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[108]:
Taxonomic rank:

Kingdom
Division
Class
Order
Family
Genus
Species
Subspecies

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:

Biovar or biotype have special biochemical or physiological properties.
Serovar or serotype have distinctive antigenic properties.
Pathovar or pathotype have pathogenic properties for certain hosts.
Phagovar or phagotype have ability to be lysed by certain bacteriophages.
Morphovar or morphotype have special morphological features.

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[1949]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 $^{o}C$ and below, up to 43 $^{o}C$ .
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 $^{o}C$ to 50 $^{o}C$ . Below 10 $^{o}C$ 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 $^{o}C$ to 30 $^{o}C$ 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 $^{o}C$ to 30 $^{o}C$ and ph between 5.5 to 6.0.

Acetic acid bacteria

[1]
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. [2]

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. [3]

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

Granulibacter bethesdensis a new pathogenic acetic acid bacteria

[4]
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

[5]
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

[6]
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

[7]
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

[8]
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

[9]
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

[10]
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. [11]

Gluconacetobacter diazotrophicus

[12]
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. [13]

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. [14]

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. [15]

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. [16]

Biotechnology application of Gluconobacter strains

[17]
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. [18]

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 $^{o}C$ . 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
Aeromonas caviae
Aeromonas sobria

Aeromonas	hydrophila	caviae	sobria
Esculin hydrolysis	+	+	-
Gas from glucose	+	-	+
Voges-Proskauer	+	-	V
Acid from arabinose	+	+	-

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 $^{o}C$ and a limited tolerance to acid. Bacillus strains capable of growing at temperatures of 65 $^{o}C$ 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 $^{o}C$ . The best growth temperatures for Bacillus stereatothermophilus is 55 to 60 $^{o}C$ . 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 D $_{121,1C}$ = 4 to 5 minutes are given.

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

Clostridium botulinum type A and B D $_{121,1C}$ = 0,1 up to 0,2 minutes.
Clostridium sporogenes D $_{121,1C}$ = 4 up to 5 minutes.
Clostridium thermosoccharolyticum D D $_{121,1C}$ = 3 up to 4 minutes.
Desulfotomaculum nigrificans D $_{121,1C}$ 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 $^{o}$ . Below of that there is no growth. To kill its spores F $_{121,1C}$ = 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:
$\includegraphics[width=300bp,height=230bp]{library/p1010034.ps}$
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.
$\includegraphics[width=300bp,height=230bp]{library/P7250060.ps}$

Aeromonas hydrophila
Aeromonas caviae
Aeromonas sobria
Aeromonas salmonicida Later studies using DNA-DNA hybridization has shown 12 genospecies of which 11 phenotypes were named:
Aeromonas caviae predominates in sewage
Aeromonas eucrenophila
Aeromonas hydrophila found in all types of water, rivers, lakes and sewage. A.hydrophila can be isolated from faeces of warm-blooded animals and in man with up to 11,7%.
Food such as fish, meat,milk , vegetables and tap water are very important place for A. hydrophila. Food can therefore be a source of human infection.In fresh unpolluted drinking waters Aeromonas hydrophila is predominant.
Aeromonas jandei
Aeromonas media
Aeromonas schbertii
Aeromonas sobria
Aeromonas trota
Aeromonas veronii
Aeromonas salmonicida being found in fish but not in water. When found in water they are associated with fish.

Aeromonas can be present in water with a ph from 5.2 to 9.8, growing by 10 $^{o}C$ up to 45 $^{o}C$ being 35 $^{o}C$ 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 coliforms 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[19]

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

Campylobacter: with 19 species and subspecies
Arcobacter : with 4 species
Helicobacter : 18 species

Description:

Campylobacter are Gram-negative, slender spiral curved rods. They need a micro-aerobic atmosphere to grow. The pathogenic species grow at 42 $^{o}C$ and 37 $^{o}C$ .

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.coli: 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. [489]
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 coli 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 $^{o}C$ but there is no growth at 25 $^{o}C$ .

The germ is very sensible to heat, chilling, and acids.
The resistance to heat in skimmed milk = D 55 $^{o}C$ is 1 to 3 minutes.
Resistent to chilling temperatures: after 5 to 8 days there were no bacteria alive left from a population of $10^{7}$ in skim milk at -20 $^{o}C$ .
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 $10^{3}$ an $10^{5}$ germs/g It is being told that even 50 bacteria can cause an infection. [1672]

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.C. Jejuni, C.coli 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.

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

[1673]
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- $\mu$ m-diameter range and high proportions in the 2- to 5- $\mu$ m-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 $^{o}C$ 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 $^{o}C$ for 48 hours under the atmosphere described above.

Biochemical confirm

Suspicious colonies are examined under a phase contrast microscope searching for comma formed bacteria.

No growth at 25 $^{o}C$ under microaerophylic conditions.
No growth under aerophylic conditions
No growth with 3,5% NaCl.
Resistant to cephalotin (30 microgram). Often sensible to nalidixic acid. (30 microgram)
Catalase and oxidase positive
Glucose negative
H2S from TSI negative

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[1420].

DNA based methods for the Campylobacter diagnostics

[1672] 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. [1674]

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. [1675]

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. [1676]

New Zealand produces update on work related to Campylobacter

[1677]
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" [1678] 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

[825]:

Poultry 20%
Red meat 0,5 - 5 %

Diseases caused by Arcobacter spp.

Acute diarrhea,appedicitis,septicemy and diarrhea in animals.

Culture of Arcobacter spp:

[826]
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:

[826]
28 g Brucella broth powder (Difco) in 910 ml A.dest. After sterilization and cooling at about 50-60 $^{o}C$ .
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 $^{o}C$ 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 $^{o}C$ .

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 $^{o}C$ 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 [1679].
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 $^{o}C$ . No growths at 25 $^{o}C$ , optimum at 37 $^{o}C$ . 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 $^{o}C$	+	-
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

[1680]
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

[1681]
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. [1682]

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.[1683]

The study of Fukase contradicts previous studies which found no relation between the bacterium and stomach cancer. [1684] [1685]

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

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 coli.
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. coli 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.coli, 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 $^{o}C$ .
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 $^{o}C$ microaerophile for 48 hours

Escherichia Coli

[97] 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.

Other denominations of 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 $^{o}C$ ) 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.

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

[98]
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 [99] [100]. 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

Heat food at at least70 $^{o}C$ .The bacteria are killed at this temperature and their verotoxins are denaturated.This is most important in case of risk groups such as babies, children, old people and immune weakened persons.
Avoid cross infection by keeping unheated food separated from heated food.
Use careful hygiene in storage and handling of food.
Infection with EHCE is bound to faecal contamination.Personal hygiene is most important, specially washing and disinfection of hands.About 90% of infections could be avoided with perfect handling of food.

Food spoiling

[21] 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 $^{o}C$ 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 $^{o}$ for 12 minutes in the presence of 15% moisture.

EU Regulation on the control of Salmonella and other zoonosis

[64]
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

[65] 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

[66] 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

[67] [68]

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. [2898]

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.

Affected Cadbury products:

[77]
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 $^{o}C$ and 44 $^{o}C$ , 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 $^{o}C$	+
Voges Proskauer	-
TSI Agar
inclined surface	basic (red)
bottom	sour ( yellow )
gas	negative
H $_{2}$ S	negative

Enriching culture:

Demonstrate the presence or absence of Vibrio parahaemolyticus
100 ml or g food are mixed with Broth.
Incubate at 37 $^{o}C$ 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 $^{o}C$ for 18 hours.

Kanagawa reaction:

Point inoculum of dried Wagatsuma-agar. Incubate at 37 $^{o}C$ 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 $^{o}C$ for 18 +- 2 hours.

Identification:

Inoculate the highest dilution on TCBS-agar. Incubate at 37 $^{o}C$ 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

[78]
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.

Pathogenic bacteria in Brazilian coastal waters

[79]
Ristori and colleagues 2007 analized oysters and estuarine water samples from coastal waters of Sao 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 vulnificus risk from untreated shellfish throughout the year

[80]
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

[81]
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

[82]
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.

Other pathogens from shellfisch

[83]
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

[84]
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 0,2 $\mu$ m to 1,5 $\mu$ m 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 $\mu$ m	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 psittaci
Chlamydia trachomatis
Chlamydia pneumoniae

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:

The elementary infectious reticular corpuscules
The reticular corpuscule with possibility of reproduction.

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[85][86]. 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[1687].

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 $^{o}C$ . Cells used for culture are HEp-2 cells and Chang cells with cycloheximide as antimetabolite [1688]

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[89][91]. 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[92][93][94][95]. 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[1686].

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[87]. 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[88].

Heart infarcts, clinical reports

Studies in Salt Lake ,Utah, USA[96] 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

[90] 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.

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

[880]
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

Normalize body weight
Avoid obesity
Avoid hipercholesterinemia
Avoid diabetes
Avoid high arterial pressure

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 [881].

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 $^{o}C$ 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:

Speed of biochemical reactions:
Temperature reduces the speed of biochemical reactions, the growing and the reproduction of bacteria.
Drying:
At freezing point water is not available to bacteria. There are irreversible enzyme inactivations. This leads to refrigeration death. However a great part of bacteria, survive refrigeration and can get to new activity when the temperature rises again.

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 $^{o}C$ 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 $^{o}C$ . Lipolytical activity of Pseudomonas in butter is present at -10 $^{o}C$ .

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:

Pseudomonas
Flavobacterium : Cytophaga is in Bergey's Manual of Systematic Bacteriology included in the Flavobacterium-Cytophaga complex (side 355 volume 1984).Cytophaga are found in fresh fish.

Water bacteria with contamination origin from sewage and other sources:

Enterobacteriaceae
Pseudomonas aeruginosa
Enterococcus
Clostridium
Spirillum volutans
Aeromonas
Bacillus punctatum
Aeromonas hydrophila
Arcobacter butzleri
Campylobacter jejuni
Escherichia coli enterotoxic ETC
Vibrio cholerae
Leptospira
Borrelia
Legionella

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 $^{o}C$ . 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 $^{o}C$ in December 1998 [694].

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 $^{o}C$ 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 $^{o}C$ . 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.

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 $^{o}C$ 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 $^{o}C$ . Escherichia coli should be incubated at 44+-1 $^{o}C$
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 $^{o}C$ 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.

Inculation:

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 ):

Glucuronidase -reaction is more sensitive compared with formation of gas from lactose. This increases diagnostic safety .
Reduced work and reduced costs
Reduced time, results are given in 24 to 48 hours compared with 96 to 144 hours.
Fluorogene substances are very sensitive. Activity of glucuronidase is a characteristic of 94% of all types of E.coli, Indol reaction is a characteristic 99% of all types of E.coli [881].

$\includegraphics[width=300bp,height=230bp]{library/MUG_methode.eps}$

To avoid false results please observe:

Glassware, other utensils and the material to be examinated should not have strong fluorescence as this might overdo the reaction of MUG.
Work always with control positive and control negative
Alkaline pH brings strong fluorescence.If the medium is acid add some droplets of 1N NaOH-solution to turn it alkaline
Indol reaction has to be done after the reading of fluorescence and not later than 24 hours.

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 $^{o}C$ . 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):

[881]
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 $^{o}C$ 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 $\mu$ l 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
K $_{2}$ HPO $_{4}$	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 $^{o}C$ .

Enrichment:

25 g from the sample are incubated at 35-37 $^{o}C$ 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.

$\includegraphics[width=250bp,height=350bp]{library/Impedance_technology.fig}$

Detection time [885]: 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 bioM�ieux.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 oof 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

[886]
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. [883]
There are 5 possible interaction of the bacteria with the substrate: The Impedance of the medium (E-value)

Molecules without charge and polar macromolecules are digested in smaller ions, increasing the conductivity of the medium.
Small molecules are digested in very small electrical charged parts which can move more easily increasing the conductivity.
Ions already existing in solution are united in great ions. The conductivity is diminished.
Small ions are united in electrical charged macromolecules. The conductivity is diminished.
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 Z $_{E}$ 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:

$\includegraphics[width=350bp, height=300bp]{library/Impedance.eps}$

The growth of Saccharomyces cerevisiae [883]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.

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. [726]
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

[728]
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

[727]
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.

[728]
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 $^{0}C$ . The colouring of tubes and additives are specified.

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

[729]
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. [730]

Pipe systems should also comply with the 3-A Sanitary Standard. [731]

Disinfection of drinking water

[732] 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 bacteria 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 m $^{3}$ 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

H $_{2}$ O + Cl $_{2}$ = HClO + HCl
Underchloric acid decays in chloric acid and oxygen:
2 HCLO = 2 HCl + O $_{2}$

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. [1180]
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. [1180]

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. [1180]

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. [1180]

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. [1180]

Chorine dioxide as disinfectant on produce, fresh fruits and vegetables:

[1181] 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.

How Salmonella attach to salad leaves

[1182]
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. [1183]

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

[1184]
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.

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

[1190]
The regulation 2073/2005 [1190], amended by the Regulation 1441/2007 in December 2007 [1186], lays down limits for specific bacteria in food. It was based on the following EU regulations:

Regulation (EC) No 178/2002 [69]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 [1188], 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

[1196]
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

[1197]
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. [1198]

Probiotics to phase out antibiotics in poultry breeding

[1199]
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

[1200]
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. [1201]

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. [1202]

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. [1203] [1206]

Bacteria, such as Klesiella pneumoniae develop resistance to carbapenems

[1204]
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 [1205]. KPC-resistace is most frequent in Klebsiella pneumoniae, and seems to have spread to the Enterobacteriaceae family, such as Proteus, Serratia, Salmonella, and Citrobacter.

Pigs as Source of Methicillin-Resistant Staphylococcus aureus CC398 Infections in Humans

[1207]
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

[1208]
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

[1208]

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.

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

[1209]
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:

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.

Viruses

Arboviruses:

Oropouche virus:

It is a tropical viral infection, a zoonosis similar to dengue fever, transmitted by biting midge (species Culicoides paraensis) and mosquitoes from the blood of sloths to humans. It occurs mainly in the Amazonic region, the Caribbean and Panama.

Venezuelan equine encephalitis virus:

It is a mosquito-borne viral pathogen that causes Venezuelan equine encephalitis or encephalomyelitis (VEE) can affect all equine species, such as horses, donkeys, and zebras. After infection, equines may suddenly die or show progressive central nervous system disorders. Humans also can contract this disease. Healthy adults who become infected by the virus may experience flu-like symptoms, such as high fevers and headaches. People with weakened immune systems and the young and the elderly can become severely ill or die from this disease.

Yellow fever:

It is also endemic; nonhuman primates are the principal reservoirs of the disease during its sylvatic cycle. Vaccination is an essential means of protection against yellow fever for both the local population and visitors. Aedes aegypti mosquitoes in Amazonian urban centers pose an ever-present risk for yellow fever transmission and are also responsible for the high incidence of dengue.

Viral hepatitis:

It is an acute liver disease with varying severity caused by the hepatitis A virus (HAV), contamination by fecal matter and ingestion of contaminated food or drinks.

Hepatitis B virus (HBV):

Contamination by contact with infectious blood, semen, and other body fluids from having sex with an infected person, sharing contaminated needles to inject drugs, or from an infected mother to her newborn.

Hepatitis C virus (HCV)

Contamination by contact with the blood of an infected person, primarily through sharing contaminated needles to inject drugs.

Hepatitis D (HDV) virus

Contamination by contact with infectious blood, similar to how HBV is spread.

Hepatitis E virus (HEV)

Contamination by ingestion of fecal matter, even in microscopic amounts; outbreaks are usually associated with contaminated water supply in countries with poor sanitation.

Kaposi sarcoma-associated herpesvirus ( HHV-8)

It is the eighth human herpesvirus. It causes Kaposi's sarcoma, a cancer commonly occurring in AIDS patients, as well as primary effusion lymphoma and some types of multicentric Castleman's disease.

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

[1210] 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 $^{o}C$ and a limited tolerance to acid. Bacillus strains capable of growing at temperatures of 65 $^{o}$ 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 $^{o}C$ the best growth temperature for Bacillus stearothermophilus is 55 to 60 $^{o}C$ . 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:

[1213]
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 $^{o}C$ , 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 $10^{5}$ 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 $^{o}C$ 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 D $_{121,1}$ = 4 up to 5 minutes are given.

D-values for other bacteria in order to draw a comparison:
Clostridium botulinum type A and B D $_{121,1}$ = 0,1 up to 0,2 min.
Clostridium sporogenes D $_{121,1}$ = 0,1 up to 1,5 min.
Clostridium thermosaccharolyticum D $_{121,1}$ = 3 up to 4,0 min.
Desulfotomaculum nigrificans D $_{121,1}$ = 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 $^{o}$ . Below of that there is no growth. To kill its spores F $_{121,1}$ 15 to 30 minutes must be used in case of canned meat.

Tetrahymena protozoan and Samonella and resistance to sanitizers

[1214] [1212] 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. [1214]

Protozoa on leafy vegetables increases risk of enteric diseases

[1215]
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:

[1216]
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. [1217]

Potentials of phages in food industry

[1218]
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.

[1219]
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.

There is no epidemiological association of Staphylococcus aureus between isolates from cows and humans

[1222]
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

[1220]
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

[1221]
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.

Listeria L-forms lost their membrane but are still highly pathogen

[1223]
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

[1224]
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)

[1225]
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

[1226]
It is a combined database of microbial responses to food environments. It is linked to the ComBase modelling toolbox, which includes :

ComBase Predictor: It is a set of 23 growth models and 6 thermal death models for food pathogenic and spoilage microorganisms including L. monocytogenes,
Perfringens Predictor: It is an application for predicting the growth of Clostridium perfringens during the cooling of meats.
DMFit; It is a fitting tool, for growth and inactivation curves. The access is free.

The Food Spoilage Predictor (FSP) and the Seafood Spoilage and Safety Predictor (SSSP)

[1227]
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

[1228]
Information from Sym'previus is available on a commercial basis.

The U.S. Food Protection Plan

[1229]
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:

Prevention of foodborne contamination: It aims to promote increased corporate responsibility to prevent illness. It also seeks to identify and assess vulnerabilities and expand understanding and use of mitigation measures.
Intervention at critical stages in the food supply chain: This includes focus inspections and risk-based sampling, risk-based surveillance, and better detection of signals that indicate contamination has occurred.
More rapid respond to problems: This is aimed to reduce the impact, and improve its communication on risks to the public, industry, and other stakeholders.

Listeria in ice cream under aging and storage and distribution conditions

[1230]
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 $^{o}C$ ), static freezing (-5 to -33 $^{o}C$ )

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

[1231]
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:

Exceptionally high numbers of rRNA and tRNA genes with regard to genome size , indicating recent phase of important size reduction.
A much higher GC content at codon position 3 than expected
The presence of a rare 47.5-kbp inverted repeat in the replication termination region

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

[1232]
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. [1233]

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%. [1233] Its genome size is according to Leong-Morgenthaler 2.3 Mbp. [1234]

Safety assessment for microorganisms added to food and feed

[1235]
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:

Gram-positive non-sporulating bacteria (GPNS)
Bacillus species
Yeasts
Filamentous fungi

Antimicrobial drug-resistant Escherichia coli from humans and poultry products

[1236]
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

[1237]
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 $^{o}C$ , 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 [1238].

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

[1239]
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

[1240]
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. [1241]
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

[1242]
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. [1243]

Raman spectroscopy to detect viruses

[1244]
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. [1245]

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

[1246]
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

[1247]
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

[1248]
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

[1249]
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

[1250]
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. [1251]

Symptoms of pneumonic plague

[8]
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. [1253]

New process explain the causes of the variability of individual cells in cell cultures

[1254]
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

[1255]
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

[1256]
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

[1257]
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.