Subsections

General bacteriology


General classification of bacteria related to food science

Great efforts were made to classify all living beings.There are three related fields of activities concerning taxonomy:
Nomenclature: Nomenclature provides names to the different groups.
Identification: Identification verifies if an organism belongs to an already described group.
Classification:
Classification tries to group bacteria on the basis of similarities or relationships. Bacteria are classified in[67]:
Taxonomic rank: intrasubspecific rank Subspecies may be divided into groups with special characters, the intrasubspecific ranks, which are very important in daily practical bacteriology but not part of the official nomenclature.

Intrasubspecific ranks are:


Sections

The Bergey's Manual of Determinative Bacteriology has grouped bacteria in different "sections" based on a few readily determined criteria.
The binary nomenclature was introduced in 1735 by the Swedish scientist C.von Linn� 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[1187]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$^{0}$C and below, up to 43$^{0}$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$^{0}$C to 50$^{0}$C . Below 10$^{0}$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$^{0}$C to 30$^{0}$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$^{0}$C to 30$^{0}$C and ph between 5.5 to 6.0.


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$^{0}$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 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 can be present in water with a ph from 5.2 to 9.8, growing by 10$^{0}$C up to 45$^{0}$C being 35$^{0}$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[1]

History

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

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


Family of Campylobacteraceae

Genus

Description:

Campylobacter are Gram-negative, slender spiral curved rods. They need a micro-aerobic atmosphere to grow. The pathogenic species grow at 42$^{0}$C and 37$^{0}$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. [296]
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$^{0}$C but there is no growth at 25$^{0}$C.

The germ is very sensible to heat, chilling, and acids.
The resistance to heat in skimmed milk = D 55$^{0}$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$^{0}$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. [967]

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.

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$^{0}$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$^{0}$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.
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[840].


DNA based methods for the Campylobacter diagnostics

[967] 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. [968]

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. [969]

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. [970]

New Zealand produces update on work related to Campylobacter

[971]
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" [972] 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[492]:
Diseases caused by Arcobacter spp.:
Acute diarrhea,appedicitis,septicemy and diarrhea in animals.

Culture of Arcobacter spp

Culture of Arcobacter spp.[493] is made with enrichment broth and isolation medium followed by biochemical and serological identification.

Enrichment in Arcobacter-Selective-Broth (ASB)

Composition of the broth:[493]
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$^{0}$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 [973].
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$^{0}$C. No growths at 25$^{0}$C , optimum at 37$^{0}$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$^{0}$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

[974]
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.

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: 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

[60] 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$^{0}$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

[61]
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 [62] [63]. 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

Food spoiling

[3] 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

[41] 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

[42] 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

[43] 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

[] [45]
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. []

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:

[47]
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$^{0}$C and 44$^{0}$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$^{0}$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$^{0}$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$^{0}$C for 18 hours

Kanagawa reaction
Point inoculum of dried Wagatsuma-agar. Incubate at 37$^{0}$C for 18 hours +- 2 hours. Colonies with positive haemolysis are labeled as Kanagawa positive.

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$^{0}$C for 18 +- 2 hours.

Identification Inoculate the highest dilution on TCBS-agar. Incubate at 37$^{0}$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

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 are Gram negative bacteria. They are nonmotile with reproduction intracellular. They cannot produce ATP so that they need other cells from eucariots as source of ATP.Artropodes are not hosts.
The evolution comprehends two types of cells: After the intracellular reproduction in vacuoles in the cytoplasm of the cells of humans, other mammals or birds the reticular corpuscules which can reproduce itself are liberated and can be assimilated by new host cells[48][49]. 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[976].

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 [977]

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[52][54].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[55][56][57][58]. 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[975].

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[50]. 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[51].

Heart infarcts, clinical reports

Studies in Salt Lake ,Utah, USA[59] 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

[53] 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.

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