones class and synthetic low molecular weight compounds and bacterial genomic information faced a fruitless time. The High-Throughput Screening (HTS) strategy failed to produce significant results.

The postgenomic strategy

now uses knowledge and libraries of foregoing researches and focuses on screening for new or underexploited natural products and structure-based drug design. Efforts to avoid bacterial resistance to antimicrobials should be further supported by alternative treatment options together with anti-virulence strategies and immunotherapeutic approaches

Alternatives to the emergence of carbapenemases in Enterobacter [265]

Looking for alternatives to the emergence of carbapenemases in Enterobacteriaceae Livermore and colleagues 2010 found that ACHN-490, a sisomicin derivative evades all plasmid-mediated aminoglycoside-modifying enzymes. Carbapenem-resistant isolates are sensitive to ACHN-490, except those strains containing 16S rRNA methylases. Apramacin evades 16S rRNA methylases and is being suggested by the authors as a starting point for future aminoglycoside development.

Cervimycin C resistance in Bacillus subtilis described [266]

Krügel and colleagues 2010 report two mutations in the intergenic region preceding the ABC transporter gene bmrA in cervimycin C-resistant clones of Bacillus subtilis, The authors stress that the cumulative of these two mutations are responsible for ABC transporter confers antibiotic resistance by the cumulative effects of two mutations in the promoter region.

Mathematical model of drug resistance predicts variation in population of pathogen bacteria [267]

Variation in the population of antibiotic non-resistant/resistant organisms such as methicillin-resistant staphylococcus aureus can be predicted by a mathematical model developed by Friedman and colleagues 2010. The model predicts that antibacterial drugs initially decrease the load of the non-resistant bacteria, reaching a low level after 6 weeks. The load of drug-resistant bacteria will also decrease at first but rise again to a high level later on. This level can be reduced with increasing drug dosis or decreased contact between health care workers and patients says the authors.

Methicillin-Resistant Staphylococcus aureus [MRSA]


Pigs as Source of Methicillin-Resistant Staphylococcus aureus CC398 Infections in Humans [268]

Methicillin-resistant Stapylococcus aureus (MRSA) is becoming increasingly recognized among persons in the community without established risk factors. MRSA primarily causes human disease and animals have not, until now, been considered a source of infection.

It has recently become apparent that animals, particularly pigs, can constitute a separate MRSA reservoir and be a source of a novel and rapidly emerging type of MRSA in humans; namely MRSA clonal complex (CC)398.

According to Robert L. Skov and colleagues 2008 an emerging subtype of methicillin-resistant Staphylococcus aureus (MRSA), clonal complex (CC) 398, was found in near 50 per cent in Danish pigs on 4 of 5 farms. The CC398 variant of the bacteria was found infecting humans. The study found that living or working on farms with animals was an independent risk factor for CC398. Based on microbiologic testing the authors stress that pigs are a source of CC398 in Denmark.

The study reinforces results of studies in France, the Netherlands, and Canada that indicated that CC398 is transmissible from animals to humans.

Methicillin-Resistant Staphylococcus aureus carriage in Belgian swine farms [269]

Denis and colleagues 2009 found a prevalence of methicillin-resistant Staphylococcus aureus ST 398 (MRSA ST 398) carriage in 37,8% of persons working on Belgian pig farms. On farms with MRSA-colonized pigs the prevalence of carriage was 50% versus 3% on farms without colonized pigs. Swine, exposed farmers and veterinarians are thus high risk factors for carriage of MRSA from Belgian pig farms. Notably these MRSA strains are genetically unrelated to hospital- or community-acquired clones.

The authors stress that risk factors for MRSA ST398 carriage was associated with being a farmer or farm co-worker, being male, having regular contact with animals (including goats, sheep, dogs, or cats) and, paradoxically, wearing gloves and apron and reporting occasional or regular hand disinfection with an antimicrobial product. This suggests that different animals could be MRSA ST398 reservoirs or vectors, at least on pig farms.

The authors call for more studies to determine other routes of transmission such as airborne transmission and contact with contaminated surfaces and companion animals which apaerntly reduce the effectiveness of the protection of gloves and apron.

Methods used for Methicillin-Resistant Staphylococcus aureus ST 398 [269]

Identification of MRSA ST398 according to Denis and colleagues 2009

Samples from anterior nares and skin lesions on hands or face of human participants were placed into Stuart transport medium (Copan, Italy), inoculated within 24 h into 7.5% NaCl brain-heart infusion enrichment broth, and subcultured after 24 h onto Chromagar MRSA (bioMérieux, Marcy l'Etoile, France) and mannitol salt agar (Becton Dickinson, Heidelberg, Germany). S. aureus isolates were identified by coagulase test and PCR for 16S rRNA, mecA, and nuc genes.

Genotyping of MRSA ST398 used by Denis and colleagues 2009

Isolates were genotyped by pulsed-field gel electrophoresis after SmaI macrorestriction, spa sequence typing (http://spaserver.ridom.de), and determination of staphylococcal cassette chromosome mec (SCCmec) type and accessory gene regulator (agr) polymorphism. Four MRSA isolates were further analyzed by multilocus sequence typing (www.mlst.net). Multiplex PCR was used to test for Panton-Valentine leukocidin, toxic shock syndrome toxin 1, and exfoliatin A and B genes.

Antimicribial drug susceptibility testing used by Denis and colleagues 2009

Antimicrobial drug susceptibility was tested by the Vitek2 system (bioMérieux). Multiplex PCR was used to test for resistance genes tetK, tetM, aac(6')-Ie + aph(2"), ant(4')-Ia, aph(3')-IIIa, ermA, and ermC.

Methicillin-resistant Staphylococcus aureus (MRSA) [270]

Deurenberg and Stobberingh 2008 stress that the resistance to methicillin and all other beta-lactam antibiotics is related to the mecA gene, situated on the staphylococcal cassette chromosome mec (SCCmec). Methicillin-resistant Staphylococcus aureus probably originated trough the transfer of SCCmec into a limited number of methicillin-sensitive lineages.

According to the authors there are seven major variants of SCCmec, type I to VII. Two clones of MRSA had been identified, the hospital-associated (HA-MRSA), and the community-associated MRSA (CA-MRSA, which includes the SCCmec type IV, V or VII and is often associated with the toxin Panton-Valentine leukocidin (PVL). However, the distinction between HA-MRSA and CA-MRSA is vanishing.

Risk factors community-associated Staphylococcus aureus [271]

Golding and colleagues 2010 report that no significant statistical differences were found between the frequency of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and community-associated methicillin-susceptible Staphylococcus aureus(CA-MSSA) infections. The most common risk factors were overcrowding, previous antibiotic usage, existing skin conditions, household exposure to someone with a skin condition, scratches/insect bites, and exposure to healthcare workers.

Standard hygienic measures and proper treatment guidelines for the control of both, CA-MRSA and CA-MSSA, in remote communities were recommended.

Methicillin-resistant Staphylococcus aureus from pigs in humans [272]

Golding and colleagues 2010 stress a high prevalence of colonization with livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 398 among pigs and pig farmers are found in the Netherlands, in Canada and in the United States. However, no human or animal infections resulting from these strains were reported in North America.

The authors identified a novel staphylococcal cassette chromosome (SCC) mecV subtype harboring clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) array in Staphylococcus aureus isolate 08 BA 02176.

Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus [273]

Christianson and colleagues 2007 pointed out that there is a high dissemination of Methicillin-resistant Staphylococcus aureus (MRSA) throughout Canadian hospitals and communities.

The authors found that the community-associated Canadian epidemic isolates (CMRSA7 and CMRSA10) contained one open reading frame (ORF) (SACOL0046) encoding a putative protein belonging to a metallo-beta-lactamase family.

The hospital-associated Canadian epidemic isolates (CMRSA1 and CMRSA2) revealed additional factors including ORFs encoding potential virulence factors involved in capsular biosynthesis, serine proteases, epidermin, adhesion factors, regulatory functions, leukotoxins, and exotoxins.

Semi-selective broth for detection of methicillin-resistant Staphylococcus aureus [274]

Böcher and colleagues 2010 describe a new method for routine diagnostic screening of methicillin-resistant Staphylococcus aureus (MRSA). The use of a semi-selective enrichment broth containing cefoxitin and aztreonam (TSB-SSI) incubated overnight followed by plating on Columbia sheep blood (5%) agar (BA) and ChromID MRSA improved significantly the detection of the bacteria, compared with non-selective enrichment broth (NB).

Meticillin-resistant Staphylococcus aureus incidence doubles in UAE hospitals

According to a study by UAE University carried out at Tawam Hospital, in Al Ain, found that the incidence of Meticillin-resistant Staphylococcus aureus (MRSA), doubled between 2003 and 2008. Sonnevend et al 2012 write in this study that the emergence of CA-MRSA clones with subsequent entry to and spread within the hospital has contributed to the increasing incidence of MRSA observed in Tawam Hospital. The study notes that 5.2 percent of analysed strains of Staphyllococcus aureus were resistant to meticillin in 2003 while this number rose to 12.3 percent in 2008. [275]

In 2003, 61,5% of typical healthcare-associated (HA-MRSA) isolates were of genotypes ST239-MRSA-III, ST22-MRSA-IV and ST5-MRSA-II. Isolates of 2008 had changed to clonal types community-associated (CA) MRSA, representing 73.1% of the strains with ST80-MRSA-IV, ST5-MRSA-IV and ST1-MRSA. Non-typable SCCmec types were the most frequent.

Community-associated (CA-) MRSA infections are acquired at home or at public places. About 75 percent of CA-MRSA are localized at skin and soft tissue where they usually can be treated effectively. But some CA-MRSA strains display enhanced virulence, spreading more rapidly and causing illness much more severe than traditional healthcare-associated (HA-) MRSA hospital infections. They can affect vital organs and lead to widespread infection (sepsis), toxic shock syndrome, and necrotizing ("flesh-eating") pneumonia.

Dr Jens Thomsen of the Health Authority Abu Dhabi (Haad) stresses that there is an ongoing antimicrobial resistance data is currently still being compiled on a Pan-Emirate level. The bacteria are spread by a combination of lack of hand hygiene and direct contact from patient to patient or from a healthcare worker to a patient. Hygiene measures are therefore to be improved. Prescription of antibiotics must be controlled to reduce excessive use where it is not needed such as in case of a common cold. Such use brings a battery of bacteria in contact with antibiotics turning them resistant. [276]

Lantibiotics as natural food preservative and bacterial disease prevention

[277] The lantibiotics are becoming interesting as natural preservatives to prevent harmful bacteria in foods. They are easy to digest, nontoxic, do not induce allergies and are difficult for dangerous bacteria to develop resistance against. Lantibiotics are a class of antimicrobial peptides that are characterised by the presence of lanthionine and/or methyllanthionine residues. Lantibiotics are produced by Gram-positive bacteria such as Streptococcus and Streptomyces Bacillus, Bifidobacterium and others. They are strong antimicrobials and may be usefull in food preservation.

Classification [278]

Type A lantibiotics: are long flexible molecules - e.g. nisin, subtilin, epidermin. Subgroup AI includes mutacin II, subgroup AII includes mutacin I & III. Type A lantibiotics act by pore formation, Type B lantibiotics: are globular - e.g. mersacidin, actagardine, cinnamycin. type B lantibiotics inhibit peptidoglycan biosynthesis. Nisin and epidermin are members of a family of lantibiotics that bind to a cell wall precursor lipid component of target bacteria and disrupt cell wall production. The duramycin family of lantibiotics binds phosphoethanolamine in the membranes of its target cells and seem to disrupt several physiological functions.

List of known lantibiotics [279]

A description of all known lantibiotics and the producer organism is given in Bactibase database.

Lantibiotic production in broth media [280]

Lee, Li and O'Sullivan 2011 report the production of lantibiotic from Bifidobacterium longum DJO10A in agar culture but suppression of its expression in broth media. The authors found that the adding an agar culture extract to a broth cultures of B. longum DJO10A induces lanA gene expression of lantibiotic in liquid media. The transcription start analysis of lanA revealed a 284 bp 5' untranslated region to be involved in repression of transcription in broth culture. An inverted repeat structure located at -75 bp relative to the transcription start functions as a binding site for the two component response regulator. Better understanding of the regulation of lanA gene is important for the production of this lantibiotic in broth cultures.

Movement of Refugees and Aid Workers

International spread of infectious diseases from conflict situations may occur through movement of refugees, relief workers, animals, goods, and private sector employees working in mining, oil, logging, or construction industries. An example of the outcomes of such conditions was the prolonged outbreak of hepatitis E virus in a camp in Darfur, Sudan, in May 2004.

Also cited is the imported case of Lassa fever which was confirmed in Germany in July 2006, after a Sierra Leonean resident, flew from Freetown to Frankfurt through Abidjan and Brussels, 5 days after symptom onset. Aid workers and British soldiers have imported Lassa fever into the Netherlands (2000) and the United Kingdom (2000 and 2003) after postings in Lassa-endemic areas of Sierra Leone.

Improving Detection and Control of Infectious Diseases in Conflict Situations

Gayer calls for a functional healthcare system for the detection and control of many emerging infectious diseases.

In such settings, good hygiene and standard infection control precautions in health facilities are needed to reduce the potential for nosocomial transmission and amplification of disease. It is imperative that the technical capacity of all humanitarian health partners and ministries of health regarding disease surveillance, prevention, and control in conflict-affected countries be enhanced to ensure effective implementation of infectious disease interventions.

Data on disease incidence and trends are essential for prioritizing risks and planning interventions and should be obtained through disease surveillance and early warning and response systems. Several of these systems have been implemented in conflict situations.

Surveillance systems rely on close partnerships with NGOs, international organizations, and community groups and are built on resources and capacities of all organizations present.

Epidemic preparedness measures to be taken should involve training staff to use surveillance tools and manage cases of epidemic-prone diseases and equipping them with reliable means of communication.

Revised International Health Regulations of 2005 provide a global legal framework to guide response to public health events of international concern.

Military forces are increasingly implementing aid programs for conflict-affected populations. These programs have a crucial role and are a valuable resource.

Infectious diseases in the Amazon region

[281] Many of the tropical diseases of the region undergo epidemiologic changes in face of a growing population, environmental, and climate changes, and the incidence of some novel diseases increase. Important diseases are:

Flagellates

Malaria

It is caused by Plasmodium spp is the most important endemic disease in the region. It is naturally transmitted by mosquitoes of the genus Anopheles. Urbanisation contributes to malaria transmission in peripheral areas of Amazonian cities.

Tegumentary leishmaniasis

It is a high-incidence disease in the region. Multiple animal species serve as reservoirs for Leishmania spp. in the rainforest, as do the disease's primary vectors, insects of the genus Lutzomya.

Chagas disease Trypanosoma cruzi

The reservoir include armadillos, dogs, cats and rodents. Chagas' disease is mainly a disease of children. It is characterized by svollen lymph nodes , fever and anemia. Cases may be acute and quickly fatal or milder and chronic.

Leishmaniasis

[282] Leishmaniasis is caused by protozoan parasites of the genus Leishmania. It is transmitted by the bite of sandflies of the genus Phlebotomus.

Only the female sandfly transmits the parasites. Female sandflies need blood for their eggs to develop, and become infected with the Leishmania parasites when they suck blood from an infected person or animal and the Leishmania can develop in the sanfly. Feeding blood from another animal or human the sandfly inoculates the Leishmania, which can than mature.

Sand flies within the genus Lutzomyia serve as the vectors for all species of the protozoan parasite Leishmania. Lutzomyia longipalpis was the predominant species collected within the Pacific plains region of western Nicaragua. Lutzomyia cruciata or Lutzomyia barrettoi majuscula were the species most frequently collected in the central highlands and Atlantic plains regions. [283]

Leishmania protozoans which infect mamals are the Leishmania donovani complex with three species (L. donovani, L. infantum, and L. chagasi), the Leishmania mexicana complex with four species (L. mexicana, L. amazonensis, and L. venezuelensis), Leishmania tropica, Leishmania major, Leishmania aethiopica, and the subgenus Viannia with four important species (L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) panamensis, and L. (V.) peruviana).

The disease may cause cutaneous, mucocutaneous or visceral forms. Cutaneous leishmaniasis is the most common form. Visceral leishmaniasis is the most severe form, in which vital organs of the body are affected. The mucocutaneous form affects the nasopharyngeal tissues with lesions of this tissue. Differention between the different species of Leishmania uses isoenzyme analysis, DNA sequence analysis, or monoclonal antibodies, because there is no difference of morphology between the species.

Cutaneous leishmaniasis is known to be endemic in south-central Texas. Wright et al.2008 report nine cases of cutaneous leishmaniasis in residents of north Texas. The authors warns that the disease may experience a northern spread. [284]

Leishmaniasis in Bolivia

In Bolivia, most cases of leishmaniasis are caused by Leishmania (Viannia) braziliensis. The parasite is transmitted zoonotically by several sandfly species and, when transmitted to humans, may cause cutaneous leishmaniasis, and potentially, mucosal leishmaniasis.

A kind of a symbiosis between Leishmania and virus increasing severity of leishmaniasis [285]

The mucocutaneous form of leishmaniasis affecting the nasopharyngeal tissues was studied by Ives et al. 2011. The authors report that metastasizing parasites are highly infected by a virus, the Leishmania RNA virus-1 (LRV1). This virus interferes with the human immune system sensor protein called Toll-like receptor 3 (TLR3) localised in macrophages of humans. The TLR3 protectve immune pathway is turned to produce proinflammatory cytokines and chemokines triggered by the LRV1 virus.

Bacterial and micotic infection

Leprosy

Bacterial infection caused by Mycobacterium Leprae. The lepromatous form of the disease is characterised by large, firm nodules in the skin of the face, hands and other exposed parts. These nodules are called lepromas contain high number of bacilli. The tuberculoid type of infections develop around peripheral nerves, leading to atrophy of the tissue, loss of sensation from sensory nerve, mutilations are therefore often not noted. Bacteria may leave or enter via the nares.

Suspected Brazilian purpuric fever

It is a Haemophilus aegyptius-caused febrile hemorrhagic illness of children that begins with conjunctivitis and has a case-fatality rate of 40%-90%.

Bartonella

Bartonella (formerly known as Rochalimaea) is a genus of Gram-negative bacteria. Facultative intracellular parasites, Bartonella species can infect healthy people but are considered especially important as opportunistic pathogens. Bartonella are transmitted by insect vectors such as ticks, fleas, sand flies and mosquitoes. At least eight Bartonella species or subspecies are known to infect humans. Bartonella bacilliformis is found in Peru, Ecuador, and Colombia and causes the Carrion's disease (Oroya fever, Verruga peruana)

Mycoses

Jorge Lobo disease

It is caused by Blastomyces loboi, are also characteristic of the region.

Adiaspiromycosis

It is a respiratory disease of humans and many animal species caused by the fungus Chrysosporium spp. and characterized by large, thick-walled spherules (adiaspores).

Intestinal parasites

Intestinal parasites high frequency of intestinal parasites are linked to contaminated food and water.

Bacillus stearothermophilus

Bacillus stearothermophilus Donk, 1920 is presumably intended to mean fat and heat-loving.In 2001 the name was chaged to Geobacillus stearothermophilus. The most distinctive diagnostic characters are capacity to grow at 65° and a limited tolerance to acid. Bacillus strains capable of growing at temperatures of 65° and above do not belong to a single species, it is however a useful diagnostic character.
Bacillus stereatothermophilus occurs in soil, hot springs, desert sand, Arctic waters, ocean sediments, food and compost.
The biochemical characteristics of Bacillus stearothermophilus are:

Catalase= negative
Voges-Proskauer test= negative
Acid from D-glucose= positive
hen Acid from L- arabinose= differ
D-xilose= differ
D-mannitol= differ
Gas from glucose= negative
Hydrolysis of casein= differ
Hydrolysis of gelatin= positive
Hydrolysis of starch= positive
Utilization of citrate= differ
Degradation of Tyrosine= negative
Desamination of phenylalanine= negative
Nitrate reduced to nitrite= differ
Formation of indole= negative
Dihydroxyacetone= negative
Sodium and potassium chloride required= negative
Alantoin or urate required= negative

Allen ( 1953) has pointed out that fresh isolates tend to diversity of characteristics. When maintained in culture for some times they are readily classifiable.

This bacterium has a vital importance for canning factories. Bacillus stearothermophilus together with Bacillus coagulans as well as other bacteria have high heat resistant spores. The temperature maximum where growth still takes place is 75° the best growth temperature for Bacillus stearothermophilus is 55 to 60°. Bacillus stearothermophilus is the bacterium whose spores can survive at temperatures higher than other bacteria. In hot springs bacteria may be found which resist higher temperatures than that.

Biological indicator for sterilisation [286]

Geobacillus stearothermophilus is a spore biological indicators, which is used by the FDA. This biological indicator is used to verify the effectiveness of pasteurisation or sterilisation processes.

The spores of the bacterium are enclosed in a glass vial, together with a culture media and an indicator. After the sterilisation process is finished the heated vial is incubated at 60°, together with a similar unheated vial. Changing colour of the media in the unheated vial but no colour change in the heated vial demonstrates successful sterilisation.

Control strips

Autoclave control strips with 105 Geobacillus stearothermophilus ATCC 7953-Sporen (former Bacillus stearothermophilus), enclosed in plastic bags, are used for the validation of steam sterilisation and dry heat sterilisation After the heating process the strips are incubated in a Caso-Broth + Bromcresol purple (Tryptic Soy Broth + Bromcresol purple) at 55-60° during 7 days. Daily control of growth seen by increasing turbidity and clour change from violet to yellow. A Gram-stain is made in case of growth. Gram-positive rods are Geobacillus, other forms are contaminants and are discarded.


D-value (Decimal reduction time)

The D-value is the time which is necessary at a specific temperature to reduce an initial population of a bacterium down to 10%. This means that it kills 90% of the bacterium. It is measured in minutes.The Temperature must be cited.

For Bacillus stearothermophilus a D-value of D121,1= 4 up to 5 minutes are given.

D-values for other bacteria in order to draw a comparison:
Clostridium botulinum type A and B D121,1= 0,1 up to 0,2 min.
Clostridium sporogenes D121,1= 0,1 up to 1,5 min.
Clostridium thermosaccharolyticum D121,1= 3 up to 4,0 min.
Desulfotomaculum nigrificans D121,1= 2 up to 3,0 min.

For tropic conserves the sterilization has to be carefully done as Bacillus stearotermophilus grows at storage temperatures higher than 37°. Below of that there is no growth. To kill its spores F121,1 15 to 30 minutes must be used in case of canned meat.

Tetrahymena protozoan and Samonella and resistance to sanitizers

[287] [288] Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages.

Microbiologist Maria T. Brandl from the Agricultural Research Service (ARS) in the US have found that Salmonella enterica serovar Thompson may be ingested by the protozoan Tetrahymena. S. enterica cannot be digested by the protozoan which is expelled encased in miniature pouches called "food vacuoles." Other food pathogens such as Listeria monocytogenes, however, are destroyed and digested.

Brandl found that twice as many Salmonella cells stayed alive in water if they were encased in expelled vacuoles than if they were not encased. Encased Salmonella cells were three times more likely than unenclosed cells to survive exposure to a 10-minute bath of two parts per million of calcium hypochlorite, the bleachlike compound often used to sanitise food and food-processing equipment being resistant to sanitising.

Brandl found that some vacuoles held as many as 50 Salmonella cells. This dense clustering might safeguard the innermost ones from environmental stresses such as ultraviolet rays or harmful temperatures.

Brandl concludes that the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health. Brandl now wants to pinpoint genes that Salmonella bacteria turn on while inside the vacuoles. Those genes may be the ones that it activates when invading humans. [287]

Protozoa on leafy vegetables increases risk of enteric diseases [289]

Brandle and colleagues 2008 studied the protozoa Glaucoma sp., Tetrahymena pyriformis, Colpoda steinii, and Acanthamoeba palestinensis on spinach and lettuce and their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes.

The authors found that vesicles production was observed during grazing on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. Also C. steinii and A. palestinensis did not produce vesicles, nor any of the enteric pathogens were trapped by both protozoa.

The authors found that E. coli O157:H7 in expelled vesicles multiplied and escaped the expelled protozoan vesicles. They conclude that protozoa and their activity on leafy vegetables with formation of vesicles may enhance the aggressivity of trapped enteric bacteria which can better resist food sanitation.

Phage potentiates antibiotic [290]

Steven Hagens found that specific bacteriophages, such as the filamentous phages Pf3 and Pf1 can make antibiotic-resistant Pseudomonas bacteria more susceptible, thus improving the attack on them by antibiotics.

According to the author of the study phage DNA sequences code for membrane proteins that create channels in the bacterium's cell wall. Antibiotics are able to enter the cell through these gateways more quickly than it can dispose of them. Tests were performed in mice. The combination therapy using the antibiotic Gentamicin and the filamentous phages was effective against a strain of P. aeruginosa containing a gene for resistance to Gentamicin.

Combination therapy using phages could then make progress when a bacterial strain was susceptible to a particular filamentous phage.

Professor Martin Loessner of the Swiss Federal Institute of Technology Zurich (ETH) researches on the use of phages against Listeria monocytogenes in foods like soft cheeses. [291]

Potentials of phages in food industry [292]

The attempts to treat diseases including dysentery, typhoid and paratyphoid fevers, cholera and pyogenic urinary tract infections have not always succeeded due to the difficulty to select the specific phage, incorrect identification of the bacterium or the mixture of bacteria which was causing the disease. Other problems were the gastric acidity which destroyed the phages, and sometimes deterioration of the patient following the release of endotoxin of the lyzed bacteria.

Research on phages becomes new priority with the spreading of antibiotic resistant bacteria. Development of phage therapy is attractive offering low cost compared with antibiotics, can be used in food industry to reduce risk contamination with pathogenic bacteria during processing. Steven Hagens and Mark L. Offerhaus, both from EBI Food Safety in the Netherlands, working the application of phages in food industry, say that phages may be used to disinfect working surfaces and even food during processing. They stress, however, that the number of phages in a solution must be very high in order to get in contact with the bacterium to be eliminated.

New phages to treat bacterial infections like multiple drug resistant strain of Staphylococcus aureus (MRSA) and Clostridium difficile [293]

A bacterial infection can be targeted using special bacteriophages which attack the exact strain of the pathogenic bacteria. Other beneficial bacteria of the body are spared, according to Ana Toribio and colleagues. Excessive use of broad-spectrum antibiotics and their use in feed as growth promoter lead to resistant strains of pathogenic bacteria.

The researchers used a cocktail of phages obtained from the River Cam to treat gut infections caused by Citrobacter rodentium in mice. Variety of phages are used to overcome bacterial mutations.

The Tbilisi Bacteriophage Institute in Georgia already uses bacteriophages for the treatment of infections such as diabetic ulcers and wounds.

Meat "blown pack" spoilage

Gaseous spoilage of vacuum-packaged chilled meats were reported in Germany. Clostridium estertheticum was found to generate the gas with nauseating odour. Health officials declared the phenomena as not health threatening. The affected meat had be destroyed. Clostridium estertheticum represents, therefore a financial threat for chilled meat producers.

Clostridium estertheticum gaseous spoilage of chilled meats [294]

Collins and colleagues 1992 performed a taxonomic study of an unknown anaerobic Gram-positive, spore-forming, psychrophilic bacterium isolated from spoiled vacuum-packed refrigerated beef and proposed it to be classified as a new species of the genus Clostridium, as Clostridium estertheticum sp. nov. The type strain is NCIMB 12511.

Contamination of vacuum-packed chilled meats with Clostridium estertheticum [295]

Clemens, Adam and Brightwell 2010 studied the contamination levels of Clostridium estertheticum spores causing gaseous spoilage of vacuum-packaged chilled meats, beef and lamb, stored at two different temperatures, -1.5 and 2 degrees C.

The authors stress that onset of blown pack spoilage is delayed storing meat at -1.5 degrees C, compared with storage at 2 degrees C. To avoid the risk of "blown pack"spoilage, contamination with spores of Clostridium estertheticum must be reduced to a minimum by strict observance of hygienic handling of meat.

PCR amplification procedure to detect clostridia causing spoilage of vacuum-packed chilled meats [296]

Broda DM, Boerema JA, Brightwell 2009 determine preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats using the polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments.

Clostridium gasigenes, C. estertheticum and C. algidicarnis/C. putrefaciens were commonly detected in farm, faeces, fleece and processing environmental samples collected at the slaughter floor operations, but only 4 out of 26 cooling floor and chiller environmental samples were positive for all of them. Frequency of C gasigenes and C. estertheticum was low, and high frequency of C. algidicarnis/C. Putrefaciens was detected in boning room. Detection frequence of C. gasigenes and C. estertheticum was high, but low for C. algidicarnis and/or C. putrefaciens in samples of faecis.

The authors concluded that control of meat spoilage by clostridia is best approached individually for each group.

Describing the bacteria isolated from blown packs of vacuum-packaged beef [297]

Yang, Gill and Balamurugan 2009 describe the bacteria recovered from the microflora of blown packs of vacuum-packaged beef identified as Leuconostoc mesenteroides, Lactococcus lactis, Carnobacterium maltaromaticum, and Clostridium estertheticum, with Leuconostoc mesenteroides predominant.

The authors stress that Clostridium estertheticum may predominate during the early stages of development of the spoilage microflora of vacuum-packaged beef but will likely be inhibited by a falling pH later on.

Inhibition by Lactobacillus sakei of spoilage bacteria on vacuum-packed meat [298]

Jones 2009 and colleagues describe the abilities of five Lactobacillus sakei strains and one Lactococcus lactis strain to retain inhibitory activity against target organisms on vacuum-packaged lamb and beef.

Among others findings, the authors report that in beef packs inoculated with Clostridium estertheticum spores and L. sakei strain 27, 44 or 63, the development of blown-pack spoilage was delayed by up to one week. The authors suggest a set of Lactobacillus sakei strains which may extended shelf life of minimally processed fresh beef and lamb.

Temperature and contamination level influences onset of vacuum-packed meat spoilage [299]

Moschonas and colleagues 2010 examined the effect of storage temperature and inoculum level on the time of onset of "blown pack" spoilage caused by psychrotolerant bacteria in vacuum-packed meats. Clostridium estertheticum ssp. estertheticum presented at all inoculum levels/storage temperature combinations examined the earliest "blown pack" spoilage of all other bacteria in test. They stress the importance contamination level control and low storage temperature to delay the onset of "blown-pack" spoilage of meat.

Isolation and source of "blown pack" spoilage bacteria identification [300]

Moschonas and colleagues 2009 report that DNA-based techniques were the most efficient methods to determine the presence and distribution of blown pack spoilage at beef abattoirs of Clostridium estertheticum and Clostridium gasigenes, compared with culture-methods which presented poor results. The authors point out that hides and faeces were found to be the main reservoirs of "blown pack" spoilage clostridia in the abattoirs.

Glucose and lactose utilisation by Clostridium estertheticum

[301] According to Yang X, Balamurugan S, Gill 2009 blown pack spoilage of vacuum packaged beef is caused by the psychrophile Clostridium estertheticum, which grow exponentially on glucose with simultaneous hydrolysis of glycogen. Growth ceased when glucose in the media was depleted; but hydrolysis of glycogen continued at a reduced rate, and lactate was consumed rapidly. The authors suggest that reducing the availiability of glucose may limit the growth of Clostridium estertheticum and other gas generating bacteria on vacuum packaged beef. Lactate fermentation, however, will continue, even after growth ceases.

PCR detection of psychrophilic Clostridium spp. [302]

Broda, Boerema and Bell 2003 developed a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause "blown pack" spoilage of vacuum-packed meat containing as few as 100 clostridial cells per gram. Clostridium gasigenes was confirmed as the causative agent of "blown pack" spoilage insome packages, and Clostridium estertheticum in others.

The described procedure may also be used in in epidemiological trace back of "blown pack" spoilage incidents in meat processing plants.

Controlling "blown pack" spoilage in meat processing plants [303]

Broda and colleagues 2002 identified abattoir sources of psychrophilic clostridia causing "blown pack" spoilage of vacuum-packed chilled meats. Restriction fragment length polymorphism analysis of the 16S rDNA gene (PCR-RFLP) and 16S-23S rDNA internal transcribed spacer (ITS) analysis were used by the authors.

The authors found that soil particles attached to hide or present in faeces are the primary reservoir of "blown pack" clostridia contaminating meat end products. The authors recommend strong dressing procedure hygiene to control the spread of psychrophilic Clostridium spp. in a meat plant.

Hygienic carcass dressing at abattoir is necessary to reduce blown pack spoilage of meat products [304]

Broda and colleagues 2009 assessed preslaughter and processing sources of psychrophilic and psychrotolerant clostridia causing spoilage of vacuum-packed chilled meats, using polymerase chain reaction (PCR) amplification of specific 16S rDNA fragments. Clostridium gasigenes, Clostridium estertheticum, Clostridium algidicarnis and Clostridium putrefaciens were detected in farm, faeces, fleece, slaughter floor operations prior to fleece removal.

Clemens, Adam and Brightwell 2010 report that storage at -1.5 degrees C significantly delays the onset of blown pack spoilage of meat in comparison with storage at 2 degrees C. The authors stress that one Clostridium estertheticum spore may initiate spoilage. They point out to the importance of hygienic carcass dressing to keep contamination to a minimum to improve shelf life of vacuum-packed chilled meat products. [305]

Description of Clostridium estertheticum subspp. laramiense and Clostridium estertheticum subsp. estertheticum growing in meat juice medium

[306]
Recently two subtypes of Clostridium estertheticum were defined. Yang, Gill and Balamurugan 2010 presented further description of these subtypes: The fermentation products of both strains of Clostridium estertheticum subsp. laramiense and Clostridium estertheticum subsp. estertheticum were butyrate, acetate, and formate from gucose and 1-butanol, ethanol, butyrate, and formate from the fermentation of lactate. Both organisms did not produce H2S. The optimum and maximum temperatures for growth of both were 10 degrees C, and 20 to 22 degrees C, respectively in a pH range for growth of 5.5 to 7.5. The authors expect blown pack spoilage of meat to be identical for both subtypes.

There is no epidemiological association of Staphylococcus aureus between isolates from cows and humans [307]

Hata and colleagues 2008 assessed the epidemiological association and bacteriological characteristics of human and animal Staphylococcus aureus isolates. Using pulsed-field gel electrophoresis the authors found that pulsotypes of isolates from bulk milk differed from pulsotypes from human isolates. They concluded that there is no epidemiological association between isolates from these 2 sources, and that a number of factors play a role in the adaptation of Staphylococcus aureus isolates to specific hosts.

Hair-like protrusions on the surface of bacteria may turn them dangerous to premature babies [308]

Giovanna Marchini and colleagues 2009 found that Staphylococcus epidermis, a coagulase-negative staphylococci, which normally reside on the skin of healthy people can cause serious infections in premature babies, attaching to the child's skin and mucous membranes and cause serious infections.

The researchers found hair-like protrusions on the surface of the bacteria which adhere the bacteria to the host's cells. According to Marchini, an antimicrobial substance LL37 is found at the skin and lungs and inhibit the growth of the bacteria in older people. This may explain the human co-existence with certain microbes, such as intestinal bacteria which produces Vitamin K and others involved in the development of an immune system.

EFSA says cases of infections with Listeria monocitogenes are rising [309]

According to the EFSA 2008 updated opinion on Listeria monocytogenes risk the Pannel stresses that the number of infections are rising and gives advices to reduce the risk.

In its advice to industry, the Panel identified the following as key areas for attention: food packaging and preparation practices in the food chain (such as the slicing of RTE meat products), storage temperatures, general industrial good hygiene practices and the education and training of food handlers. The lack of an effective HACCP system may pose another risk.

Food poisoning outbreaks linked to cantaloup

Every growing season bacterial and viral outbreaks linked to cantaloupe are reported Cantaloupe-linked infections are top 5 of the list of fruits and vegetables for outbreaks. This is largely due to their growing conditions and the potential to support the growth of bacteria. Listeria monocytogenes contamination is known associated deli meat, unpasteurized cheese, raw milk, fresh-cut fruit, and fresh-cut vegetables. Reservoirs for Listeria monocytogenes include ruminant animals, such as cattle, goats, and deer, decaying vegetation, and cold, wet, and difficult to clean environments. CDC and food safety authorities publish advices to reduce the infection risk.

Deadly bacteria or viruses can be pushed into the meat of the cantaloupe during slicing, Washing the cantaloupe before cutting is therefore highly recommended. Cantaloup has a very rough rind which rends the fruit prone to harbour and multiply pathogenic bacteria and viruses. A scrub brush should be used during washing under running water to keep bacterial number low. Cool cantaloupe and its slice in refrigerator.

High incidence of outbreaks related to cantaloupe demonstrate the need to increase safety measures at farm, transportation, storage and in kitchen [310]:
1990 - 1991 two Salmonella outbreaks
1993 Escherichia coil O157:H7 outbreak
1997 Salmonella saphra and Escherichia coil O157:H7 outbreaks
1999 - 2003 Three Norovirus and seven Salmonella outbreaks
2005 - 2008 Five Salmonella outbreaks
2011 Linsteria monocitogenes

Invasive listeriosis, deadly cantaloupe outbreak in late 2011 [311]

CDC highlights the risk of listeriosis outbreak which started in July 2011 in Colorado, U.S.A. The disease spread over 28 states, 146 cases of invasive listeriosis were confirmed, 30 patients died and one miscarriage was reported until December. The source of this outbreak was tracked down to Jensen Farms cantaloupe.

Cantaloupes have been linked to listeriosis illness caused by Listeria bacteria that contaminated the fruit while they were being stored and sorted in cold conditions after harvest. The U.S. Centers for Disease Control and Prevention is investigating the link between cantaloupe Listeria contamination and human sewage sludge that may have become airborne from a nearby farm.

Infection incidence

An estimated 1,600 cases of invasive listeriosis and 260 related deaths occur annually in the United States. Listeria monocytogenes ranks third as a cause of death due to major known food born pathogens in the United States.

Groups at higher risk for invasive listeriosis

Higher-risk groups include older adults, patients undergoing transplants or receiving immunosuppressive medications, persons with immunocompromising conditions, patients with liver or kidney disease, diabetes and alcoholism. Pregnant women may suffer foetal loss, premature labour, neonatal sepsis and meningitis.

Safety precautions

Listeria monocytogenes is able to grow at refrigeration temperatures. This means that storage times should be as short as possible, particularly for foods that are known to spread the disease such as soft cheeses, unpasteurized dairy products (including raw milk); deli and lunch meats, cold cuts, hot dogs, p$\hat{a}$té, and meat spreads.

Persons at higher-risk for invasive listeriosis should not eat deli and lunch meats, cold cuts, or hot dogs, unless they are heated to an internal temperature of 165° or until steaming hot just before serving. Persons at higher risk also should not eat soft cheeses unless they are labelled as made from pasteurized milk.

No one should drink raw milk or eat un-aged cheeses or other products made from raw milk. In addition to Listeria monocytogenes, a wide variety of pathogens that are sometimes found in raw milk can make people sick.

Diagosis of listeriosis

Food products, such as smoked salmon, cheese, sandwiches, and lettuce/leafy greens, may be recalled because testing identified Listeria monocytogenes contamination, even without any associated case of illness. However, CDC says that no testing or treatment is indicated for an asymptomatic patient who ate a recalled product, unless symptoms appear within 2 months of eating the recalled product.

According to CDC the diagnosis is confirmed only after isolation of Listeria monocytogenes from a normally sterile site, such as blood, or from amniotic fluid or the placenta in the setting of pregnancy. Listeria monocytogenes can be isolated readily on routine media, but care must be taken to distinguish this organism from other Gram-positive rods, particularly diphtheroids. Selective enrichment media improve rates of isolation from contaminated specimens. Serological tests are unreliable, and not recommended at the present time.

FDA performed an environmental assessment [312]

The environmental assessment including on-site visits to the farm, packing facility, and cold storage, and environmental and finished product sampling of cantaloup.

Growing environment

Environmental samples collected in the growing fields such as soil, wild animal excreta, perimeter and furrow drag swabs, agricultural water, pond water, and cantaloupe were negative for Listeria monocytogenes. The environmental samples collected in the packing facility and cantaloupe collected in cold storage were positive for Listeria monocytogenes. The growing fields are not a likely means of contamination. However, FDA has determined that the growing environment cannot be eliminated as a potential contributor in the introduction of Listeria monocytogenes contamination. Specifically, low-level sporadic Listeria monocytogenes contamination from the agricultural environment and incoming cantaloupes may have allowed for establishment of a harbourage or niche for Listeria monocytogenes in the packing facility and cold storage.

There are a number of factors that are likely to have contributed to the introduction, growth, or spread of Listeria monocytogenes at Jensen Farms, such as facility and equipment design packing facility cold Storage and postharvest practices, free moisture or increased water activity of the cantaloupe rind from postharvest washing procedures.

Recommendations for prevention of Listeria monocytogenes contamination

Fresh fruit and vegetable producers should employ good agricultural and management practices recommended for the growing, harvesting, washing, sorting, packing, storage, and transporting of fruits and vegetables sold to consumers in an unprocessed or minimally processed raw form. These practices are set forth in FDA and USDA's "Guidance for Industry - Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables." [313]

FDA's findings regarding this particular outbreak highlight the importance for the industry to employ good agricultural and management practices in their packing facilities as well as in growing fields.Read "Tips for Fresh Produce Safety: Safe Handling of Raw Produce and Fresh-Squeezed Juices" [314] www.foodsafety.gov/keep/types/fruits/tipsfreshprodsafety.htm

Listeria L-forms lost their membrane but are still highly pathogen [315]

Listeria (Listeria monocytogenes), a gram positive bacteria causes often fatal food-borne infections They were found in milk and soft cheese. Listeria can cross the blood-brain barrier and the placenta barrier.

Loessner and colleagues 2009 report that listeria cells, usually in rod form, may loose their cell walls, being replaced by a thin cytoplasmic membrane. They become spherical and are small or greatly enlarged, the so called L-form. The stress regulating genes were found to be activated while metabolic and energy balance genes were downregulated in the L-form.

L-form listeria bacteria form protoplast-like vesicles bearing daughter cells which are released when the vesicles bust. These L-forms can grow in milk but are difficult to cultivate under laboratory conditions and may remain undetected in milk.

Microbiological criteria [316]

Growth of L. monocytogenes is a function of the type of food, the storage time and the storage temperature.

Microbiological criteria have been implemented in Europe according to the categories of ready-to-eat foods. Microbiological criteria will assist in controlling the levels of L. monocytogenes e.g. absence in 25 g or 100 cfu/g at the point of consumption.

The Codex alimentarius

Codex alimentarius document on microbiological criteria for L. monocytogenes in ready-to-eat foods suggests a zero tolerance throughout the shelf life of the product for ready-to-eat foods in which growth of this microorganism can occur. Applying this criterion close to the end of shelf life could classify products as unsatisfactory, although they are of low risk. An additional option proposed in this Codex document is therefore to tolerate 100 cfu/g throughout the shelf life provided that the manufacturer is able to demonstrate that the product will not exceed this limit throughout the shelf life.

Recommendations EU Pannel

For ready-to-eat foods that support growth of L. monocytogenes, it is impossible to predict with high degree of certainty that the level will or will not exceed 100 cfu/g during the shelf life of these products. Thus, applying this option may result in accepting a probability that foods with more than 100 cfu/g will be consumed. The impact on public health would depend whether the levels markedly above 100 cfu/g are reached.

The Pannel recommends to investigate listeriosis cases more thoroughly and generate and analyse data on the consumption in the EU of ready-to-eat foods in which Listeria can be found.

The Panel also advised that consumers should continue to observe recommended storage temperatures and keep food appropriately chilled at all times, and take note of the shelf-life of food in their refrigerators. Good food hygiene and preparation principles also play an important role in the prevention of Listeria and other food-borne infections.

New predictive microbiology tools for Listeria monocitogenes

The Pathogen Modeling Program (PMP) [317]

It is the most widely used predictive microbiology application software, and includes more than 35 models for 11 bacterial pathogens including L. monocytogenes. Download is free.

The ComBase [318]

It is a combined database of microbial responses to food environments. It is linked to the ComBase modelling toolbox, which includes :

The Food Spoilage Predictor (FSP) and the Seafood Spoilage and Safety Predictor (SSSP) [319]

Include models for different seafood spoilage bacteria, and a model to predict the simultaneous growth of L. monocytogenes and spoilage microorganisms in sliced and vacuum packed cold-smoked salmon.

Sym'previus [320]

Information from Sym'previus is available on a commercial basis.

The U.S. Food Protection Plan [321]

The FDA developed a new Food Protection Plan in late 2007 to address the changes in food sources, production, and consumption that we face in today's world. This plan is intended to cope with new infections like listeriosis.
The Plan is based on three strings:

Listeria in ice cream under aging and storage and distribution conditions [322]

Gougouli, Angelidis and Koutsoumanis 2008 simulated conditions of the aging process and of normal or abuse conditions during distribution and storage of commercial ice cream products to study the kinetic behaviour of Listeria monocytogenes.

The bacteria was inoculated and the samples stored under static chilling (4 to 16°), static freezing (-5 to -33°)

Under chilling conditions, L. monocytogenes grew well at all temperatures tested. Under freezing conditions, no significant changes in the population of the pathogen were observed throughout a 90-day storage period, however freezing did not cause a severe stress in L. monocytogenes cells which were able to initiate growth within a very short time after a temperature upshift from freezing to chilling temperatures.

The authors developed mathematical models, which can be used by the dairy industry as effective tools for predicting the behaviour of the pathogen during the manufacture, distribution, and storage of ice cream products

Changing genome sequence of yoghurt bacteria [323]

Van de Guchte and colleagues studying the genome sequence of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) found that the sequence shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions.

The author point out signs of a rapid evolution of the genome: The authors conclude that L. bulgaricus is in ongoing adaptation from a plant-associated habitat to the stable protein and lactose-rich milk environment, such as yoghurt,through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

Genome sequence analysis of Lactobacillus delbrueckii [324]

Lactobacillus delbrueckii subsp. bulgaricus, together with Lactobazillus helveticus and Streptococcus thermophilus are part of starter cultures for fermented dairy products such as yoghurt, Swiss cheese and Italian cheese.

The genome sequence of L.delbrueckii subsp bulgaricus is of interest for the dairy industry because strains must be resistant to bacteriophage, have stable fermentation properties, and consistently produce products with acceptable flavour and texture attributes. A complete genome sequence analysis will provide the knowledge of the enzymes and metabolic pathways helping to enhance these strains.

Lactobacillus delbrueckii species contains three subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus. L. delbrueckii subsp. Bulgaricus. The species are acid tolerant, cannot synthesize porphyrins, and possess a strictly fermentative metabolism with lactic acid as the major metabolic end product, and according to Axelsson, 1998, are part of the obligately homofermentative ("Group I") cluster, which produce D-lactic acid from hexose sugars via the Embden-Meyerhof pathway and are incapable of fermenting pentoses and, according to Hammes and Vogel, grow on a restricted number of carbohydrates and require pantothenic acid and niacin. [325]

Hammes and Vogel found that the GC ratio of L. delbrueckii subsp. bulgaricus with 49-51%, is higher than that found among other species of this phylogenetic tree with 34 to 46%. [325] Its genome size is according to Leong-Morgenthaler 2.3 Mbp. [326]


Safety assessment for microorganisms added to food and feed [327]

EFSA introducted the use of Qualified Presumption of Safety (QPS) as a safety assessment tool for microorganisms added to food and feed.

A wide variety of bacterial and fungal species are used in food and feed production, either directly or as a source of additives. Some of these have a long history of apparent safe use, while others are less well understood and may represent a possible risk for consumers. To capture important risk aspects without committing resources to thorough investigations of organisms known to be safe, there is a need for a tool for setting priorities within the risk assessment of microorganisms in the production of food and feed.

In 2002/3 the introduction for selected microorganisms of a Qualified Presumption of Safety (QPS) was proposed. This concept shared some of the elements and purpose of the USA GRAS (Generally Recognised As Safe) system. In essence this proposed that a safety assessment of a defined taxonomic group (e.g. genus or group of related species) would be made independently of any particular pre-market authorisation process. If the taxonomic group did not raise safety concerns or, if safety concerns existed, but could be defined and excluded (the qualification) the grouping would be granted QPS status.

Thereafter a strain of microorganism whose identity could be unambiguously established and assigned to a QPS group would be freed from the need for further safety assessment other than meeting any qualifications specified. Microorganisms not considered suitable for QPS status would remain subject to a full safety assessment.

Strains representing approximately 100 species of microorganisms have been or are expected to be referred to EFSA for a safety assessment (see Annex 1 for those already notified to EFSA). Individual species may be the subject of a single notification but more usually are found in several notifications. This list includes both live organisms deliberately introduced into the food chain and those used as a source of food/feed additives. A large majority of these  100 species fall within four broad groupings:

  1. Gram-positive non-sporulating bacteria (GPNS)
  2. Bacillus species
  3. Yeasts
  4. Filamentous fungi

Antimicrobial drug-resistant Escherichia coli from humans and poultry products [328]

According to Johnson and colleagues 2007 the food supply, including poultry products, may transmit antimicrobial drug-resistant Escherichia coli to humans. Drug-resistant human isolates from US population were phylogenetic and virulence gene profile similar to poultry isolates, and drug-susceptible and drug-resistant poultry isolates were largely indistinguishable.

Human-source drug-resistant fecal E. coli isolates more likely originated in poultry than in humans, whereas drug-resistant poultry isolates likely derive from drug-susceptible poultry isolates. However, avoidance of poultry consumption may not reliably provide personal protection.

Microbiology of raw milk is important for dairy product quality [329]

Hantsis-Zacharov and Halpern 2008 investigated milk spoilage during cold storage after milk collection. The source of the bacteria are the teat of the cow, the udder, milk equipment and the milking environment. They found that psychrotrophic bacteria and their extracellular proteases and lipases, even refrigerated at 7°, contribute to the spoilage of dairy products. The heat-stable enzymes still affect the flavour quality of milk and its products. Lipases release free fatty acids causing flavour defects in the milk, and proteases may cause bitter flavour.

The authors classified the psychrotrophic isolates in seven classes:

The microbiologic isolates related to the seasons of the year

Gammaproteobacteria in spring and winter, Bacilli in summer, and Actinobacteria in autumn. The four minor classes were Alphaproteobacteria, Betaproteobacteria, Flavobacteria, and Sphingobacteria.

The enzymes

The dominant genera, Pseudomonas and Acinetobacter (Gammaproteobacteria) were lipolytic, Microbacterium (Actinobacteria) was lipolytic and proteolytic, and the lactic acid bacteria (Lactococcus and Leuconostoc) displayed had only minor enzymatic activity.

Hantsis-Zacharov and Halpern stress that about 20% of the isolated bacteria were novel species and more studies are needed. They published the classification of a new species, the Chryseobacterium oranimense sp. Nov., which, together with two other novel bateria, Chryseobacterium haifense and Chryseobacterium bovis produce adverse effect on milk quality [330].

The authors conclude that monitoring the dominant psychrotrophic species could become a sensitive and efficient quality control in the dairy industry.


Non-culture techniques to detect difficult to cultivate bacteria [331]

Uncultivated and difficult-to-cultivate bacteria, such as Fusobacterium nucleatum, Leptotrichia (Sneathia) spp., Bergeyella sp., Peptostreptococcus sp., Bacteroides spp., and a species of the order Clostridiales may have clinical relevance.

Dr. Yiping W. Han and colleagues 2009 stress that 60% of the microbes present in cases of intra-amniotic inflammation, leading to preterm birth, are missed with traditional culture testing. The authors compared the results of 16S rRNA-based culture-independent methods with standard culture testing.

Two-thirds of the species detected by the culture-independent methods were not isolated by culture. These results correlated with increased amniotic fluid levels of interleukin-6, histological chorioamnionitis, funisitis, and delivery of neonates with early-onset sepsis. The authors point to the fact

Pathogenicity of Bordetella hinzii [332]

According to Register and Kunkel 2009 Bordetella hinzii was believed to be non-pathogenic in poultry. However, recent studies say that the bacterium caused severe disease causing 100 percent morbidity in turkeys which was formerly attributed to Bordetella avium, a pathogenic bacterium that causes upper respiratory disease in poultry and wild birds.
To distinguish between B. hinzii and B. avium DNA-based tests were used. [333]
Register and Kunkle found four bordetella hinzii strains which were able to to grow and persist in the trachea and also caused clinical disease. The strains varied in severity, although none demonstrated 100 percent morbidity.
Another study found that Bordetella hinzii is not pathogenic for chickens.
Further researches will clarify how pathogenic strains differ from non-pathogenic once. And will look after the virulence factors affecting turkey poults.


Bordetella hinzii pathogenicity to humans [334]

Funke and colleagues 1996 highlight the importance of the genus Bordetella comprising species such as Bordetella pertussis and Bordetella parapertussis which are the causative agents of whooping cough in humans, Bordetella bronchiseptica is primarily a respiratory pathogen found in animals but may also cause pneumonia and bacteremia in humans. B.avium causes coryza in poultry but has never been described as causing infections in humans. Bordetella holmesii have been isolated from young adults with septicemia. Bordetella hinzii was proposed as the species of some strains isolated from poultry with respiratory disease. The authors report two isolates of Bordetella hinzii which, together with two other cases demonstrate that Bordetella hinzii can cause disease in humans. They caution, however, the role of Bordetella hinzii in human infections but is still not completely understood.

Fry and colleagues 2007 describe a clinical isolate of Bordetella hinzii in the UK from a patient which had no known avian exposure, and the source of the organism remains unknown. The authors stress that human infection with Bordetella hinzii is rare. Genotypic methods, and the greater mutational variation of the ompA gene compared to other genes (e.g. 16S rRNA gene) is being suggested by the authors to differentiate Bordetella hinzii from Bordetella avium and other non-classical Bordetella species. [335]

Raman spectroscopy to detect viruses [336]

A new rapid system for detecting and identifying viruses in near-real time is being developed by Tripp and colleagues. It is based on surface-enhanced Raman spectroscopy to measure the frequency of near-infrared laser light. It detects and classifies microRNAs (miRNAs), which are regulators of gene expression during development and cell differentiation as well as biomarkers of disease.

miRNAs were first described in 1993 by Lee and colleagues, and the term microRNA was only introduced in 2001. [337]

The authors stress that the SERS-based sensor can detect extremely low number of viruses and provides its molecular fingerprint. The rapid response system can detect viruses from a nasal swab in one minute or less. A device which is now being developed can help to control disease outbreaks and bioterrorism, monitoring passengers at airports or speed up a diagnosis.

Fourier reflection infrared spectroscopy (FTIR-ATR) detection of bacteria [338]

Li and Tripp 2008 used an alumina-coated ZnSe internal reflection element (IRE) to detect spores of Bacillus globigii by attenuated total reflection infrared spectroscopy (FTIR-ATR). The interaction between the carboxylate groups of the bacterium and the positively charged sites on the alumina modulates the adsorption which is highly dependent on pH having ist maximum value at pH 5 a detection limit of 10(7) spores per cm2 is reported by the authors.

Multidrug-Resistant strains of Streptococcus pneumoniae [339]

Mingtao Zeng and colleagues 2009 report that Streptococcus pneumoniae 19A is resistant to all approved antimicrobial drugs for treatment of acute otitis media

in children. Other S. pneumoniae isolates expressing 19A capsule were serotype 19A The authors describe the ST-2722 strain which belongs to a cluster which have 6 alleles of ST-156 expressing different capsular serotypes, such as 9V, 14, 11A, 15C, and 19F, They are antimicrobial resistant. The authors point out that such multidrug-resistant Streptococcus pneumoniae strains are of serious concern.

The use of seven-valent pneumococcal conjugate vaccine [340]

Dagan accentuates the reduction of penicillin-resistant Streptococcus pneumoniae carriage following the use of seven-valent pneumococcal conjugate vaccine (PCV7), However, a replacement by non-vaccine pneumococci serotypes was observed, with unknown impact on the disease.

Penicillin-intermediate serotype 19A isolates increased with the use of the PCV7 vaccine. This serotype is found worldwide, is highly multidrug-resistant and cause invasive pneumococcal disease in children and the elderly after the use of vaccine. The author stresses that infections with serovar 19A increases also without vaccination in regions with heavy use of antibiotics which turn strategies to contain antibiotic resistance so important.

Routine use of vaccine [341]

Cohen 2009 reports that the Kaiser Pemanente study found a reduction of 7.8% of otitis after the introduction of the seven-valent vaccine (PCV7). In Israel a nine-valent PCV was used with a reduction of 17% in antibiotic use, and 41.9% less antibiotics treating acute otitis media were prescribed in USA and 10% less in France on account of vaccine PCV7.

According to the author the penicillin-resistant pneumococci carriage reduced from 15.4% to 6.7% and penicillin-non-susceptible strains from 47.7% to 30.4%. Vaccine-serotype pneumococci carriage dropped from 44.3% to 28.9%.

These studies, however, report a rise of carriage of non-vaccine serotypes from 9.6% to 15.8%. The serotype 19A increased from 8.6% to 12.6%, and highly penicillin-resistant strains decreased from 15.6% to 1.1%.

Cohen points to the fact that supportive education on restricting the use of antibiotics should be part of an implementation of pneumococcal conjugate vaccine.

Pneumonic plague in China worse than the svine flu pandemic [342]

Yersinia pestis (formerly Pasteurella pestis) is a Gram-negative rod-shaped bacterium belonging to the family Enterobacteriaceae. It is a facultative anaerobe that can infect humans and other animals.

Human Y. pestis infection takes three main forms: pneumonic, septicemic, and the notorious bubonic plagues. All three forms have been responsible for high mortality rates in epidemics throughout human history, including the Black Death (a bubonic plague) that accounted for the death of at least one-third of the European population in 1347 to 1353. Depending on circumstances, these forms may occur separately or in combination.

Recently Y. pestis has gained attention as a possible biological warfare agent and the CDC has classified it as category A pathogen requiring preparation for a possible terrorist attack.

Pneumonic plague erupted in the Chinese town of Ziketan in the Qinghai Province in July 2009. Three people died and ten others were also infected, all closely associated with the diseased. The Town was shut off in an effort to avoid the spread of the disease. The authorities tracked down people who came within germ-spreading distance of the infected people. A travel alert asked all visitors to keep an eye out for flu-like symptoms of the pneumonic plague. [343]

Symptoms of pneumonic plague [344]

The first signs are fever, headache, weakness, and rapidly developing pneumonia with shortness of breath, chest pain, cough, and sometimes bloody or watery sputum. The pneumonia progresses for 2 to 4 days and may cause respiratory failure and shock. Without early treatment, patients may die.

Transmission

The transmission is different for the three forms of plague.

- Pneumonic plague

Yersinia pestis infects the lungs. Infection spreads from person to person through the air in respiratory droplets from a person (or animal) with pneumonic plague.

- Bubonic plague

It is the most common form of plague. This occurs when an infected flea bites a person or when materials contaminated with Y. pestis enter through a break in a person's skin. Patients develop swollen, tender lymph glands (called buboes) and fever, headache, chills, and weakness. Bubonic plague does not spread from person to person.

- Septicemic plague

It occurs when plague bacteria multiply in the blood. It can be a complication of pneumonic or bubonic plague or it can occur by itself. When it occurs alone, it is caused in the same ways as bubonic plague; however, buboes do not develop. Patients have fever, chills, prostration, abdominal pain, shock, and bleeding into skin and other organs. Septicemic plague does not spread from person to person.

Early treatment of pneumonic plague is essential

To reduce the chance of death, antibiotics must be given within 24 hours of first symptoms. Streptomycin, gentamicin, the tetracyclines, and chloramphenicol are all effective against pneumonic plague. Antibiotic treatment for 7 days will protect people who have had direct, close contact with infected patients. Wearing a close-fitting surgical mask also protects against infection. A plague vaccine is not currently available for use in the United States.
Rodents are the reservoir of Yersinia pestis in western North America, southern South America, southern Africa, the Middle East, and central Asia. Infected fleas infect rats and man. [345]

New process explain the causes of the variability of individual cells in cell cultures [346]

Pelkmans and colleagues 2990 studied the causes of the causes of single-cell heterogeneity in cell adherent populations. They found that the properties of a cell population determine the different cell activities observed in cells of the same type, explaining that the reasons behind the different reactions seen in cells of the same type are certain causes that lead to predictable distribution patterns.

The researchers developed a process aided by a image-based screening centre to observe the variability of individual cells in cell cultures, what they called as "heterogeneity signature". The process and the special computer program allow automatic quantification and description of the phenotypes of the cells, showing how individual cell properties develop and affect each other. The authors found that cell properties were predetermined by characteristics of the population of the cell culture, such as the size of the population, the local cell density, the size of an individual cell, whether the cell is on the edge of the cell culture. Also the progression of the infection by three different viruses were analysed with this method.

The authors stress that their study may be important for the pharmaceutical industry studying effects of new substances, as many effects do not influence the cell, but act on the population as a whole, which then influences the individual cells.

Recombinant lactic acid bacteria protects against Yersinia pseudotuberculosis [347]

Daniel and colleagues 2009 report an immune response triggered by a recombinant Lactococcus lactis strain. It protected mice against both oral and systemic Yersinia pseudotuberculosis infections. The authors wrote that the Lactococcus strain secrets the Yersinia pseudotuberculosis low-calcium response V (LcrV) antigen which protects against the infection. The authors stress that recombinant lactic acid bacteria may provide an anti-Yersinia vaccination strategy.

Hydrogen from wastewater

[348] Bruce E. Logan of the Penn State Institutes of Energy and the Environment, in connection with the Napa Wine Company in Oakville developed a demonstration microbial electrolysis plant producing hydrogen from wastewater of the production of wine.

Microbial electrolysis cells consist of one carbon anode and one stainless steel cathode in his system instead of platinum or gold electrodes. This keeps the cost low. The organic material of the are converted by the bacteria into electrical current which hydrolyses water into oxygen at the carbon electrode and hydrogen on the stainless steel cathode.

The natural bacteria that work in the electrolysis cells can cope with wastewater composition such as high in sugar or shifting to remnants of the fermentation process.

Electromethanogenesis capturing CO2 as biofuel [349]

In their researches concerning the production of hydrogen Logan and colleagues 2009 found how to convert carbon dioxide in methan in a process called electromethanogenesis.
This research shows that methanogenic microorganisms produce methane in marshes and dumps not by utilising hydrogen or organic materials, such as acetate, but it is carbon dioxide and water which is converted to methane.

The researchers found that the Archaea may convert carbon dioxide and water to methane without any organic material, bacteria or hydrogen. The microorganism Archaeon, Methanobacterium palustre, can accept electrons directly, and use them to create methane in a two-chambered cell with an anode immersed in water on one side of the chamber and a cathode in water, inorganic nutrients and carbon dioxide on the other side of the chamber.

The authors stress that the system uses carbon dioxide as feed stock. Using solar energy or wind power to operate the system the produced methane would be carbon neutral. Methane is preferred over hydrogen because a large infrastructure of methane as fuel is already available.

'Hero Baby' brand baby milk contaminated with Enterobacter sakazakii [350]

Enterobacter sakazakii infection has been associated with the use of powdered infant formula, with some strains able to survive in a desiccated state for more than 2 years.

The Kuwait Municipality has ordered the withdrawal of the 'Hero Baby' brand of baby milk formula from co-operative societies. Traces of the potentially fatal entro bacter sakazakii bacteria were found in these products.

This decision was based on similar moves made by the United Arab Emirates (UAE) and Saudi Arabia.

Mycoplasma [351]

Mycoplasma is a genus of bacteria which lack a cell wall. Without a cell wall, they are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprotrophic. Several species are pathogenic in humans, including Mycoplasma pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and Mycoplasma genitalium, which is believed to be involved in pelvic inflammatory diseases.

Mycoplasma contamination is a problem in cell culture

Mycoplasma species are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients, such as serum or trypsin. Mycoplasma cells are physically small - less than 1 µm - and cannot be eliminated by sterile filtration. Mycoplasmas may induce cellular changes, including chromosome aberrations, changes in metabolism and cell growth. Severe Mycoplasma infections may destroy a cell line. Detection techniques include PCR, plating on sensitive agar and staining with a DNA stain including DAPI or Hoechst.

Mycoplasma pneumoniae reduced its genome size by discarding the complete anabolic and metabolic pathways acquiring an obligate parasitic lifestyle.

Mycoplasma detection using PCR method validated according European Pharmacopoeia standards [352]

Zhi and colleagues 2010 report the completion of the validation of a nucleic acid amplification technique (NAT) for use in detection of mycoplasma contaminants in biologics drugs, meeting or exceeding European Pharmacopoeia compliance requirements. This method may replace or supplement the existing culture-based methods with a PCR-based method and will become important for the biopharmaceutical industry.

Mycoplasma detection in fermenters [353]

Asarnow and colleagues 2010 describe a method based on a simple extraction procedure followed by a qualitative "touchdown" (TD) PCR protocol with primers specific to the 16S rRNA gene. The method was developed and validated an assay to monitor rFVIII production fermenters for mycoplasma contamination. The method was validated according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, and may replace the culture-based mycoplasma detection.

Mycoplasma gallisepticum infection detection in chicken [354]

Kahya and colleagues 2010 compared a real-time PCR (rPCR) test to serology and culture for the detection of Mycoplasma gallisepticum infection in chicken breeder flocks. Agreements between serology and culture, and serology and rPCR were 83.9% and 90.3%, respectively.
Based on the data of their study the authors advise the use of rPCR and/or culture for confirmation of serology results during screening infection of flocks.

Detection and eradication of mycoplasma in cell lines [355]

Molla Kazemiha and colleagues 2010 assessing the mycoplasma infection status of 200 cell lines using PCR assay with generic-specific universal primers found 20% of the cell lines contaminated with mycoplasma. Contaminating species were M. hyorhinis, M. fermentas, M. arginini, M. orale and A. laidlawii. Cell lines were treated with BM-Cyclin eradicating 100 % of the contamination. Mycoplasma removal agent eliminated 70% of mycoplasma, and Ciprofloxacin 42%. The authors noted a regrowth after 4 month with 12% of BM-Cyclin treatment, 62.5% of mycoplasma removal agent and 82.5% Ciprofloxacin.

Genotyping of Mycoplasma pneumoniae [356]

Schwartz and colleagues 2009 stress the importance of Mycoplasma pneumoniae, accounting for up to 25% of community-acquired pneumonia. Mycoplasma pneumoniae are classified in type 1 and type 2 based on sequence variation within the gene encoding the major adhesion molecule P1. The authors present a real-time PCR assay that can distinguish between the two types using high-resolution melt-curve analysis with reference strains M129 (type 1) and FH (type 2). The authors claim that this assay is rapid, reliable and convenient.

A rapid and sensitive flow cytometric technique quantifying viable mycoplasma [357]

Mohammadpour and colleagues 2010 developed a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable Mycoplasma agassizii cells. This mycoplasma threatened desert tortoise of the Southwestern United States, causing upper respiratory tract disease.

Detection of living Mycoplasma agassizii was performed using nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester, which penetrates the cell membranes of the mycoplasma, and is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be counted in less than 1 h. The authors stress the importance of this technique in the field of basic immunological, biochemical and pharmacological studies.

Brachyspira digestve infections in pigs and birds

Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia
indexBrachyspira hyodysenteriaeare helicoidal Gram negative, mobile and anaerobic bacteria. Brachyspira are responsible for diverse digestive infections in animals and humans, causing economic losses due to the delay of growth of the infected animals. These bacteria are responsible for the grey diarrhoea syndrome. It is estimated that 70% of the porcine breeding farms are affected by this syndrome.

Detection of Brachyspira [358]

The diagnostic is based on the microorganism isolation by culture followed by its biochemical characterisation, however, the results are not always conclusive. Molecular techniques (real time PCR) are the methods of choice for detecting the presence of the different Brachyspira after extraction and purification of the bacterial DNA.

The molecular detection system Brachyspira Ceeram Tools uses real-time PCR with a detection limit is 5 genome copies. No cross-reaction was observed with other organisms susceptible to infected pigs and in samples of the avian industry.

La, Phillips and Hampson 2003 described a duplex PCR amplifying portions of the Brachyspira hyodysenteriae NADH oxidase gene and the B. pilosicoli 16S rRNA gene for the faecal detection of these spirochaetes. [359]

Comparing ordinary PCR with real time PCR methods to detect Brachyspira [360]

Akase and colleagues 2009 compared ordinary PCR with real time PCR methods to detect Brachyspira infections in swine dysentery cases. The authors found that real time PCR using nox primers was more effective than ordinary PCR and culture.

Genome sequence of Brachyspira hyodysenteriae [361]

Hampson and Ahmed 2009 stressed that the genus Brachyspira contains important gut pathogens of pigs, birds and other animal species, including human beings. The genome sequence of the pathogen Brachyspira hyodysenteriae is characterised by extensive genome adaptation to the environment of the colon. Comparing it with the genome sequences of other Brachyspira may explain their involvement in colitis and diarrhoea.

High prevalence of spirochetosis in cholera patients [362]

Spirochetes from the genus Brachyspira were identified in more than one third of cholera patients in Bangladesh by Nelson and colleagues. The authors recommend that spirochetosis be tracked in cholera outbreaks.

Pathogenicity of Brachyspira pilosicoli [363]

Studies on the physiology of Brachyspira pilosicoli are difficult because it is an anaerobic spirochete which requires a specialized culture, and its growth is slowly. To study the mechanism of colitis induced by Brachyspira pilosicoli Naresh and colleagues 2009 incubated strains of the bacterium with a human colonic adenocarcinoma cell line (Caco-2 cells).

They found that the effect on the Caco-2 cells was an up-regulation of interleukin-1beta (IL-1beta) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1beta, TNF-alfa, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression. The authors concluded that B. pilosicoli is pathogenic.

Human intestinal spirochetosis [364]

A histological diagnosis of human intestinal spirochetosis were performed inpatients with abdominal pain, bloody stools, diarrhea or bowel symptoms, and in patients which showed only occult fecal blood. PCR analysis found more patients infected with Brachyspira aalborgi than B. pilosicoli. Infestation with B. aalborgi was detected over a 6-year period. Sato and colleaguies 2010 suggest that these spirochetes may be harmless commensals in humans.

Antagonistic effect of gut bacteria on swine enteropathogens [365]

Klose and colleagues 2010 screened beneficial strains from the animal gut to control pathogens related to dysentery in pig, such as Clostridium perfringens type A, various serovars of enterotoxigenic Escherichia coli and Salmonella enterica, as well as Brachyspira pilosicoli.

The authors report that Enterobacteriaceae were effectively inhibited by Lactobacillus salivarius and Lactobacillus reuteristrains. Lactobacillus mucosae, Lactobacillus amylovorus and Bifidobacterium thermophilum, were less effective and their effect was based on the production of organic acid.

The Bacillus subtilis strain was found to have anti-clostridial and anti-Brachyspira pilosicoli effect and homofermentative lactobacilli and Bifidobacterium thermophilum could suppress the growth of Brachyspira pilosicoli. Heterofermentative lactobacilli, such as Lactobacillus reuteri and Lactobacillus mucosae had no effect. Enterococcus faecium and L. amylovorus strain presented antagonism producing lactate and hydrogen peroxide. The authors call for more researches on gut bacteriia as antagonist in pig production.

Probiotic feed additive to inhibit Brachyspira hyodisenteriae which causes dysentery in swine [366]

Close and colleagues isolated strains of Bifidobacterium thermophilum, Enterococcus faecium and Bacillus subtilis which may be used as probiotic feed additives for prevention of swine dysentery. Their characteristics are a well-established identity, antibiotic susceptibility and antagonistic activity against Brachyspira hyodisenteriae.

Probiotic inhibition of Brachyspira hyodisenteriae and Brachyspira pilosicoli [367]

Bernardeau and colleagues 2009 studied the inhibition activity of the probiotic Lactobacillus rhamnosus CNCM-I-3698 and Lactobacillus farciminis CNCM-I-3699 on Brachyspira hyodysenteriae and Brachyspira pilosicoli, agents of Swine Dysentery and Porcine Intestinal Spirochaetosis. The in vitro research demonstrated the trapping of spirochaetal cells in a physical network, and the inhibition of the motility of Brachyspira. The authors call for in vivo studies regarding the use of probiotic lactobacilli as feed addsitive for the prevention of Brachyspira hyodysenteriae and Brachyspira pilosicoli.

Human intestinal spirochetosis [368]

According to Tsinganou and Gebbers 2010 Brachyspira aalborgi and Brachyspira pilosicoli predominate in human intestinal spirochetosis. Rates of the disease are low where living standards are high, and common in poor populations. Invasion of spirochetes beyond the surface epithelium may be associated with gastrointestinal symptoms, but is asymptomatic invasion remains at the surface of gut epithelium. Rare cases of spirochetemia and multiple organ failure have been reported in critically ill patients with IS.

Brachyspira murdochii is low pathogenic for pigs [369]

Jensen, Christensen and Boye 2009 describe a Brachyspira murdochii catarrhal colitis in pigs, applying fluorescent in situ hybridization and species-specific oligonucleotide probe targeting 23S rRNA. The bacterium was closely associated with the surface epithelium in diseased pigs. The authors concluded that high numbers of Brachyspira murdochii is low pathogenic for pigs.

Pathogenicity of Brachyspira among poultry [370]

Brachyspira intermedia and Brachyspira pilosicoli were found to be affect layer and breeder flocks in Europe and Australia. Myers and colleagues 2009 tested flocks of Pennsylvania for Brachyspira intermedia and Brachyspira pilosicoli using a duplex PCR and Brachyspira genus-specific PCR. The authors found colonization of layer flocks, older than 40 weeks of age, with pathogenic and call for further investigation.

Brachyspira intermedia and other indole-positive Brachyspira species [371]

Phillips and colleagues 2009 using the multilocus sequence typing assessed the population structure of Brachyspira intermedia isolates from pigs and chicken, and the relationship of the species to the other two indole-positive but strongly haemolytic Brachyspira species-B. hyodysenteriae and "B. suanatina".

The results of sequence types, amino acid types and clonal complexes indicate that cross-species may occur. Some isolates were found to be separated from others by large genetic distances and ongoing minor genetic change amongst isolates at the farm level are taking place. The authors conclude that their findings concerning isolates of B. hyodysenteriae, and "B. suanatina", suggest that it will not be possible to classify all weakly haemolytic indole-positive Brachyspira isolates as Brachyspira intermedia.

Relations between strains of Spanish Brachyspira spp and isolates from Germany and Belgium [372]

Hidalgo and colleagues 2010 described the genetic and phenotypic diversity of Spanish Brachyspira hyodysenteriae, and confirmed the presence of tiamulin-resistant isolates in Spain.

The PCR analysis was used for the identification of Brachyspira spp. and for the detection of the smpA/smpB gene. The combination of Random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) protocol was used to determine the epidemiological relationships.

The authors stress that indole-negative and tiamulin-resistant isolates of B. hyodysenteriae were found, and the genetic findings indicated a relationship between Spanish isolates and strains of Germany and Belgium.

Understanding coral leaching and susceptibility to disease [373]

Low immunity response may increase the risk of coral leaching and its susceptibility to disease.

Palmer, Bythell and Willis 2010 found an inverse correlation of the content of melanin, size of melanin-containing granular cells, and phenoloxidase activity, as well as concentrations of fluorescent proteins to thermal bleaching and disease susceptibility, in hard (Scleractinia) and soft (Alcyonacea) corals. These indicators are known to be related to immunity in invertebrates.

The authors stress that pheniloxidase activity, melanin-containing granular cell size, and fluorescent proteins concentration are predictors of susceptibility and are important in coral immunity. The authors call for a holistic approach to study coral reef bleaching and disease susceptibility.

Wound healing properties of honey [374]

Salomon and colleagues 2010 describe the wound healing properties of honey. The high concentration of sugar constitute a hyperosmotic medium with antimicrobial properties. The authors also stress that different enzymes, including glucose-oxidase that generates hydrogen peroxide and gluconic acid in the presence of glucose and water. In addition, honey presents favourable viscosity and the hygroscopic qualities allowing spread on the wound bed. The authors concluded that honey is an efficient treatment of chronic wounds of the lower leg and also of abdominal wounds.

Bacteriostatic effects of honey [375]

Kwakman and colleagues 2010 described all bactericidal factors in a medical-grade honey. Bacillus subtilis, methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamase producing Escherichia coli, ciprofloxacin-resistant Pseudomonas aeruginosa, and vancomycin-resistant Enterococcus faecium, were killed by 10-20% (v/v) honey, whereas more than 40% (v/v) of a honey-equivalent sugar solution was required for similar activity.

Activity against all other bacteria tested depended on sugar, H2O2, methylglyoxal, and bee defensin-1, contributing to the effects of honey in medicine, whereas bee defensin-1 was the most active compound.

According to Boukraa and Sulaiman 2009, honey, propolis, royal jelly and bee venom have a strong antibacterial activity, but considerably variability is found within the same product and its botanical origin. Propolis presents the strongest antibacterial activity based on its richness in flavonoids. The authors stress that food quality of honey and royal jelly contain pollen and other particles which might cause allergies when used in wound care. Fine filters must therefore be used in the production of medical products. A safety issue of honey and their products for medical use is the presence of viable spores which includes clostridia. The growing number of licensed medical bee products will increase understanding of its use, which, however, should be limited to those which are safe and with certified antibacterial activities, say the authors. [376]

Chernev and colleagues 2010 report benefits of combined, noncontact, low-frequency ultrasound and topical application of medical honey in treatment of chronic and delayed healing wounds. [377]

No manuka honey resistant mutants found so far [378]

Cooper and colleagues 2010 report that honey-resistant bacteria have not been isolated from wounds treated with honey. However, bacteria isolated from wounds, exposed to sub-lethal concentrations of manuka honey presented a stepwise resistance training, but changes were not permanent and honey-resistant mutants were not detected. The authors concluded that high concentrations of honey will keep the risk of development of honey resistant mutants low.

Antibacterial properties of tualang honey [379]

Mohd Nasir compared the properties of tualang honey with that of manuka honey in treatment of burn wounds. The authors found that tualang honey has a bactericidal as well as bacteriostatic effect, it is less sticky compared to Manuka honey. Tualang honey was less effective for Gram positive bacteria as silver-based dressing or medical grade honey dressing.

Classic treatment of burn wounds recommended instead of honey products [380]

Topical antimicrobials are employed for prophylaxis and treatment of burn wound infections. Glasser and colleagues 2010 point out that no defined susceptibility breakpoints exist and standards need to be established for topical antimicrobial. The authors recommend continuing to use silver products for prophylaxis against gram-negatives and mafenide acetate for treatment, and mupirocin for methicillin-resistant Staphylococcus aureus.


Susceptibility beakingpoints

Susceptibility beakingpoints are used to define susceptibility and resistance to antibacterials.
Depending on the testing method, they are expressed as either a concentration (in mg/liter or g/ml) or a zone diameter (in mm). Susceptibility breakpoints allow communication from the clinical laboratory to the prescriber regarding the likelihood that a particular antibacterial regimen will be clinically useful in the treatment of patients with infections. [381]

Fungal invasion pathway of plant and animal cells [382]

Kale and colleagues 2010 relate that special proteins from fungi and oomycetes, known as effectors are transferred to the interior of host cells suppressing its natural defences. This pathway may explain the Irish potato famine in the nineteenth century, actual soybean diseases and fatal infectious diseases in humans.

The effector proteins bind a specific lipid molecule found on the cell surface, the lipid phosphatidylinositol 3-phosphate (PI3P), and can enter the cell using the lipid raft, a region of the cell's outer membrane. The PI3P lipid acts as a bridge between the effector protein and the lipid raft.

Bacteria puncture the host cell's membrane and then inject their effectors into the host cell's membrane with a needle-like structure. Fungi and oomycetes lack such an injection structure. They slip their effectors into plant cells by means of the PI3P found at the surface of plant cells, animal cells, and some human cells. The effector proteins N-terminal RXLR and dEER motifs enable oomycetes to bind with the PI3P to enter into host cells via the lipid raft of the cells wall. Fungi, contain functional variants of the RXLR motif which may also enter human cells and may be targeted by new therapeutic measures, which act on the RXLR terminal or on the PI3P lipid of the cell, say the authors.

The lipid raft of cell walls [383]

The plasma membrane of cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid rafts. These specialized membrane microdomains compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules, influencing membrane fluidity and membrane protein trafficking, and regulating neurotransmission and receptor trafficking. Lipid rafts are more ordered and tightly packed than the surrounding bilayer, but float freely in the membrane bilayer.

Bacteriological methods

Bacteriological Analytical Manual (BAM) [384]

FDA's Bacteriological Analytical Manual (The BAM) is a collection of procedures preferred by analysts in U.S. Food and Drug Administration laboratories for the detection in food and cosmetic products of pathogens (bacterial, viral, parasitic, plus yeast and mould) and of microbial toxins.

Methods development has always been driven by the demand for tests that are faster, cheaper, easier, and more accurate. Pressure for improved procedures is particularly apparent in microbial food safety, because traditional tests may involve many steps which take much time.

Ways proposed to accelerate the procedure included, initially, improved media and compacted culturing. Then, automation began to replace manual execution. Also, indirect identification, i.e., by biochemical (e.g., fatty acid profiles, nucleic acid sequences) or biophysical shortcuts (FT-IR) that reveal organisms' pertinent biomarkers or genetic fingerprints, began to make the isolation of viable microbes not as necessary. These newer tests - known as "rapid methods" if they took hours rather than days and as "real-time" testing if they took minutes - have not yet, however, made traditional testing obsolete.

There are good reasons why analysts should continue to have the traditional skills to resuscitate, enrich, isolate, and identify microorganisms. Often, some culturing is necessary before there is enough material for the application of a rapid method or real-time test. Then, too, foods may contain substances that interfere with biochemical/molecular test shortcuts. Furthermore, having a viable microbial isolate may still provide quantitative and infectivity information not otherwise available, or be mandatory because of regulatory requirements and legal issues, or be useful later for retrospective investigations such as the characterization of new biomarkers. And, since no two types of test have the same sensitivity, the old ones serve as convenient standards for false positive and false negative rates. Kit versions of rapid methods are interpreted differently depending on whether the results are positive or negative: negative results are considered definitive but positive results require confirmation by another test.
See index of Bacteriological Analytical Manual at:
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

Aerobial plate count (APC) [385]

The aerobic plate count (APC) is intended to indicate the level of microorganism in a product. The FDA presents conventional plate count method for examining frozen, chilled, precooked, or prepared foods for a suitable colony counting range of 25-250.
An automated spiral plate count method for the examination of foods and cosmetics, are also described at:
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/UCM063346

Microbiology Laboratory Guidebook [386]

The Guidebook contains current protocols for analytical tests required by FSIS regulatory activities on meat, poultry and egg products. Specifically, microbiological methods are presented for sample preparation, isolation and identification of the major foodborne pathogenic microorganisms and their toxins, meat tissue species identification, and the detection of extraneous materials and antimicrobial residues. Media and reagent formulations, and Most Probable Number Tables are contained in an appendix.
See index of the Gudebook at:
http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp


Genomics and Rapid Diagnostic Tests

Diagnostic medical bacteriology is based on microscopy, Gram stain, culture of microorganisms and antibiotic susceptibility testing. Molecular methods re evolving and genome sequencing has causes deep impact on the study of bacterial pathogens and tracing the source of epidemics,. Genomics may cause a revolution in diagnostic and public health microbiology. Says Pallen and Loman 2011. Genomic testing platforms will develop the rapid diagnoses of infectious. [387]

Rapid Diagnostic Tests (RDTs)

Some rapid diagnostic test have been developed to spare time and costs. However, accuracy must be determined to avoid false results. Accuracy involves sensitivity and specificity, positive and negative predictive values, or positive and negative diagnostic likelihood ratios. The Rapid Diagnostic Tests can be classified in 5 groups [388]:
Antigen detection: This group includes enzyme immunoassay and immunoproteins like protein A and clumping factor from Staphylococcus aureus, and detection of PBP2a to identify Methicillin-resistant Staphylococcus aureus (MRSA) with low protein A. [389]
Molecular detection: Molecular detection includes genomic testing methodologies, such as nucleic acid hybridization with RNA or DNA probes, amplification, polymerase chain reaction (PCR) technologies, or nucleic acid sequencing. Such PCR platforms were developed for detection of infections of MRSA, Clostridium difficile, and Neisseria gonorrhea.However, PCR is still costly and time-consuming.
Rapid biochemical tests: This includes the famous urine dipsticks for nitrite and leukocyte esterase.
Special stains: Stains for direct microscopy such as calcofluor white stain, Gram stain for detection of gram-positive diplococcic (Streptococcus pneumoniae) in a sputum smear, gram-negative diplococcic (Neisseria gonorrhoea) in a urethral smear. The Ziehl- Neelsen for Mycobacteria Blood smears stained with acridine orange, and examined with a fluorescence microscope reveal the presence of trypanosomes that cause sleeping sickness (Trypanosoma brucei) or Chagas disease (Trypanosoma cruzi).

Serologic testing

Immunoelectrophoresis: In the current era of advanced technology, it is very easy to disregard the value of basic light microscopy in the rapid diagnosis of infections. A Gram stain can confirm within minutes the presence of, or gram-negative rods in a spun specimen of urine. Mycobacteria can be identified rapidly by microscopy of specimens stained with Ziehl-Neelsen. Kinyoun, or rhodamine auromine stains. Are used to detct Giardia lamblia and Entamoeba histolytica in stuhl. India ink (nigrosin) wet mounts prepared from cerebrospinal fluid (CSF) detects encapsulated cells of Cryptococcus species.
Special culture media: selective and differential solid media for the qualitative direct detection of VRE and MRSA Methicillin-resistant Staphylococcus aureus (MRSA) [390]

Specyfic Rapid Diagnostic Tests (RDTs)

Herpes simplex virus: Real-time PCR is faster and more sensitive than previous technologies, such as cell cultures or immunofluorescence microscopy, for detecting and genotyping herpes simplex virus (HSV) in clinical specimens.

Septicemia: Molecular tests were developed to detect septicemia using whole blood specimens. Multiplex-PCR, a modification of PCR, is such a test. However, low sensitivity, difficulties to identify bacteria involved and the inability to give antimicrobial susceptibility testing information makes the broth-based blood culture indispensable. Sutherland used Affymetrix array and multiplex tandem (MT)-PCR to evaluate transcriptional profiles in circulating white blood cells applying a set of 42 molecular markers that had been identified a priori to predict sepsis. This molecular biomarker test can detect sepsis by monitoring molecular reaction of blood white cells in respond to a bacterial invasion. [391]

Tuberculosis: Diagnosis includes culture of the organism, drug-susceptibility testing, using standard biochemical analyses, nucleic acid probes, or 16S rRNA gene sequencing. Real-time PCR assays that rapidly and specifically detect Mycobacterium tuberculosis complex directly from acid-fast, smear-positive respiratory specimens and broth cultures are now routinely conducted in various reference laboratories, giving results within hours compared with the average of 2 weeks required for traditional susceptibility testing methods. Rapid tests for TB do not replace acid-fast smears or mycobacterial cultures. However, smear microscopy combined with most rapid tests improved diagnostic sensitivity from 75% (smear alone) up to 89% (smear plus rapid test) but also caused an unacceptable false positive rate of 42%. [392] DNA specific probes, hsp65 PRA and hsp65 gene sequencing are described by Esparcia et al 2011 to identify non-tuberculous mycobacteria. [393]

Clostridium difficile: Available EIA and glutamate dehydrogenase tests are easy to perform and offer results within 2 hours, but lack sensitivity. Shetty, Wren and Coen 2011 report that the glutamate dehydrogenase test to detect Clostridium difficile, yields a specificity of 80-100% with a false positivity rate around 20%, detecting toxigenic and non-toxigenic strains of the organism, and test has high sensitivity and negative predictive value when combined with a test to detect toxin. [394]

All real-time PCRs can be applied in screening for Clostridium difficile infections, but cannot be applied as stand-alone test, because of its low positive predictive value. However the test may be used to reduce the spread of the epidemic Clostridium difficile PCR ribotype 027. [395]

Influenza:Tests for influenza include rapid antigen testing, reverse transcription-PCR, Real-time PCR, and immunofluorescence assays that identify influenza A and B viral nucleoprotein antigens in respiratory specimens. Outbreaks of H1N1 influenza strains demonstrate the need for more sensitive RDTs to differentiate between influenza and other respiratory viruses. No one approach is applicable to all conditions, says Lee et al 2011, and a combination of different tests may be necessary. The authors stress that costs should not reduce quality of results on which clinical decisions depend on. [396]

The real-time reverse transcription polymerase chain reaction (rRT-PCR) technique has been used as the reference technique for the diagnosis of pandemic (H1N1)2009 virus infections. Ciblak et al 2010 compared the sensitivity of the reference rRT-PCR technique with rapid influenza diagnostics tests (RIDTs).The sensitivity of the rapid tests ranged from 31.7% to 50%, compared to 97.7% to 100% of the rRT-PCR technique.The authors stressed that available RIDTs are not sensitive enough and could lead to delay in treatment of patients. [397]

Sexually transmitted infections: Culture of Treponema pallidum is not possible. All available syphilis RDT kits use T pallidum recombinant antigens to detect treponeme specific antibodies. Non-treponemal screening tests, such as the rapid plasma reagin (RPR) test does not look for antibodies against the actual bacterium, but rather for antibodies against substances released by cells when they are damaged by T. pallidum. Accurate detection of T pallidum antibodies is carried out using lateral-flow immunoassay. Syphilis lateral flow tests become available within 30 minutes and do not require a laboratory or other instrumentation. [398]

The CDC proposed to test serum samples using a treponema-specific test and positive samples should be analyzed with a nontreponemal assay. Binnicker, Jespersen and Rollins 2011 compared the performance of different treponemal assays, fluorescent treponemal antibody assay, Treponema pallidum particle agglutination, Trep-Sure enzyme immunoassay, Trep-Chek EIA , Trep-ID EIA, Treponema ViraBlot IgG, rapid plasma reagin (RPR) assay and a treponemal IgM Western blot assay. These techniques were compared to the FTA assay as the gold standard, and data may be used to decide between these tests. [399]

MRSA and VRE: Currently, the standing screening method of detection of these pathogens is culture. Chromogenic media offer enhanced bacterial identification over tradition culture media through colour differentiation. Nearly all studies that evaluated PCR (either conventional or real-time) have shown improved sensitivities for detecting VRE from faecal specimens compared with culture.

Kilic and Basustaoglu 2011 developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles targeting tuf, nuc, mecA, atlE, gap and mvaA genes.The whole test is perfomed in 83 minutes. [400]

Rapid test using chemiluminescence differentiates between bacterial or viral infection [401]

Antibiotics are ineffective in treating viral infections. A new test based on the immune system's response to the infection, differentiates between bacterial or viral infection allowing a clinical decisions promptly upon admission. Standard bacteriological methods need 24-48 hours to provide results which sometimes are not sufficient for a diagnosis.

The test adds luminol to a blood sample of the patient. The glow is measured. Luminol is a luminescent chemical substance used in crime scenes to locate traces of blood. Polymorphonuclear leukocytes or phagocytes, responding to infections, undergo functional changes that differ between bacterial or viral origin. These changes may be assessed by chemiluminescent byproduct reaction. Luminol: Luminol exhibits chemiluminescence, with a striking blue glow, when mixed with an appropriate oxidizing agent.Luminol is used by forensic investigators to detect trace amounts of blood left at crime scenes as it reacts with iron found in hemoglobin. It is used by biologists in cellular assays for the detection of copper, iron, and cyanides, in addition to the detection of specific proteins by Western Blot. [402]

The authors claim that the method is timesaving, easy to perform and can be commercially available, and has predictive diagnostic value.

Bacteria from dog faeces are important source of airborne bacteria in cities [403]

Faecal bacteria from dog faecal material are important source of airborne bacteria in US cities, particularly in winter, says a new study. Airborne bacteria may impair human health, triggering allergic asthma and seasonal allergies.

According to Bowers et al. 2011 airborne bacterial communities of Detroit and Cleveland were found to be similar to communities found in dog excrements which are an important source of bacteria at the atmosphere of both cities. Included in this study were Chicago, Cleveland, Detroit, and Mayville, Wis. The authors used high-throughput pyrosequencing to analyze bacterial present in the PM2.5 aerosol fraction (fine particulate matter <2.5 micro mm) and compared their data against a database of bacteria from leaf surfaces, soil, and human, cow and dog faeces.

The authors stress that airborne bacteria community varied significantly. In summer, airborne bacteria come from a variety of sources including soil, dust, leaf surfaces, lakes and oceans, however in winter, leaves drop and snow covers the ground. It is when airborne bacteria from dog faecal material predominate.

The authors call for more studies to determine the types of bacteria in the air, how these bacteria vary by location and season, and where they are coming from.

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