Wednesday, July 29, 2009

Thesis of Shigella

Thesis submitted to
ONAN BIOTECH PVT.LTD
HYDERA BAD

For the award of degree of
MASTER OF BIOTECHNOLOGY



SUBMITED BY
SUSHMA GINJALA (M.Sc)



DEPARTMENT OF BIOTECHNOLOGY,
GOVERNMENT CITY COLLEGE,
HYDERABAD.






DECLARATION




I G.SUSHMA here by declare that the project report entitled “DEVELOPMENT OF A DIAGNOSTIC KIT FOR DETECTION OF FOOD BORNE PATHOGENS (Clostridium botulinum, E.coli, Campylobacter jejuni, Shigella sps, Listeria monocytogens)’’ done by me under the guidance of Mr.T. Sri Venu Madhav (M.Phil) at ONAN BIOTECH PVT. LTD,HYDERABAD. This is submitted in partial fulfillment of the requirement for the award of degree in M.Sc (Bio technology)






Signature of the guide Signature of the candidate














AKNOWLEDGEMENT




I thank Dr.Narsimha Rao N CEO for guiding me during the duration of my project wark and giving me valuable information about the basics of laboratory with a proper schedule for providing a friendly environment with my Bio Analytical Lab staff and my co-trainees.

I thank Mr.T. Sri Venu Madhav (M.Phil) of Molecular Investigations ONAN BIOTECH PVT.Ltd for giving valuable knowledge about the PCR.

I Very thankful to Dr.RAMAKRISHNA(Ph.d.) HOD, Dept of Biotechnology for his guidance and encouragement in pursuing this project.

Finally I would like to thank my parents for their moral and financial support, without which I cannot complete this valuable training successfully.



















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INTRODUCTION


1.1Taxonomic classification
1.2History
1.3Classification
1.4Description
1.5Genomic structure
1.6Metabolism
1.7LIFE CYCLE
1.8Symptoms
1.9prevention
1.10DIAGNOSIS

REVIEW OF LITARATURE

OBJECTIVES

MATERIALS AND METHODS

6.1 Polymerase chain reaction
6.2 Principle and standardization
6.3 Primer designing
6.4 Data base
6.5 Preparation of genomic DNA from Bacteria
6.6 Materials
6.7 Isolation of genomic DNA by QIAgen kit method

RESULT

7.1 Reaction 1
7.2 Reaction 2
7.3 Reaction 3
7.4 Sensitivity detection

CONCLUSION

REFERENCE

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FOOD BORNE PATHOGENS


Introduction:
Food microbiology can be divided into three focus areas; beneficial microorganisms, spoilage microorganisms, and disease causing microorganisms. Beneficial microorganisms are those used in food fermentation to produce products such as cheese, fermented meat (pepperoni), fermented vegetables (pickles), fermented dairy products (yogurt), and ethnic fermented products such as sauerkraut, idli and kimchi. In fermented products (produced by natural or control fermentation), microorganisms metabolize complex substrates to produce enzymes, flavor compounds, acids, and antimicrobial agents to improve product shelf-life and to prevent pathogens growth and to provide product attributes. Microorganisms with their enzymes also breakdown indigestible compounds to make the product more palatable and easy to digest.

In addition, the beneficial microorganisms also serve as probiotics to impart direct health benefit by modulating the immune system to provide protection against chronic metabolic diseases, bacterial infection, atherosclerosis, and allergic responses. Examples of beneficial microorganisms are Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactococcus lactis, and Pediococccus acidilactici Food spoilage microorganisms are those which upon growth in a food, produce undesirable flavor (odor), texture and appearance, and make food unsuitable for human consumption. Sometimes uncontrolled growth of many of the beneficial microorganisms can cause spoilage. Food spoilage is a serious issue in developing countries because of inadequate processing and refrigeration facilities. Examples of food spoilage microorganisms are Listeria, Salmonella, Shigella, Clostridium, Campylobacter, Brocothrix, Lactobacillus, Bacillus, Pseudomonas spp., and some molds.

The microenvironment created in a spoiled food generally discourages the growth of the pathogenic microorganisms, which are considered poor competitors.Foodborne pathogenic microorganisms when grown in a food may not alter the aesthetic quality of products and, thus may not be easy to asses the microbial safety of a product without performing multiple microbiological test
Shigella sps :

INTRODUCTION:


Shigella is a genus of Gram-negative, non-spore forming rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agents of human shigellosis, Shigella cause disease in primates, but not in other mammals. It is only naturally found in humans and apes During infection, it typically causes dysentery.

Taxonomic Classification:











History:
Shigella organisms are a group of gram-negative pathogens, which were initially recognized as the causal agents of shigellosis (also known as bacillary dysentery) in the 1890s and became an official genus in the 1950s. Each species has their own “niche”, which represents what the species main function or area is. S. dysenteriae serotype 1 causes deadly epidemics mainly in developing countries, S boydii is restricted to the Indian subcontinent, and S. flexneri and S. sonnei are prevalent in developing and developed countries, respectively. S. flexneri is also responsible for the worldwide endemic form of bacillary dysentery.
Classification:
Shigella species are classified by four serogroups:
Serogroup A: S. dysenteriae (12 serotypes)
Serogroup B: S. flexneri (6 serotypes)
Serogroup C: S. boydii (23 serotypes)
Serogroup D: S. sonnei (1 serotype)
Group A–C are physiologically similar; S. sonnei (group D) can be differentiated on the basis of biochemical metabolism assays. Three Shigella groups are the major disease-causing species: S. flexneri is the most frequently isolated species worldwide and accounts for 60% of cases in the developing world; S. sonnei causes 77% of cases in the developed world, compared to only 15% of cases in the developing world; and S. dysenteriae is usually the cause of epidemics of dysentery, particularly in confined populations such as refugee camps.

Description:
Shigella is a non spore-forming gram-negative bacterium which aids in the facilitation of intracellular pathogens. It is able to survive the proteases and acids of the intestinal tract, which allows the bacteria to infect in very small amounts, down to as few as 10 bacteria being necessary.




Genome structure:
Shigella is a non spore-forming gram-negative bacterium which aids in the facilitation of intracellular pathogens. It is able to survive the proteases and acids of the intestinal tract, which allows the bacteria to infect in very small amounts, down to as few as 10 bacteria being necessary.
The four different species of Shigella vary greatly in the genomic structure. The largest species, S. sonnei, contains 4,825,265 base pairs. S. flexneri contains 4,607,203 base pairs, S. boydii contains 4,519,823 base pairs and the smallest species, S. dysenteriae, contains 4,369,232 base pairs.

Metabolism:
Shigella pathogens use a mixed acid fermentation pathway to metabolize substrates. Products of this anaerobic pathway include ethanol, acetic acid, lactic acid, succinic acid, formic acid, and carbon dioxide.
Life Cycle
The Shigella life cycle begins with penetration of colonic mucosa. This results in degradation of the epithelium and acute inflammatory colitis in the lamina propria. This causes leakage of blood, inflammation in the colon, and mucus into the intestinal lumen.


Shigella species is the causative agent of human shigellosis. Shigella is a highly infectious disease, and it enters our body though gastrointestinal ducts with contaminated food to cause illness. The outbreak of the disease is quick, and it is usually found in areas especially with shortages of tap water or bad hygienic environment regions. Shigella spreads widely and causes large-scale infection of the areas. Most the patients are infected because of direct contact with their unwashed hands and stitch nail when shaking hands with carrier of stool fever, and indirect contact with the unclean food contaminated , and fly could also transmit virus foods. In general, an amount of 10-100 bacteria is sufficient to induce symptoms.
Typically, shigella enters our body directly though the gastrointestinal cells and release endotoxin to induce illness; pathologically, its target is colon, sigmoid colon, and rectum. Primary symptoms of shigella are diarrhea which comes along with fever, nausea, and abdominal pain. Different from typical bacteria or virus-induced diarrhea, diarrhea caused by shigellosis is more serious whether you take food or not, and it would even cause mucosal ulceration, rectal bleeding, and drastic in aggressive shigellosis. The disease can be resolved within 4-7 days to several weeks, while the mortality rate is associated with the health conditions of patients such as ages, serotypes of pathogen, and nutrient conditions.
Most patients suffering from shigella will take antibiotics, and if the disease is become serious you should request for medical treatment as soon as if you start to have mucosal ulceration or rectal bleeding symptoms. The spread of shigellosis is associated with environmental sanitation, as a result, the more crowded and unclean environment, the more possible it is to have infection, especially in jail, nursery, sanatorium, refugee camp, and among homosexuality populations. In the tropics and subtropics area, it is considered to be of regional epidemic disease. Shigella is a highly infectious disease which transmits though gastrointestinal ducts. In order to respond quickly to an outbreak of shigellosis, if you find any carrier, all of the relevant people have to be examined by the health authorities so as to spot the source of infection as early as possible to protect the health of yourself and your family.

Symptoms of Shigella:
1.Diarrhea, fever, nausea, spasm, and. Tenesmus (an urgent desire to defecate without significant production of stools)
2.Patients are suffering with mucosal ulceration, rectal bleeding in their stools, approximately 1/3 patients have watery stools.
Prevention of shigellosis:
Food fully boiled, and do not eat anything raw and contaminated food.
To drink boiled water would be the safety policy.
Frequently wash hands to prevent infection from your fingernails.
Maintain environmental sanitation where you live, and carefully keep disposal of centralized garbage, and replace the cap of the trash can.
Carefully administer stool disposal to prevent water contaminations.
Don’t leave the water-pipe away in drainage ditch to prevent water contamination. The tap water should be boiled thoroughly for safety.
If one starts to have diarrhea, mucosal ulceration, or rectal bleeding symptoms, you should receive medical treatment as soon as possible.


Diagnosis:
A stool specimen is Gram-stained to show Gram-negative rods, with no particular arrangement. Enrichment is performed by growing the organisms on Selenite-F broth. Then, since the specimen is not sterile, the use of selective plates is mandatory. XLD agar DCA agar, or HE agar are inoculated and colonies are colorless on all of them as the organism is non-lactose a fermentor. Inoculation of a TSI slant shows an alkaline slant and acidic butt with no gas or H2S production. Following incubation on SIM, the culture appears non-motile with no H2S production. Addition of Kovac's reagent to the SIM tube following growth typically indicates no indole formation (serotypes 2, 7 and 8 produce indole).
It's noteworthy that Shigella flexneri will produce acid and gas from glucose, and Shigella sonnei is mannitol and ornithine positive, and is also late lactose fermentor (ONPG positive). Some Shigella species are capable of producing indole.






Clostridum botulinum:

INTRODUCTION:

C. botulinum is a rod-shaped microorganism. It is an obligate anaerobe, meaning that oxygen is poisonous to the cells. However, they tolerate very small traces of oxygen due to an enzyme called superoxidedismutase (SOD) which is an important antioxidant defense in nearly all cells exposed to oxygen. Under unfavorable circumstances they are able to form endospores that allow them to survive in a dormant state until exposed to conditions that can support their growth. In laboratory the microorganism is usually isolated in Tryptose Sulfite Cycloserine (TSC) growth media, always in an anaerobic environment with less than 2% of Oxygen. This can be achieved by several commercial kits that use a chemical reaction to replace O2 with CO2 (E.J. GasPak System). C. botulinumis lipase negative microorganism, it grows between pH values of 4.8 and 7 and it can't use lactose as a primary carbon source, characteristics important during biochemical identification
Taxonomy of botulinum:
C. botulinum was first recognized and isolated in 1896 by Emile van Ermengem from home cured ham implicated in a botulism outbreak. The isolate was originally named Bacillus botulinus. However, isolates from subsequent outbreaks were always found to be anaerobic spore formers, so Bengston proposed that the organism be placed into the genus Clostridium as the Bacillus genus was restricted to aerobic spore-forming rod
Since 1953 all species producing the botulinum neurotoxins (types A-G) has been designated C. botulinum. Substantial phenotypic and genotypic evidence exist to demonstrate heterogeneity within the species. This has led to the reclassification of C. botulinum type-G strains to a new species Clostridium argentinense.
C. botulinum strains that do not produce a botulin toxin are referred to as Clostridium sporo genes.
The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Food-borne botulism probably has accompanied mankind since its beginning.
Botulism is today divided into 5 clinical forms: classic or food-borne botulism, infant botulism, wound botulism, hidden botulism, and inadvertent botulism. BoNTs are serologically differentiated according to their neutralization with type-specific antitoxins into seven serotypes, designated by the letters A through G. Based on the toxin type produced, C. botulinum strains are divided in groups I to IV, with groups I and II being the main human pathogens. Group I consists of proteolytic types A, B, and F, and group II consists of nonproteolytic types B, E, and F. The two groups are completely different in their phenotypical characteristics, such as temperature requirements, biochemical profile, and production of metabolites. Types C and D provoke botulism in animal species, including the avian form
Clostridium botulinum is a soil dwelling, heterotrophic bacteria which metabolizes food strictly through fermentation.

FIG: The formation of endospore in Clostridium botulinum
Classification of botulinum:
Clostridium botulinum is also separated into four groups and seven types depending on chemical and physiological characteristics, as well as susceptibility in species to the toxin.









Table: Classification of Clostridium botulinum based on Physico-chemical properties.








Over the years, Clostridium botulinum has been separated into four groups (I – IV), depending on how proteolytic the strains are as well as other physiological traits. The term proteolytic refers to the ability of the strains to hydrolyze proteins, such as egg albumen or casein. These differences within the species, signifies adaptations made by the organism, depending on its location. The proteolytic research is not the only type of research done on C. botulinum adaption.
There have also been studies on three particular resistances that spores possess –
1. Heat resistance,
2. Radiation resistance, and
3. Resistance to other agents.

1. Heat Resistance:
Generally, with food processing, heat is a key element to preservation and the destruction of bacteria. Since C. botulinum does quite enjoy the lovely habitat that improperly preserved foods provide, resistances in types A, B, and E have developed. In particularly, group I (strains A and B in this case), resistance to temperature is high. Spores in these strains can survive in temperatures in the upwards of 120 degrees Fahrenheit. This resistance to heat is largely attributed to the temperature at which the cultures are grown.
2. Radiation Resistance:
The concept of using radiation to eliminate potential dangerous bacteria is a double-edged sword. With too much radiation, the bacteria are indeed eliminated, but the food itself changes so much it tastes terrible. With too little, weak bacteria are destroyed, but stronger ones, such as C. botulinum have free reign.

3. Resistance to other other agents:
There are a number of other resistances that different types of C. botulinum possess. One example would be the resistances to detergent-chlorine complexes and iodophor compared to those in regular chlorine. In this instance, the resistance for the impure chlorine were much higher than those in regular chlorine (the chemicals still killed the bacteria – it just took longer). Another example would be the resistance that C. botulinum spores have to ultraviolet light. For an unknown reason, all strains tend to possess some sort of protection against harmful ultraviolet rays.
These resistances make C. botulinum one tough organism.
Neurotoxin types
Neurotoxin production is the unifying feature of the species C. botulinum. Seven types of toxins have been identified and allocated a letter (A-G). Most strains produce one type of neurotoxin but strains producing multiple toxins have been described. C. botulinum producing B and F toxin types have been isolated from human botulism cases in New Mexico and California. The toxin type has been designated Bf as the type B toxin was found in excess to the type F. Similarly, strains producing Ab and Af toxins have been reported.
Organisms genetically identified as other Clostridium species have caused human botulism; Clostridium butyricum producing type E toxin and Clostridium baratii producing type F toxin. The ability of C. botulinum to naturally transfer neurotoxin genes to other clostridia is concerning, especially in the food industry where preservation systems are designed to destroy or inhibit only C. botulinum but not other Clostridium species.





Indian population suffering from Clostridium botulinum:
Food borne botulism, particularly associated with Clostridium butyricum, is rare; no cases had been reported in India before this outbreak.

The term 'prevalence' of Infant botulism food poisoning usually refers to the estimated population of people who are managing Infant botulism food poisoning at any given time. The term 'incidence' of Infant botulism food poisoning refers to the annual diagnosis
rate, or the number of new cases of Infant botulism food poisoning diagnosed each year. Hence, these two statistics types can differ: a short-lived disease like flu can have high annual incidence but low prevalence, but a life-long disease like diabetes has a low annual incidence but high prevalence.

World population suffering from Clostridium botulinum: The prevalence of Clostridium botulinum type E gene in fish and fishery products of commercial importance in Finland was. The contamination level in 438 raw fish samples from intestines, surface and whole fish and 208 fish roe samples varied from 10-40% and from 4-14% respectively, depending on the fish species studied. The presence of C. botulinumin European wild freshwater fish and roe was demonstrated for the first time by isolation of the organism from PCR-positive samples. Five percent of 214 vacuum-packed and 3% of 123 air-packed fishery product samples examined at retail level were positive forthe botulinum neurotoxin type E gene. A contamination level of 10% in vacuum-packed hot-smoked whitefish was detected. The results demonstrate that C. botulinumtype E poses a serious health risk for those consuming fishery products from the Baltic Sea area.
Diagnosis:

A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins. In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected. The specific PCR products were obtained from toxigenic C. botulinumtypes A to F.A monoclonal antibody has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody.

Listeria monocytosis:

INTRODUCTION:
Listeria monocytogenes is a Gram-positive rod-shaped bacterium. It is the agent of listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects primarily pregnant women, newborns, and adults with weakened immune systems.
Listeriosis a serious disease for humans; the overt form of the disease has a mortality greater than 25 percent. The two main clinical manifestations are sepsis and meningitis. Meningitis is often complicated by encephalitis, a pathology that is unusual for bacterial infections.
Microscopically, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains. In direct smears they may be coccoid, so they can be mistaken for streptococci. Longer cells may resemble coryne-bacteria. Flagella are produced at room temperature but not at 37°C. Hemolytic activity on blood agar has been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization may be necessary to distinguish between the different Listeria species.
As Gram-positive, non-sporeforming, catalase-positive rods, the genus Listeria was classified in the family Coryne bacteriaceae through the seventh edition of of Bergey's Manual. 16S rRNA cataloging studies of Stackebrandt et al. demonstrated that Listeria monocytogenes was a distinct taxon within the Lactobacillus-Bacillus branch of the bacterial phylogeny constructed by Woese. In 2001, the Famiiy Listeriaceae was created within the expanding Order Bacillales, which also includes Staphylo coccaceae, Bacillaceae and others. Within this phylogeny there are six species of Listeria. The only other genus in the family is Brochothrix.
Description:
The retention of bacteria on food contact surfaces increases the risk of potential transmission of pathogens to food products. Exposure to pathogenic bacteria on surfaces may occur either by direct contact with contaminated surfaces or indirectly by atomization. As a group, bacteria are well adapted to life on surfaces and may have a selective advantage when attached. Listeria monocytogenes is a prominent food borne pathogen known for its ability to withstand less than favorable environments. It is ubiquitous and psychotropic in nature, making control of it especially difficult. This organism ability to attach to various food contact surfaces such as stainless steel, polypropylene, and rubber compounds is well documented. The presence of Listeria monocytogenes in RTE foods is widely believed to occur due to post process procedures rather than being due to survival during the processing itself. In the food plant, it can survive on almost every surface. In the onset of Listeria monocytogenes contamination in the processing environment, control of the organism has proven to be difficult (Smoot and Pierson, 1998). The recovery pathogenic organisms such as Listeria monocytogenes from specific food manufacturing environments strongly suggests that contamination may occur during the production of other foods in the processing plant and it may be a hygiene / cleaning protocol problem. Food borne illness could be reduced through increased efforts to prevent these organisms from attaching to food contact surfaces and improved methods for the removal of these organisms from food contact surfaces. Accurate detection and enumeration of microbial contaminants, either by use of conventional sampling techniques or novel methods employing sonication or ultrasonics, relies initially upon mechanical action to effectively dislodge and remove the contaminant from the surface. It is also imperative that the method employed also releases the microorganisms from potential devices used for subsequent recovery and cultivation. Some methods are inherently more effective at the removal from the soiled surface and recovery is optimized. Meaning, technician- to-technician variation in the sampling procedure may potentially play a significant role in the recovery and enumeration of the sampled surface. This can lead to misleading determinations of the initial cleanliness of the surface or the effectiveness of the cleaning procedure used in processing plant. Such risks play a major factor in cross contamination, which can ultimately lead to premature spoilage of food products and in certain cases, lead to food borne diseases. While many surface sampling techniques are available, their effectiveness as a reliable quantitative technique varies greatly. The purpose of the study is to make a contribution to the methodology in the field of quantitative microbiological sampling of food contact environments, using Listeria monocytogenes attached onto stainless steel. It will also consider effectiveness of experimental design, particularly monitoring for results that may not be as sensitive in enumeration as other sampling techniques and also for results that may be invalidated due to the presence of excessive, uncontrolled variability and data scattering due to technician
to technician variability.



Taxonomy:










History:
L. monocytogenes was first described by E.G.D.Murray in 1926 based on six cases of sudden death in young rabbits. Murray referred to the organism as Bacterium monocytogenes before J.H. Harvey Pirie changed the genus name to Listeria in 1940. Although clinical descriptions of L. monocytogenes infection in both animals and humans were published in the 1920s, not until 1952 in East Germany was it recognized as a significant cause of neonatal sepsis and meningitis. Listeriosis in adults would later be associated with patients living with compromised immune systems, such as individuals taking immunosuppressant drugs and corticosteroids for malignancies or organ transplants, and those with HIV infection.
It wasn't until 1981 however that L. monocytogenes was identified as a cause of food borne illness. An outbreak of Listeriosis in Halifax, Nova Scotia involving 41 cases and 18 deaths, mostly in pregnant women and neonates, was epidemiologically linked to the consumption of coleslaw containing cabbage that had been treated with L. monocytogenes contaminated raw sheep manure .Since then a number of cases of food borne Listeriosis have been reported, and L. monocytogenes is now widely recognized as an important hazard in the food industry.
Classification:
L. monocytogenes is a gram positive, non-spore forming, motile, facultative anaerobic, rod shaped bacterium. It is catalase positive, oxidase negative, and expresses a Beta hemolysin which causes destruction of red blood cells. This bacterium exhibits characteristic tumbling motility when viewed with light microscopy. Although L. monocytogenes is actively motile by means of peritrichous flagella at room temperature (20-250C), the organism does not synthesize flagella at body temperatures (370C).
The genus Listeria belongs to the Clostridium sub-branch, together with Staphylococcus, Streptococcus, Lactobacillus and Brochothrix. The genus Listeria includes 6 different species (L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seegligeri, and L. grayi). Both L. ivanovii and L. monocytogenes are pathogenic in mice, but only L. monocytogenes is consistently associated with human illness. There are 13 serotypes of L. monocytogenes which can cause disease, but more than 90 percent of human isolates belong to only three serotypes: 1/2a, 1/2b, and 4b




Escherichia coli:



INTRODUCTION:

Escherichia coli, is a Gram negative bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some, such as serotype O157:H7, can cause serious food poisoning in humans, and are occasionally responsible for costly product recalls. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, or by preventing the establishment of pathogenic bacteria within the intestine.
E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily-manipulated or duplicated through a process of metagenics, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology and microbiology.


DESCRIPTION:
E. coli was discovered by German pediatrician and bacteriologist Theodor Escherich in 1885, and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria.
A strain of E. coli is a sub-group within the species that has unique characteristics that distinguish it from other E. coli strains. These differences are often detectable only on the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity, the ability to use a unique carbon source, the ability to inhabit a particular ecological niche or the ability to resist antimicrobial agents. Different strains of E. coli are often host-specific, making it possible to determine the source of fecal contamination in environmental samples. For example, knowing which E. coli strains are present in a water sample allows to make assumptions about whether the contamination originated from a human, another mammal or a bird.
New strains of E. coli evolve through the natural biological process of mutation, and some strains develop traits that can be harmful to a host animal. Although virulent strains typically cause no more than a bout of diarrhea in healthy adult humans, particularly virulent strains, such as O157:H7 or O111:B4, can cause serious illness or death in the elderly, the very young or the immuno compromised.





HISTORY:
E. coli O157:H7—a food borne pathogen
One of hundreds of strains of the bacterium Escherichia coli. E. coli O157:H7 is an emerging cause of food borne and waterborne illness. Although most strains of E. coli are harmless and live in the intestines of healthy humans and animals, this strain produces a powerful toxin and can cause severe illness.
E. coli O157:H7 was first recognized as a cause of illness during an outbreak in 1982 traced to contaminated hamburgers. Since then, most infections are believed to have come from eating undercooked ground beef
E. coli O157:H7 was first recognized as a food borne pathogen in 1982 during an investigation into an outbreak of hemorrhagic colitis (bloody diarrhea) associated with consumption of contaminated hamburgers (Riley, et al., 1983). The following year, Shiga toxin (Stx), produced by the then little-known E. coli O157:H7, was identified as the real culprit.
In the ten years following the 1982 outbreak, approximately thirty E. coli O157:H7 outbreaks were recorded in the United States (Griffin & Tauxe, 1991). The actual number that occurred is probably much higher because E. coli O157:H7 infections did not become a reportable disease (required to be reported to public health authorities) until 1987 (Keene et al., 1991 p. 60, 73). As a result, only the most geographically concentrated outbreaks would have garnered enough attention to prompt further investigation (Keene et al., 1991 p. 583). It is important to note that only about 10% of infections occur in outbreaks, the rest are sporadic.
The CDC has estimated that 85% of E. coli O157:H7 infections are food borne in origin (Mead, et al., 1999). In fact, consumption of any food or beverage that becomes contaminated by animal (especially cattle) manure can result in contracting the disease. Foods that have been sources of contamination include ground beef, venison, sausages, dried (non-cooked) salami, unpasteurized milk and cheese, unpasteurized apple juice and cider (Cody, et al., 1999), orange juice, alfalfa and radish sprouts (Breuer, et al., 2001), lettuce, spinach, and water (Friedman, et al., 1999).
Shiga toxin is one of the most potent toxins known to man, so much so that the Centers for Disease Control and Prevention (CDC) lists it as a potential bioterrorist agent (CDC, n.d.). It seems likely that DNA from Shiga toxin-producing Shigella bacteria was transferred by a bacteriophage (a virus that infects bacteria) to otherwise harmless E. coli bacteria, thereby providing them with the genetic material to produce Shiga toxin.
Although E. coli O157:H7 is responsible for the majority of human illnesses attributed to E. coli, there are additional Stx-producing E. coli (e.g., E. coli O121:H19) that can also cause hemorrhagic colitis and post-diarrheal hemolytic uremic syndrome (D+HUS). HUS is a syndrome that is defined by the trilogy of hemolytic anemia (destruction of red blood cells), thrombocytopenia (low platelet count), and acute kidney failure.





Escherichia Coli infection:
Escherichia coli is commonly responsible for urinary tract infections, bacteraemia, neonatal meningitis and peritoneal and biliary infections.
Four main categories of E. Coli produce diarrhea in humans:
1.Enteropathogenic (childhood diarrhea): Primarily affects children less than 2 years of age. This is caused by the bacteria attaching itself to the cells of the intestines and causing destruction of the cells and diarrhea as a result.
2.Enterotoxigenic (cholera): Produces a toxin mediated diarrhea that is indistinguishable from severe cholera, traveler's diarrhea and maybe childhood diarrhea.
3.Enterohemorrhagic: produces a shigellalike toxin that causes bloody diarrhea and colitis.
4.Enteroinvasive (bacillary dysentery): Produces a disease similar to that produced by Shigellaorganisms.

















Campylobacter jejuni:







Introduction:
The genus Campylobacter, (meaning 'twisted bacteria') first discovered in 1963describes Gram-negative, spiral, microaerophilic bacteria. Motile, with either uni- or bi-polar flagella, the organisms have a characteristic spiral/corkscrew appearance (see photo) and are oxidase-positive. Campylobacter jejuniis now recognized as one of the main causes of bacterial foodborne disease in many developed countries. At least a dozen species of Campylobacterhave been implicated in human disease, with C. jejuni and C. coli the most common.C. fetus is a cause of spontaneous abortions in cattle and sheep, as well as it is an opportunisitic pathogen in humans.

History:
Awareness of the public health implications of Campylobacterinfections has evolved over more than a century. In 1886, Escherich observed organisms resembling Campylobactersin stool samples of children with diarrhea. In 1913, McFaydean and Stockman identified Campylobacters(called related Vibrio) in fetal tissues of aborted sheep .In 1957, King described the isolation of related Vibrio from blood samples of children with diarrhea, and in 1972, clinical microbiologists in Belgium first isolated Campylobactersfrom stool samples of patients with diarrhea .The development of selective growth media in the 1970s permitted more laboratories to test stool specimens for Campylobacter. Soon Campylobacterspp. were established as common human pathogens. Campylobacter jejuniinfections are now the leading cause of bacterial gastroenteritis. In 1996, 46% of laboratory-confirmed cases of bacterial gastroenteritis reported in the Centers for Disease Control and Prevention. Department of Agriculture/Food and Drug Administration Collaborating Sites Foodborne Disease Active Surveillance Network were caused by Campylobacterspecies. Campylobacteriosis was followed in prevalence by salmonellosis (28%), shigellosis (17%), and Escherichia coli O157 infection (5%)
Description:
Campylobacter jejun iis a species of curved, rod-shaped, non-spore forming, Gram-negative micro aerophilic, bacteria commonly found in animal feces. It is one of the most common causes of human gastroenteritis in the world. Food poisoning caused by Campylobacter species can be severely debilitating but is rarely life-threatening. It has been linked with subsequent development of Guillain-Barré syndrome (GBS), which usually develops two to three weeks after the initial illness.
C. jejuni is commonly associated with poultry and naturally colonises the GI tract of many bird species. It has also been isolated from wombat and kangaroo feces, being a cause of bushwalkers' diarrhea. Contaminated drinking water and unpasteurized milk provide an efficient means for distribution. Contaminated food is a major source of isolated infections, with incorrectly prepared meat and poultry normally the source of the bacteria.
Infection with C. jejuni usually results in enteritis, which is characterised by abdominal pain, diarrhea, fever, and malaise. The symptoms usually persist for between 24 hours and a week, but may be longer. Diarrhea can vary in severity from loose stools to bloody stools. No antibiotics are usually given as the disease is self-limiting, however, severe or prolonged cases may require ciprofloxacin, erythromycin, azithromycin or norfloxacin. 90% of cases respond to ciprofloxacin treatment. Fluid and electrolyte replacement may be required for serious cases.
The first full-genome sequence of C. jejuni was performed in 2000 (strain NCTC11168 with a circular chromosome of 1,641,481 base pairs).
Genome:
The genomes of several Campylobacter species have been sequenced, providing insights into their mechanisms of pathogenesis. The first Campylobacter genome to be sequenced was C. jejuni, in 2000.
Campylobacter species contain two flagellin genes in tandem for motility, flaA and flaB. These genes undergo intergenic recombination, further contributing to their virulence. Non-motile mutants do not colonize.
Diagnosis:
Campylobacteriosis is an infection by campylobacter. The common routes of transmission are fecal-oral, person-to-person sexual contact, ingestion of contaminated food or water, and the eating of raw meat. It produces an inflammatory, sometimes bloody, diarrhea, periodontitis or dysentery syndrome, mostly including cramps, fever and pain. The infection is usually self-limiting and in most cases, symptomatic treatment by reposition of liquid and electrolyte replacement is enough in human infections. The use of antibiotics, on the other hand, is controversial.

Cause:
The exact cause of campylobacteriosis has not been clarified yet, but seem to involve multiple mechanisms. One mechanism is invasion into epithelial cells of the gut, which is generally low, but damages these cells. The sites of tissue injury include the jejunum, the ileum, and the colon. Most strains of C jejuni produce a toxin (cytodistending toxin) that hinders the cells to divide and activate the immune system. This helps the bacteria to evade immune system and survive for a limited time in the cells. For a while, it was thought that also a cholera-like enterotoxin was made, but this appeared not to be the case. The organism produces diffuse, bloody, edematous, and exudative enteritis. In a small number of cases, the infection may be associated with hemolytic uremic syndrome and thrombotic thrombocytopenic purpura through a poorly understood mechanism.
Pathogenesis:
The pathogenesis of C. jejuni infection involves both host- and pathogen-specific factors. The health and age of the host and C. jejuni-specific humoral immunity from previous exposure influence clinical outcome after infection. In a volunteer study, C. jejuni infection occurred after ingestion of as few as 800 organisms. Rates of infection increased with the ingested dose. Rates of illness appeared to increase when inocula were ingested in a suspension buffered to reduce gastric acidity .
Many pathogen-specific virulence determinants may contribute to the pathogenesis of C. jejuni infection, but none has a proven role) Suspected determinants of pathogenicity include chemotaxis, motility, and flagella, which are required for attachment and colonization of the gut epithelium Once colonization occurs, other possible virulence determinants are iron acquisition, host cell invasion, toxin production, inflammation and active secretion, and epithelial disruption with leakage of serosal fluid .

Survival in the Environment :
Survival of C. jejuni outside the gut is poor, and replication does not occur readily) C. jejuni grows best at 37°C to 42°C, the approximate body temperature of the chicken (41°C to 42°C). C. jejuni grows best in a low oxygen or microaerophilic environment, such as an atmosphere of 5% O2, 10% CO2, and 85% N2. The organism is sensitive to freezing, drying, acidic conditions (pH < 5.0), and salinity.





REVIEW OF LITERATURE :
Shigella sps:


In the year 2007 a research was conducted to develop a rapid and simple multiplex polymerase chain reaction (PCR) method which discriminates extended-spectrum beta-lactamases (ESBLs) genes in sporadic Shigellaisolates from 1998 to 2007 in Hangzhou city, China. After ESBLs screening according to the Clinical and Laboratory Standards Institute (CLSI) method, CTX-M, TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification. Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigellaisolates. With similar trend of national surveillance data, the proportion of sporadic Shigellaisolates harbouring ESBLs genes might probably be on increase.(Wng H Q etal)


In the year 2009 a research was conducted a rapid and sensitive method based on microchip capillary electrophoresis with condition optimization of genetic algorithm-support vector regression (GA-SVR) was developed and applied to simultaneous analysis of multiplex PCR products of four foodborne pathogenic bacteria. Four pairs of oligonucleotide primers were designed to exclusively amplify the targeted gene of Vibrio parahemolyticus, Salmonella, Escherichia coli (E. coli) O157:H7, Shigellaand the quadruplex PCR parameters were optimized. At the same time, GA-SVR was employed to optimize the separation conditions of DNA fragments in microchip capillary electrophoresis. The proposed method was applied to simultaneously detect the multiplex PCR products of four foodborne pathogenic bacteria under the optimal conditions within 8 min. The levels of detection were as low as 1.2 x 10(2) CFU mL(-1) of Vibrio parahemolyticus, 2.9 x 10(2) CFU mL(-1) of Salmonella, 8.7 x 10(1) CFU mL(-1) of E. coli O157:H7 and 5.2 x 10(1) CFU mL(-1) of Shigella, respectively. The relative standard deviation of migration time was in the range of 0.74-2.09%. The results demonstrated that the good resolution and less analytical time were achieved due to the application of the multivariate strategy. This study offers an efficient alternative to routine foodborne pathogenic bacteria detection in a fast, reliable, and sensitive way.(Zheng B etal)

In the year 2009 a research was conducted Foodborne diseases due to well-recognized pathogens have emerged as an important and growing public health problem with a significant impact on health. Molecular methods for subtyping these microorganisms have become a valid adjunct to the traditional techniques employed in most laboratories. One such molecular technique for the detection and In identification of food pathogens is pulsed-field gel electrophoresis (PFGE). This method separates large DNA molecules by the use of an alternating electrical field, such that greater size resolution can be obtained when compared to normal agarose gel electrophoresis. PFGE is often employed to track pathogens, such as Salmonella, Shigella, Escherichia coli (including O157), Campylobacter, and Listeriaspecies through the food chain. The contour-clamped
homogeneous electric field (CHEF) PFGE system is considered to be the gold standard for use in epidemiological studies of these organisms.(Health protection Agency ,UK)


In the year 2009 a research was conducted Laboratory-acquired infections due to a wide variety of bacteria, viruses, fungi, and parasites have been described. Although the precise risk of infection after an exposure remains poorly defined, surveys of laboratory-acquired infections suggest that Brucella species, Shigellaspecies, Salmonella species, Mycobacterium tuberculosis, and Neisseria meningitidis are the most common causes. Infections due to the bloodborne pathogens (hepatitis B virus, hepatitis C virus, and human immunodeficiency virus) remain the most common reported viral infections, whereas the dimorphic fungi are responsible for the greatest number of fungal infections. Because of the increasing attention on the role of the laboratory in bioterrorism preparation, I discuss the risk of laboratory-acquired infection with uncommon agents, such as Francisella tularensis and Bacillus anthracis. Physicians who care for a sick laboratory worker need to consider the likelihood of an occupationally acquired infection while advising exposed laboratory workers about postexposure prophylaxis. In addition, physicians should be aware of the importance of alerting the laboratory if infection with a high-risk agent is suspected (singh K)

In the year 2009 a research was conducted to develop a new, rapid and accurate reverse dot blot (RDB) method for the detection of intestinal pathogens in fecal samples. The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum, Bacillus cereus, Clostridium perfringens, Vibrio parahaemolyticus, Shigellaspp., Yersinia enterocolitica, Vibrio cholerae, Listeriamonocytogenes and Staphylococcus aureus. The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay. The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%. The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.(xing JM etal)


In the year 2009 a research was conducted Two glycopeptide chimeras corresponding to the Shigellaflexneri Y O-polysaccharide and its peptide mimic were designed in an attempt to improve the binding affinity by increasing the entropy of binding relative to the original octapeptide mimic of the O-polysaccharide. The design was based on the X-ray crystal structures of a monoclonal antibody SYA/J6 in complex with its cognate ligands, a pentasaccharide corresponding to the S. flexneri Y O-polysaccharide and the octapeptide mimic, MDWNMHAA. Both chimeric molecules consist of a rhamnose trisaccharide linked through an alpha- or beta-thioglycosidic linkage to a MDW moiety in which the W unit has been modified. We predicted that omission of the NMHAA moiety would obviate the bound water molecules that provided complementarity with the antibody-combining site, and the conformational restriction resulting from imposition of an alpha-turn at the C-terminus of the peptide. The glycopeptides were then docked into the active site of SYA/J6 using the program autodock 3.0, and the structures were optimized. The best models obtained in each case showed that the chimeric molecules, with either an alpha- or beta-thioglycosidic linkage, might be reasonable surrogate ligands for the antibody. We report here the synthesis of the alpha-glycopeptide employing solution and solid-phase strategies. Immunochemical characterization indicated that the alpha-glycopeptide unfortunately did not inhibit binding of SYA/J6 to the S. flexneri Y lipopolysaccharide(hossany BR etal).

In the year 2009 a research was conducted Gram-negative bacterial foodborne pathogens are a worldwide cause of morbidity and mortality. The ability to carry out epidemiological investigations to determine the primary sources of bacterial contamination is important to improve public health. Multiple methods are available for bacterial source tracking and to determine the distribution of pathogens isolated from sick patients. The molecular based typing methods available fall into three general categories: those based on restriction analysis of the bacterial DNA; those based on polymerase chain reaction (PCR) amplification of particular genetic targets; and those based on the identification of DNA sequence polymorphisms. The techniques that are examined in this review include: plasmid analysis, restriction fragment length polymorphism methods, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, PCR-based genotyping, and variable number of tandem repeat analysis, multilocus sequence typing, and single nucleotide polymorphism analysis. These methods are described along with a discussion of the strengths and weaknesses of the techniques for genotyping the major Gram-negative foodborne pathogens--Campylobacterspp., Salmonella enterica, Shigella(foley SL etal)


In the year 2009 a research was conducted Studies of microbial pathogens and the toxins they produce are important for determining the mechanisms by which they cause disease and spread throughout a population. Some bacteria produce secretory enterotoxins (such as cholera toxin or the heat-labile or stable enterotoxins produced by Escherichia coli) that invade cells directly. Others invade cells or produce cytotoxins (such as those produced by Shigella, enteroinvasive E coli, or Clostridium difficile) that damage cells or trigger host responses that cause small or large bowel diseases (such as enteroaggregative or enter pathogenic E coli or Salmonella). Viruses (such as nor viruses and rotaviruses) and protozoa (such as Cryptosporidium, Guardia, or Endameba histolytic) disrupt cell functions and cause short- or long-term disease. Much epidemiologic data about these pathogens have been collected from community- and hospital-acquired settings, as well as from patients with travelers or persistent diarrhea. These studies have led to practical approaches for prevention, diagnosis, and treatment (pawlowski SW etal).

In the year 2009 a research was conducted Bromelia pinguin L. is a plant native to Sinaloa, Mexico, where its fruit is used as food or as a phytotherapeutic agent. The fruits of B. pinguin were characterized and they could be considered as a functional food. These fruits show an average weight of 13.7 g and a yellow color of high luminosity (b* = 43.2, L* = 74.5). The values for acidity (4.6%, as citric acid) and pH (3.7) of B. pinguin fruit are similar to those of citrics. The edible portion is characterized by a high content of vitamin C (126 mg/100 g), ash (10.6 g/100 g d.w.), crude fiber (3.4 g/100 g d.w.), calcium (1290 mg/100 g d.w.), magnesium (500 mg/100 g d.w.), manganese (2.95 mg/100 g d.w.) and it is a good source of zinc (2.8 mg/100 g d.w.). Polar fractions extracted from the pulp fruit showed activity against several genera of human pathogenic-bacteria (Staphylococcus, Streptococcus, Enterococcus, Salmonella, Shigella, Escherichia and Pseudomonas). (pio-leon JF etal)

In the year 2009 a research was conducted Shigelladysenteriae Type 1 dysentery is a major cause of morbidity and mortality in children from less developed and developing countries. The present study explores the hypothesis that lactobacilli protect the host cell during S. dysenteriae Type 1 infection and its mechanism of action. Caco-2 cells incubated for 1h with Lactobacillus rhamnosus or Lactobacillus acidophilus at the multiplicity of infection of 100, either alone or in combination followed by addition of Shigellaat the same multiplicity of infection for 5h served as treatment groups. Cells incubated with Shigellawithout lactobacilli addition served as infected cells. At the end of experimental period, cells were processed suitably to enumerate adherent and internalized Shigella.

In the year 2009 a research was conducted Results of a study conducted to assess the degree of airborne microbial contamination generated by a wastewater treatment plant (WWTP) with bioreactor "BIO-PAK" closed treatment system and evaluation of the dispersion of potential pathogens are described. Over the year aerosol samples were collected simultaneously with sedimentation and impact methods from several plant sites and the surroundings. External upwind sites were used as control. Total colony-forming counts of heterotrophic psychrophilic, psychrotrophic, mesophilic, haemolytic bacteria, as well as members of the Enterobacteriaceae family, Staphylococcus, Enterococcus and Pseudomonas genera, actinomycetes and fungi (moulds, yeasts and yeast-like fungi) were determined.

Clostridium botulinum:
Hocking, A.D. et al. (1997). Food borne Microorganisms of Public Health Significance. 5th ed. North Sydney. AIFST NSW Branch Food Microbiology Group.
Doyle, M.P. (1989). Food borne Bacterial Pathogens. Marcel Dekker; New York.
















Listeria monocytosis:
Characteristics of the organism:
The genus Listeriahas six species currently recognized: L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. grayi). Listeria species are Gram positive, non-spore forming short rods (0.5 x 1-2 µm) occurring singly or in short chains that are widely distributed in the environment. It is catalase positive, oxidase negative, and shows β-hemolysis on blood agar. Listeriaspecies are characterized as being motile, facultative anaerobes which prefer microaerophillic conditions. Listeria monocytogenes grows well on nutritionally rich media such as brain heart infusion agar (BHIA) and tryptic soy agar (TSA). It can propagate up to 10% NaCl, however, L. monocytogenes is able to survive in salt concentrations as high as 25% at 4°C . Listeria monocytogenes is psychrotrophic in nature. It can grow at temperatures ranging from 2°C to 45°C, but the optimum temperature range is 30-37°C. Listeria grown at 37°C show little or no motility but tumbling motility can be detected by the incubation of cultures at approximately 20°C
with motile organisms having peritrichous flagellae . Listeria may swim with 1 to 5 flagella to invade human cells . Its ability to survive and grow at low temperatures in food products and food contact surfaces provides an advantageous niche from other food contaminant species. Once present in the processing facility, L. monocytogenes has been hard to control. Food facilities and processing plants that operate under low temperatures may be more susceptible to the presence of L. monocytogenes. Although refrigeration conditions and high osmotic strength environments are strategies commonly used to control bacteria in ready-to-eat foods, the conditions mentioned provide an advantage for survival and growth Listeria monocytogenes over other micro-flora. L. monocytogenes can also grow at a relatively wide pH range, from 4.8 to 9.6. It has been documented to survive for long durations of time at a pH of 5 in such products as cheddar cheese.
Distribution:
Listeria monocytogenes is ubiquitous in nature, and can be found in water samples, soil, feces, as well as plants. Moreover, it can be found on a wide spectrum of foods, with it having being associated with fresh produce, poultry, and dairy products and it has been isolated in various animals. It has been found in 37 species of mammals and 17 species of birds. Listeria have been identified in fish, shellfish, and molluscans as well. Through the FSIS Nationwide Sampling Programs, prevalence of Listeria monocytogenes have been reported as 30.5% for raw ground turkey, 41.5% for raw ground chicken, 11.7% for raw ground beef, 5.9% for turkey carcasses, 15% for broiler chicken carcasses, 7.4% for market hog carcasses, and 11.3% for cow/bull carcasses.
Listeriosis:
Listeriosiswas initially recognized as a disease of animals following the initial isolation and description by J. Pirie in 1925 and Murray and colleagues in 1926, who were working independently of each other. In the 1980s, however, it was described as a food borne human disease that fueled research of Listeria monocytogenes as a food borne pathogen. Infection may present itself as meningitis, sepsis, or more rarely, encephalitis. Listeriosisis also of great concern due to the possible onset of abortion of the fetus (50% of cases) in pregnant women. AIDS patients have an estimated 100-500 fold increased risk over the normal population . Other patients that are immunocompromised, such as those who have had organ transplants, diabetes, chronic liver disease, renal disease, and cancer patients undergoing chemotherapy have an elevated risk of Listeriosisthan the normal population. Although food borne Listeriosishas a low incidence rate, it is of great concern due to the high case fatality rate ñ at the low end of the spectrum to be about 20% to the mortality rate being suggested to be as high as 50%. The infective dose of L. monocytogenes is unknown, but is believed to vary with the strain type and overall susceptibility of the host. From cases contracted through raw or under pasteurized milk, it is presumable that fewer than 1000 total organisms may cause disease in susceptible populations. How ever due to lack of reporting for typical foodborne illnesses, the incidence rate may be much higher. The onset of symptoms of Listeriosismay be as short as one day to as long as 70 days for some patients. The Center for Food Safety and Applied Nutrition of the FDA states, ìThe onset time to serious forms of Listeriosisis unknown but may range from a few days to three weeks. The onset time to gastrointestinal symptoms is unknown but is probably
greater than 12 hours. Host susceptibility plays a key role in Listeriosisas well as the bacteria virulence. Abnormalities in T-cell immunity are clearly associated with an increased risk of
listeriosis. Incidences as described above reflect this and the age of the potential host weigh in as a factor for susceptibility as well. The immunosuppression associated with pregnancy and the multiple immune defects of even normal newborn for being among the highest at risk population reinforce this concept. Listeriamonocytogenes associated gastroenteritis. Non-invasive Listeriosiscaused by ListerIa monocytogenes associated gastroenteritis has also been described. It affects healthy individuals and usually presents symptoms more than 12 hours after the ingestion of the contaminated food. Symptoms of L. monocytogenes associated gastroenteritis are primarily those associated with gastrointestinal illness: chills, diarrhea, headache, abdominal pain and cramps, nausea, vomiting, fatigue, and myalgia. A variety of foods have been implicated as the vehicle of infection. A high potential for underreporting listerial gastroenteritis exist because symptoms are mild, and it is more likely that foodborne diseases in general often go unreported by the sufferer. Data is not currently available through foodborne surveillance mechanisms such as Food Net to record the incidence of listeria gastroenteritis since routine stool cultures do not include evaluation for Listeria monocytogenes, nor do other parts of the world report gastroenteritis from exposure to the pathogen (FDA, 2005; Makino, et al. 2005). Gastrointestinal and other mild symptoms were reported in individuals with no known underlying predisposition, and in examining the product that caused the disease, there was evidence of very high levels of food contamination.

Pathogenesis:
An infective dose of Listeriamonocytogenes most commonly enters the body through consumption of contaminated food. The bacteria are capable of entering the host cell, evading the host cell ís defense mechanism, multiplying within the cytoplasm, and spreading between the cells. Though the mechanisms of intracellular multiplication have been identified, the means for bacterial adherence to cells are still of great interest. It has been proposed that a cellular surface protein of Listeriamonocytogenes interacts with receptors in the intestinal lining and induces phagocytosis of the bacterium. Movement within the host cytoplasm follows envelopment of the bacterium by polymerizing act in filaments from the host cytoskeleton to propel them towards the host cell membrane which they push on and create elongated protrusions into neighbouring cells. Other bacteria such as ShigellaFlexner and Rickettsia rickettsiae use similar methods of evasion of immune response phagocytic cells and infection of the host body (Brooks et al., 1998). Although there are six species of Listeria, only L. monocytogenes is highly regarded as a foodborne pathogen; and within the species, pathogenicity in man has been correlated to a few distinct serotypes


Food borne transmission:

The general consensus in the scientific community is that the consumption of contaminated foods is the principle route of transmission for listeriosis, although rarely Listeriosiscan be transmitted through direct contact with infected animals or cross infection during the neonatal period.Most cases of Listeriosisoutbreaks are attributed to foods that were generally designed as read-to-eat (RTE) foods. RTE food products are routinely heat treated and receive a high degree of processing. The presence of Listeriamonocytogenes in RTE foods is widely believed to occur due to post process procedures rather than being due to survival during the processing itself. Food items that have been linked to the outbreak of Listeriosisinclude dairy, soft cheeses, coleslaw; meat products such as undercooked poultry, uncooked hotdogs and deli meats; seafood; and vegetable based products. The recovery of the strains from specific food
manufacturing environments further solidify suggestions that contamination is highly likely to occur during the production of other foods in the processing plant and it may be a hygiene / cleaning protocol problem.

Presence in food and food processing environment :
Of those cases, 58 of the cases were among mother-infant pairs. Twenty-nine deaths occurred with eight neonatal deaths, 13 stillbirths, and eight non-neonatal deaths. The consumption of Mexican style soft cheese was implicated in the illness. Resultant of the outbreak, the manufacturer instituted a voluntary recall of the implicated cheese products. Television, radio, and newspaper announcements were made warning the public against ingestion of cheeses manufactured in the Jalisco plant.. Seven fatalities and three stillbirths were resultant from the outbreak.

Bacterial attachment to food contact surfaces :
The presence of pathogenic bacteria on food contact surfaces may present great health risks, since as few as ten colony forming units (CFUs) of certain pathogens can lead to life-threatening infections. The initial aggregation and formation of surface contaminants such as dental plaques and biofilms is the attraction and adherence of the bacteria onto a surface. Cellular surface components such as fimbriae, pili or outermembrane polysaccharide layers are used by the bacteria in attaching to the surface as well as to other cells. Compared to the bacterial cell membrane, the structures are relatively fragile. Attached bacteria on a surface produce extracellular polymeric substances , that plays an important role in initial attachment as well as facilitate the formation of biofilms by providing a firm anchorage to solid surfaces. Once formed, biofilms are highly resistant to disinfectants and other cleaning agents. Bacterial attachment and biofilm formation on stainless steel pipes, vessels, valves, tables, utensils or other equipment in contact with food products impose serious trepidations to the food processing industry (Arnold and Bailey, 2000). Many factors influence the adhesion of a microorganism. Attachment may be affected by the structural and physiological characteristics of the cell as well as the nature and temperature of the suspension. The physical and chemical properties of contact surface, such as geometry, porosity, roughness, composition, and hydrophobicity dictate the strength of adhesion. Attachment of microorganisms to surfaces is metabolically favorable due to the concentration of organic molecules being more concentrated at the interface. After attachment, the microorganismís exocellular polysaccharides provide means for it to transition to irreversible attachment. This transition occurs very rapidly on surfaces such as stainless steel, with irreversible attachment being reported to start occurring in less than a minute, and increasing with the elapse of time. The type of bacteria, its adherence strength, and the bacterial concentration on the surface varies with the type of surface. The type of surface is divided into two categories; one being high surface energy materials that are hydrophilic often carrying a negative charge such as glass, minerals, and metals; and the other type being low surface energy that shows hydrophobicity with low charge (either positive or negative) such as plastics and rubber surfaces. Smoot and Pierson (1998) noted that the growth conditions prior to exposure influenced the level of attachment occurred. They concluded that exposing cells of L.

E.coli:

CHARECTERS OF ORGANISM:
Infection with E. coli O157:H7 presents with a wide spectrum of clinical manifestations, including asymptomatic carriage, nonbloody diarrhea, hemorrhagic colitis, the hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. Not only is E. coli O157:H7 an important agent for hemorrhagic colitis, it is also one of the leading causes of bacterial diarrhea. Patients at extremes of age have an increased risk for infection and associated complications.

TRANSMISSION OF E.coli:
Transmission of E. coli O157:H7 is primarily food-borne. Undercooked meat is the most common culprit, and secondary person-to-person spread is also important. The organism produces at least two Shiga-like toxins that differ antigenically, physicochemically, immunologically, and in their biological effects. These toxins are thought to have direct pathogenic significance in E. coli O157:H7 infection.

Pathogenesis:
This infection is usually diagnosed from a positive stool culture, from the presence of Shiga-like toxins, or both. Timely collection (within 7 days of illness onset) of a stool sample for culture is imperative for a high recovery rate. Treatment is primarily supportive and includes the management of complications as necessary. Antibiotic therapy has not been proved beneficial. Important public health measures include educating the public on the danger of eating undercooked meat, increasing physician awareness of E. coli O157:H7 infection, and mandating case reporting.
A comprehensive review was conducted of a preliminary farm-to-table process risk assessment of the microbial pathogen E. coli O157:H7 in ground beef prepared by department of Agriculture. The risk assessment comprises an examination of the occurrences and levels of the pathogen at points along the farm-to-table continuum and an attendant empirical model. The review included evaluations of the overarching logical structure of the risk assessment and the model, the validity and appropriateness of all input data used, the reasonableness of the assumptions made in the assessment and the anchoring approach that was taken, and the model's mathematics and equations.
Escherichia coli are commensal bacteria that can account for up to 1% of the bacterial population of the gut. Ruminant animals are reservoirs of the pathogenic bacteria E. coli strain O157:H7, and approximately 30% of feedlot cattle shed E. coli O157:H7. Feedlot and high-producing dairy cattle are fed high grain rations in order to increase feed efficiency. When cattle are fed high grain rations, some starch escapes ruminal microbial degradation and passes to the hindgut where it undergoes fermentation. Ten years ago researchers demonstrated that populations of total E. coli were higher in grain-fed than in forage-fed cattle, and when cattle were abruptly switched from a high grain diet to an all hay diet, total E. coli populations declined 1000-fold within 5 days and reduced the ability of the surviving E. coli to survive an acid shock mimicking passage through the human gastric stomach.







Campylobacter jejuni:


Campylobacter jejuni: molecular biology and pathogenesis;


Campylobacter jejuniis a foodborne bacterial pathogen that is common in the developed world. However, we know less about its biology and pathogenicity than we do about other less prevalent pathogens. Interest in C. jejuni has increased in recent years as a result of the growing appreciation of its importance as a pathogen and the availability of new model systems and genetic and genomic technologies. C. jejuni establishes persistent, benign infections in chickens and is rapidly cleared by many strains of laboratory mouse, but causes significant inflammation and enteritis in humans. Comparing the different host responses to C. jejuni colonization should increase our understanding of this organism.

Bacteriology and ecology

Campylobacterwas first described in 1880 by Theodore Escherich
The name Campylobacteris derived from the Greek word “kampylos,” which means curved.
Campylobacterare Gram negative, slender, spiral curved rods having dimensions of 0.2 _m to 0.8 _m wide and 0.5 _m to 5_m long. Extremely rapid, darting, reciprocating motility can be seen with a phase contrast microscope, with comma-shaped, S, or gull wing–shaped cells.
As Campylobactercells begin to age, they become coccoid in shape (Moran and Upton 1987). Several investigations have shown an association between the transition from the spiral to coccoid morphology with a nonculturable state (Moran and Upton 1986; Rollins and Colwell 1986; Jones and others 1991; Stern and others 1994).
Recent studies, however, suggest no correlation between culturability and cell morphology (Medema and others 1992; Hazeleger and others 1995; Lazaro and others 1999).
There are mixed reports on the existence and characteristics of theviable but not culturable (VBNC) state of Campylobacterjejuni


Characters of an organism:

Campylobacteris a fastidious organism that is capable of survivingin a wide range of environments. It has been isolated fromrivers, estuarine, and coastal waters, at populations ranging from10 to 230 colony-forming units (CFU)/100 mL (Bolton and others1982, 1987).

Campylobacteris a commensal organism routinelyfound in cattle, sheep, swine, and avian species. The avian speciesare the most common host for Campylobacter, probably because of their higher body temperature (Skirrow 1977).


A study byBolton and others (1982, 1987) investigated the effect of environmental temperatures over different seasons and nutrients on thesurvival of C. jejuni. They found peak isolation during the late falland winter months

. Willis and Murray (1997) found Campylobacterbe at their highest populations on poultry during thewarmer months (May through October). During these months,87% to 97% of the samples tested were positive for C. jejuni. Theyalso reported substantial variability in the intestinal colonizationof C. jejuni across different broiler flocks at different ages in theproduction cycle

.
Campylobacter jejuniand Campylobactercoli account for themajority of human infections (Friedman and others 2000) and arecommonly referred to as “thermophilic” campylobacters, beingable to grow at 37 °C to 42 °C with an optimum growing temperatureof 42 °C but incapable of growth below 30 °C. However, astudy by De Cesare and others (2002) found that C. jejuni survivedin excess of 4 h at 27 °C and 60% to 62% relative humidityon some common clean or soiled food contact surfaces.


sensitivity of organism:

unusually sensitive to oxygen and dehydration.
Enzymes present in C. jejuni such as superoxide dismutase(SOD), catalase, peroxidase, glutathione synthetase, andglutathionereductase are believed to play a vital role in providingprotection against oxygen toxicity (Pesci and others 1994;Purdyand Park 1994). Campylobacterrequires a special atmosphere,which usually consists of 5% oxygen, 10% carbon dioxide, and85% nitrogen for growth in or on laboratory media
.
Doyle and Roman (1982b) examined the sensitivity of C. jejuni
to drying. They demonstrated that several factors influenced the
rate of inactivation of Campylobacterdried on a glass surface, including bacterial strain, temperature, humidity, and the suspension
medium.
In all instances, greater survival occurred when organisms were dried in Brucella broth rather than in skim milk. Theresults of Doyle and Roman (1982a) suggest that C. jejuni is quitesensitive to drying and storage at room temperature, but at refrigerationtemperatures and appropriate humidity, large numbersmay survive drying and remain viable for several weeks. Otherstudies have also found C. jejuni to be quite sensitive to drying atroom temperature Campylobacteris oxidase- and catalase-positive and contains asingle polar unsheathed flagellum at one or both ends. C. jejuni hydrolyzeshippurate, indoxyl, and acetate and reduces nitrate, but isunable to oxidize or ferment carbohydrates. Most strains are resistantto cephalothin. Many are also resistant to fluoroquinolones, acategory of antibiotics used to treat animal and human illness

.




Sources and infection:

Campylobacterhas long been recognized as a cause of diarrhea in cattle and of septic abortion in both cattle and sheep. Only in the last 25 y has Campylobacterbeen recognized as an important cause of human illness (Friedman and others 2000). The Centers for Disease Control and Prevention reported that Campylobacteris a major cause of bacterial diarrheal illness in the United States, with
40000 cases documented annually (CDC 2003). Disease control studies have demonstrated that 50% to 70% of human Campylobacterillness is attributed to consuming poultry and poultry products,thus the value of reducing levels associated with raw poultry has drawn considerable attention Children
less than 1 y of age and young adults aged 15 to 25 y are more susceptible to developing this disease, and individuals with immunosuppression can develop prolonged or unusually severe cases of illness (Friedman and others 2000). Deaths attributed to Campylobacterinfection in the United States are estimated at 680 to 730/y (Saleha and others 1998). Doses as low as 500 organisms have been reported to cause illness There are several species of Campylobacter(C. jejuni, C. coli, C. lari, and C. uppsaliensis) capable of causing human illness. However, C. jejuni is implicated in about 85% of the cases of human campylobacteriosis, with the remaining cases being primarily caused by C. coli





Prevention:

Some simple food handling practices can help prevent Campylobacterinfections.
• Cook all poultry products thoroughly. Make sure that the meat is cooked throughout (no longer pink) and any juices run clear. All poultry should be cooked to reach a minimum internal temperature of 165 °F.
• If you are served undercooked poultry in a restaurant, send it back for further cooking.
• Wash hands with soap before preparing food
AIM:
To develop a PCR based kit for the identification of shigella sps, clostridium botulinum, Listeria monocytosis, e.coli,and Campylobacter jejuni by targeting species specific genes.



OBJECTIVES


To isolate Shigella sps, Clostridium botulinum, Listeria monocytosis, E.coli, Campylobacter jejuni from the food materials

Design Primers for Shigella sps, Clostridium botulinum, Listeria monocytosis, E.coli, Campylobacter jejuni species

Standardized and Optimization of Primers designed

To check the sensitivity of the primers designed




















MATERIALS AND METHODS


Polymerase chain reaction principle and standardization:

Components and reagents of PCR:

DNA template- That contains the DNA region (target) to be amplified. Primers- Which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA. DNA polymerase, such as Taq polymerase (Lawyer et al., 1976) or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs)- Also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution- Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations- Magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.



Cycling conditions:

Initialization step- This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes.
Denaturation step-This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step- The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step- The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, (Lawyer FC et.al 1993) and commonly a temperature of 72°C is used with this enzyme.
Final elongation- This single step is occasionally performed at a temperature of70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold- This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler for about 20 t0 40 cycles.

Primer designing:
A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. These primers are designed(Compton T 1990) to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal (Figure2).

Fig 1: Primer annealing principle.
When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction. The following program will perform these calculations on any primer sequence or pair.

Considerations for designing primers-
1.Primers should be 17-28 bases in length
2. Base composition should be 50-60% (G+C)
3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming
4. Tms between 55-80oC are preferred
5.3’-ends of primers should not be complementary (i.e. base pair), as otherwise primer dimers will be synthesised preferentially to any other product
6. Primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided
7. Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.
Optimization of PCR reaction components:

Initially, there was some variation from test to test when the same PCR program was used. Solving this reproducibility problem required adjustments of PCR components.
Amount of primer- Initially equimolar primer concentrations of each can be used in the multiplex PCR, if there was uneven amplification, with some of the products barely visible even after the reaction was optimized for the cycling conditions, changing the proportions of various primers in the reaction, with an increase in the amount of primers for the “weak” amplicons and a decrease in the amount for the strong”amplicons.
MgCl2 concentrations- A recommended magnesium chloride concentration in a standard PCR is 1.5 mM at dNTP concentrations of around 200 mM each. If the amplification was not complete for all the primers varying concentrations of MgCl2 ranging from 1.5mM to 3mM concentrations.
Amount of template DNA- Recommended Template DNA concentrations for Normal PCR is 10 ng to 50 ng. For a multiplex PCR depending on the intensity of the amplicons template concentrations can vary between 50 ng to 200ng.
Amount of Taq DNA polymerase- Different concentrations of Taq DNA polymerase can be used for performing PCR reaction the optimum concentration per reaction is 0.3 mL or 1U/25 mL reaction volume. Too much. enzyme, possibly because of the high glycerol concentration in the stock solution, can result in an unbalanced amplification.
Use of adjuvants- Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific roducts) of PCR, when used in concentrations varying between 5%–10% (vol/vol).





Primer Designing:
The process of designing the primers was initiated by entering the site of NCBI through which a data base GENOME was selected where the prescribed organism(bacteria) is opted. The given bacteria Staphylococcus aureus is been clicked for further assumptions.The related genes concerned with the bacteria Staphylococcus aureus are viewed in which few types of chosen genes were reared from the following number of genes. There are many types of genes like Heat shock genes, Cell division genes, T-RNA specific genes, Ribosomal genes, Hypothetical genes etc…
The specific genes were taken into the consideration and were traced through nucleotide links by the help of FASTA. The sequenced primers are obtained for a particular selected gene and these sequences are transferred are imported through the clipboard and then it is sent into bioedit sequence. These genes were named and the primer sequences were sent for BLAST (Basic Local Alignment Search Tool) nucleotide sequencing.The following primer sequences is pasted on the respective block of the nucleotide blast by clicking OTHERS and algorithm parameters are taken with a expect threshold of about 10 and the respective genes related to staphylococcus aureus are traced and were taken as a soft copy.Then the particular gene is taken and clicked under human genomic and transcript whether the following gene is specific in human or not with a expect threshold of 0.01.
Primer3 tool, was used in designing PCR primers. Primer3 tool has a very powerful PCR primer design program permitting one considerable control over the nature of the primers, including size of product desired, primer size and Tm range, and presence/absence of a 3’-GC clamp.It is a new improved web interface to the popular Primer3 primer design program.The selected primers were the sequenced and they are clicked for pick primers.
Primer3 tool is a comprehensive web primer design program using Primer3 core program as a major primer design engine to design different types of PCR primers and sequencing primers in a high-through manner. BatchPrimer3 allows users to design several types of primers including generic primers, hybridization oligos, SSR primers together with SSR detection, and SNP genotyping primers (including single-base extension primers, allele-specific primers, and tetra-primers for tetra-primer ARMS PCR), as well as DNA sequencing primers. A batch input of large number of sequences and a tab-delimited result output greatly facilitates rapid primer design and ordering process.
Oligonucleotide primers are widely used in various molecular biology techniques like DNA sequencing and the polymerase chain reaction (PCR). Since a primer serves as the starting point for DNA replication, specific binding of the oligonucleotide to the target sequence on the template strand is essential for a successful experiment. The binding specificity of a primer is determined by several of its properties, like the melting temperature (Tm), GC-content and self complementarity. Designing primers is usually done with the help of computer programs, among which Primer3 is most widely used judging from the hundreds of citations of the primary publication (1). Primer3 is popular since the program can be used online and is redistributed free of charge. Recently, a SourceForge project was started for Primer3 in which several improvements are being discussed and implemented. Subsequently Primer3 was updated to include an additional method for Tm calculation [SantaLucia model, (2)], since it was argued that primer design based on the Breslauer mode.
The primers ware synthesized commercially (Bioserve India Pvt, Ltd.)






PREPARATION OF GENOMIC DNA FROM BACTERIA
MINI PREPARATION OF BACTERIAL GENOMIC DNA:
Bacteria from a saturated liquid culture were lysed and proteins were removed by digestion with proteinase K. Cell wall debris, polysaccharides, and remaining proteins were removed by selective precipitation with CTAB, and high-molecular-weight DNA is recovered from the resulting supernatant by isopropanol precipitation.


MATERIALS:
TE BUFFER
1ml of 1M tris (pH 8) + 200micro/lit 0.5 M EDTA in 100ML of deionized water. Incubator or water bath at 50c, with shaker
10% sodium dodecyl sulfate(SDS) (10g of SDS to make 100ml of solution)
20mg/ml proteinaseK (5micro/lit of 20mg/ml proteinase K per 1ml)
5M Nacl(29.25g to make 100ml of solution)
CTAB/Nacl Solution(10% CTAB in 0.7M Nacl)
Dissolve 4.1 Nacl in 80ml water and slowly add 10g CTAB (hexadecyltrimethyl ammonium bromide) while heating and stirring. If necessary, heat to 65c to dissolve.Adjust final volume to 100ml.24:1 chloroform/isoamyl alcohol. Isopropanol 70% ethanol (70ml EtoH + 30ml deionized water)
CTAB method……………….
Isolation of Genomic DNA from Curd:
Take 25mL of contaminated Curd. To it add 25mL of 1XPBS buffer and mix it thoroughly.
Now centrifuge the tube at 1000 rpm for 15 min and remove the solid contents (of curd) present. Now collect the supernatant and centrifuge it at 10,000 rpm for 30 min.
CTAB method or kit method…….

Isolation of Genomic DNA from Potato:
Take 25gm of baked potato salad and grind it by using 1%PBS buffer. Centrifuge the obtained solution at 1000 rpm for 15min and collect the supernatant leaving the pellet as such. Now again centrifuge the supernatant obtained at 10,000 rpm for 30 min and collect the supernatant obtained.
CTAB method or kit method………..

Isolation of Genomic DNA from Honey:
Take 20mL of Honey. To it add 20mL of 1%PBS and mix it thoroughly. Now centrifuge it for 10629 rpm for 30 min.
Discard the supernatant. Add 10mL of 1X PBS buffer to the pellet and again centrifuge at 10,629 rpm for 10 min.(Repeat this step for two times)

CTAB method……………….

Extraction of Genomic DNA by CTAB method:

Take 50microL of deionised free water into 1.5mL tube. Transfer loop full of bacterial culture into the deionised water. Lyse the bacterial cells by heating at 95C for 30 min.Remove the cell debris by spinning at 5000 rpm for 5 min.
Transfer the supernatant into fresh tube.
1. Inoculate a 5-ml liquid culture with the bacterial strain of interest. Grow in conditions appropriate for that strain (i.e, appropriate medium, drug selection, temperature) until the culture is saturated. This may take several hours to several days, depending on the growth rate.
2. Spin 1.5ml of the culture in a microcentrifuge for 2min, or until a compact pellet forms. Discard the supernatant.
3. Resuspend the pellet in 567micro/lit TE buffer by repeated pipetting. Add 30micro/lit of 10%SDS and 3micro/lit of 20mg/ml proteinaseK to give a final concentration of 100mg/ml proteinase K in 0.5% SDS.Mix thoroughly and incubate 1hr at 37c.
4. Add 100micro/lit of 5M Nacl and mix thoroughly.
5. Add 80micro/lit of CTAB/Nacl solution. Mix thoroughly and incubate 10min at 65c.
6. Add an approximately equal volume (0.7to0.8) of chloroform/isoamyl alcohol, mix thoroughly, and spin 4to5min in a microcentrifuge.
7. Remove aqueous, viscous supernatant to a fresh microcentrifuge tube, leaving the interface behind. Add an equal volume of phenol/chloroform/isoamyl alcohol, extract thoroughly, and spin in a microcentrifuge for 5 min.
8. Transfer the supernatant to a fresh tube. Add 0.6 vol isopropanol to precipitate the nucleic acids (there is no need to add salt since the Nacl concentration is already high). Shake the tube back and forth until a stringy white DNA precipitate becomes clearly visible. At this point it is possible to transfer the pellet to a fresh tube containing 70% ethanol by hooking it onto the end of a micropipette that has been heat-sealed and bent in a Bunsen flame.Alternatively,the precipitate can be pelleted by spinning briefly at room temperature.
9. Wash the DNA with 70% ethanol to remove residual CTAB and respin 5min at room temperature to repellet it. Carefully remove the supernatant and briefly dry the pellet in a lyophilizer.
10. Redisslove the pellet in 100ml TE buffer.









Isolation of Genomic DNA by Qiagen Kit Method
1.5ml culture was centrifuged at 10,000rpm for 10min to get the bacterial pellet

Discard the supernatant and resuspend the pellet 200 micro/litre AL buffer
Add 40l of proteinase K (20mg/ml) and 20micro/litre of RnaseA (10mg/ml)
Incubate at 56c for about 10 minutes
Add 200l of ethanol (96-100%) and mix it by pulse vertexing for 15 sec

Transfer the mixture on to column and centrifuge at 8,000rpm for 1minute
Discard the Flow-though
Add 500l wash buffer1 to the column and centrifuge at 8,000rpm for 1min

Discard the Flow-though

Add 500l wash buffer 2 to the column and centrifuge at 13,000rpm for 3min

Discard the Flow-though

Centrifuge the empty column for one min at 13,000rpm

Transfer column into fresh tube and elute the DNA in 50l of de-ionized water by centrifuge at 8,000rpm for 1min




Quantification of DNA:
Isolated DNA was quantified by measuring the absorbance at 260 and at 280 nm. The ratio of absorbance 260/280 was used to determine the quality of the isolated DNA .The concentration was calculated using the following formula
Concentration of the DNA = OD260 X 50 X dilution factor = g of DNA /ml
Quantified DNA was diluted to 150 ng/l and used for polymerase chain











RESOLUTION OF DNA FRAGMENTS ON STANDARD AGAROSE GELS:

Materials:
Electrophoresis buffer (1X TAE)
Ethidium bromide solution 0.5 mg/ml
Electrophoresis-grade agarose
6 X loading buffer
DNA molecular weight markers
Horizontal gel electrophoresis apparatus
Gel casting platform
Gel combs (slot formers)
DC power supply


Preparing the gel

1. Prepare an adequate volume of electrophoresis buffer fill the electrophoresis tank and prepare the gel.

Caution - Ethidium bromide is a mutagen and potential carcinogen. Gloves should be worn and care should be taken when handling ethidium bromide solutions.

1.Add 0.25g of the Agarose in 25 ml of 1XTAE to prepare 1% gel.
2. Dissolve agarose by heating at 70C in a micro-wave.
3. Allow it to cool and add 1l of the Ethidium bromide
4.Pour the melted agarose onto the gel-casting platform which has comb for the creation of the wells
5.Remove the comb after solidification of the gel
6.Place the gel into the gel loading tank and fill with the 1 X TAE till the gel submerged into the buffer.
7.Load the samples mixed with gel loading dye which has bromophenol blue (Front dye) and Xylene cyanol (Tracking dye) and the Reference size standard (to know the size) in the gel and run at 75 volts till the time the bromophenol blue reaches 3/4th of the gel.
8.Visualize the DNA by placing on a UV light source and can be photographed directly by gel documentation unit.













Polymerase chain reaction:
Polymerase chain reaction was performed using primers that were designed from the specific gene of the ShigellaSps Primers were commercially obtained from Bio-serve India pvt, Ltd. All the primers designed were 20 base pairs in length with GC content in the range of 40-80%. Tm was calculated according to the formula Tm(C) = 2(A+T) + 4(G+C)  5. The annealing temperature for each pair of PCR primers was optimized experimentally.
PCR amplification was carried out with following reaction parameters
dNTPs 200 µM
PCR reaction buffer - 1X
Magnesium chloride - 1.5 -2.5 mM
Primer (forward) - 10 pmoles
Primer (reverse) - 10 pmoles
Templet DNA - 100 ng
Taq DNA polymerase - 1U
The final reaction volume was 15µl.
The cycling conditions used:
Initial denaturation - 94oC, 2 min
Denaturation - 94oC, 45 sec
Annealing - 55 oC, 40sec
Elongation - 72oC, 40 sec
Cycles - 35

Final elongation - 72oC, 5 min

Amplification of the primers was confirmed for the following parameters:

1)PCR primers have to standardize initially with the DNA isolated from the streptococcus Pneumonia.
2)Specific amplification of the primers can be confirm by performing PCR with the human genomic DNA as a negative control and performing PCR with DNA OF other staphylococcus species
3)Performing PCR with the PCR negative control confirms presence of contamination or not
4)Repeat the experiment to check the experimental reproducibility








RESULTS:

Results of Shigella sps:

DNA Isolation- The DNA isolated with CTAB method and the kit method was analyzed by the gel electrophoresis to check the integrity of the DNA as shown in figure


DNA isolated and their spectroscopic Results:
After isolation of DNA, we have taken the O.D values to identification and purification of DNA….

O.D values of Shigella:
1. 82.9 260/280 = 1.10
2. 80.3 260/280 = 1.31
3. 81.0 260/280 = 1.30
Average:
(82.9+80.3+81.0)/3 = 81.4µg/ml
For 1000µl - 81.4 µg/ml
For 60 µl - (30*81.4)/1000
= 2.442 µg
For 1 µl = 2.442/30
= 0.081 µg/µl
For the identification of DNA present in the isolated sample from shigellaculture, we performed the gel electrophoresis. After performing gel electrophoresis the bands obtained as follows:













We performed the PCR reactions, after identification of DNA.,
Reaction1:
Reaction volume 25µl:
Number of reactions 1
Buffer - 2.5µl
Mgcl2 - 1.5 µl
dNTP’s - 2.5 µl
Forward primer - 2 µl
Reverse primer - 2 µl
Template - 5 µl
Enzyme - 0.33 µl
Water - 9.17 µl
Annealing Temperature: 580
Now we perform the PCR reaction with this reaction mixture.
After performing the PCR reaction we obtained the PCR product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands and are shoed in figure 1 below















SENSITIVITY DETECTION:
To identify the sensitivity and efficiency of the PCR for amplification of DNA at low template concentrations, so template can be diluted to minimal concentrations as follows,
Template dilution:
5 µl template in 45 µl water (diluted)

Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 0.33µl*2 = 0.66µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 5 µl
Water - 9.17 µl

Sample 2:
template - 2.5 µl
Water - 11.67 µl





µl template in 49 µl water (diluted)
Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 0.33µl*2 = 0.66µl

We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 10 µl
Water - 4.17 µl


Sample 2: template - 5 µl
Water - 9.17 µl
Now we perform the PCR with this reaction mixture for DNA amplification.
After performing the reaction we obtained the pcr product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands as follows.







































Results of Clostridium botulinum:

DNA Isolation- The DNA isolated with CTAB method and the kit method was analyzed by the gel electrophoresis to check the integrity of the DNA as shown in figure

DNA isolated and their spectroscopic Results:

After isolation of DNA, we have taken the O.D values to identification and purification of DNA….

O.D values of Clostridum:
1. 80.7 260/280 = 1.12
2. 74.0 260/280 = 1.12
3. 79.0 260/280 = 1.12


Average:
(80.7+74.0+79.0)/3 = 77.9µg/ml
For 1000µl - 77.9 µg/ml
For 30 µl - (30*77.9)/1000
= 2.337 µg
For 1 µl = 2.337/30
= 0.0779 µg/µl
For the identification of DNA present in the isolated sample from Clostridum culture, we performed the gel electrophoresis. After performing gel electrophoresis the bands obtained as follows:













We performed the PCR reactions, after identification of DNA.,
Reaction1:
Reaction volume 25µl:
Number of reactions 1
Buffer - 2.5µl
Mgcl2 - 1.5 µl
dNTP’s - 2.5 µl
Forward primer - 2 µl
Reverse primer - 2 µl
Template - 3 µl
Enzyme - 1 µl
Water - 11.5 µl
Annealing Temperature: 560
Now we perform the PCR reaction with this reaction mixture.
After performing the PCR reaction we obtained the PCR product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands and are showed in figure 1 below












SENSITIVITY DETECTION:
To identify the sensitivity and efficiency of the PCR for amplification of DNA at low template concentrations, so template can be diluted to minimal concentrations as follows,

Template dilution:

5 µl template in 45 µl water (diluted)

Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 1 µl*2 = 2 µl
Reverse primer - 1 µl*2 = 2 µl
Enzyme - 1 µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 10 µl
Water - 3.5 µl

Sample 2: template - 5 µl
Water - 8.5 µl
Now we perform the PCR with this reaction mixture for DNA amplification.
After performing the reaction we obtained the pcr product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands as follows.
































Results of Listeria monocytosis:
DNA Isolation- The DNA isolated with CTAB method and the kit method was analyzed by the gel electrophoresis to check the integrity of the DNA as shown in figure

DNA isolated and their spectroscopic Results:
After isolation of DNA, we have taken the O.D values to identification and purification of DNA….
O.D values of Listeria:
1. 119.6 260/280 = 1.10
2. 116.1 260/280 = 1.12
3. 121.8 260/280 = 1.17


Average:
(119.6+116.1+121.8)/3 = 119.2 µg/ml
For 1000µl - 119.2 µg/ml
For 20 µl - (20*119.2)/1000
= 0.238 µg
For 1 µl = 0.238/20
= 0.0119 µg/µl
For the identification of DNA present in the isolated sample from Listeriaculture, we performed the gel electrophoresis. After performing gel electrophoresis the bands obtained as follows:













We performed the PCR reactions, after identification of DNA.,
Reaction 1:
Reaction volume 25µl:
Number of reactions 1
Buffer - 2.5µl
Mgcl2 - 2.5 µl
dNTP’s - 2.5 µl
Forward primer - 2 µl
Reverse primer - 2 µl
Template - 3 µl
Enzyme - 1 µl
Water - 11.5 µl
Annealing Temperature: 580
Now we perform the PCR reaction with this reaction mixture.
After performing the PCR reaction we obtained the PCR product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands and are shoed in figure 1 below
















SENSITIVITY DETECTION:
To identify the sensitivity and efficiency of the PCR for amplification of DNA at low template concentrations, so template can be diluted to minimal concentrations as follows,
Template dilution:

5 µl template in 45 µl water (diluted)

Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 2.5 µl*2 = 5 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 1 µl*2 = 2 µl
Reverse primer - 1 µl*2 = 2 µl
Enzyme - 1 µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 10 µl
Water - 2.5 µl
Sample 2: template - 5 µl
Water - 7.5 µl


µl template in 49 µl water (diluted)

Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 2.5 µl*2 = 5 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 1 µl*2 = 2 µl
Reverse primer - 1 µl*2 = 2 µl
Enzyme - 1 µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 10 µl
Water - 2.5 µl
Now we perform the PCR with this reaction mixture for DNA amplification.
After performing the reaction we obtained the pcr product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands as follows.

































Results of E.coli:


DNA Isolation- The DNA isolated with CTAB method and the kit method was analyzed by the gel electrophoresis to check the integrity of the DNA as shown in figure

DNA isolated and their spectroscopic Results:

After isolation of DNA, we have taken the O.D values to identification and purification of DNA….
O.D values of Ecoli:
1. 30.5 260/280 = 1.29
2. 30.9 260/280 = 1.31
3. 34.3 260/280 = 1.30


Average:
(30.5+30.9+34.3)/3 = 31.9µg/ml
For 1000µl - 31.9 µg/ml
For 60 µl - (60*31.9)/1000
= 1.914 µg
For 1 µl = 1.914/60
= 0.031 µg/µl

For the identification of DNA present in the isolated sample from Clostridum culture, we performed the gel electrophoresis. After performing gel electrophoresis the bands obtained as follows:















We performed the PCR reactions, after identification of DNA.,
Reaction1:
Reaction volume 25µl:
Number of reactions 1
Buffer - 2.5µl
Mgcl2 - 1.5 µl
dNTP’s - 2.5 µl
Forward primer - 2 µl
Reverse primer - 2 µl
Template - 6 µl
Enzyme - 1 µl
Water - 8.5 µl
Annealing Temperature: 56.50
Now we perform the PCR reaction with this reaction mixture.
After performing the PCR reaction we obtained the PCR product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands and are shoed in figure 1 below













SENSITIVITY DETECTION:
To identify the sensitivity and efficiency of the PCR for amplification of DNA at low template concentrations, so template can be diluted to minimal concentrations as follows,
Template dilution:
5 µl template in 45 µl water (diluted)

Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 1 µl*2 = 2 µl

We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 5 µl
Water - 8.5 µl


µl template in 49 µl water (diluted)
Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 1 µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 2.5 µl
Water - 11 µl


Sample 2: template - 5 µl
Water - 8.5 µl
Now we perform the PCR with this reaction mixture for DNA amplification.
After performing the reaction we obtained the pcr product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands as follows.
:


















RESULTS OF Campylobacter jejuni:

DNA Isolation- The DNA isolated with CTAB method and the kit method was analyzed by the gel electrophoresis to check the integrity of the DNA as shown in figure

DNA isolated and their spectroscopic Results:

After isolation of DNA, we have taken the O.D values to identification and purification of DNA….

O.D values of Campylobacter:
1. 86.3 260/280 = 1.11
2. 81.4 260/280 = 1.11
3. 84.3 260/280 = 1.12


Average:
(86.3+81.4+84.3)/3 = 84.0µg/ml
For 1000µl - 84.0 µg/ml
For 60 µl - (30*84.0)/1000
= 2.52 µg
For 1 µl = 2.52/30
= 0.084 µg/ml
For the identification of DNA present in the isolated sample from Clostridum culture, we performed the gel electrophoresis. After performing gel electrophoresis the bands obtained as follows:

















We performed the PCR reactions, after identification of DNA.,
Reaction1:
Reaction volume 25µl:
Number of reactions 1
Buffer - 2.5µl
Mgcl2 - 1.5 µl
DNTP’s - 2.5 µl
Forward primer - 2 µl
Reverse primer - 2 µl
Template - 4.5 µl
Enzyme - 1 µl
Water - 9 µl
Now we perform the PCR reaction with this reaction mixture.
After performing the PCR reaction we obtained the PCR product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands and are shoed in figure 1 below











SENSITIVITY DETECTION:

To identify the sensitivity and efficiency of the PCR for amplification of DNA at low template concentrations, so template can be diluted to minimal concentrations as follows,
Template dilution:
5 µl template in 45 µl water (diluted)

Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 1µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 2.5 µl
Water - 11 µl


Sample 2: template - 5 µl
Water - 8.5 µl
Now we perform the PCR with this reaction mixture for DNA amplification.


µl template in 49 µl water (diluted)
Reaction:
Reaction volume - 25 µl
Number of reactions - 2
Buffer - 2.5 µl*2 = 5 µl
Mgcl2 - 1.5 µl*2 = 3 µl
dNTP’s - 2.5 µl*2 = 5 µl
Forward primer - 2 µl*2 = 4 µl
Reverse primer - 2 µl*2 = 4 µl
Enzyme - 1 µl*2 = 2 µl
We taken the template in different volumes, in above two reactions and finally make the reaction volume to 25 µl with water.
Sample 1: template - 10 µl
Water - 3.5 µl

Sample 2: template - 5 µl
Water - 8.5 µl
Now we perform the PCR with this reaction mixture for DNA amplification.
After performing the reaction we obtained the pcr product.
By using this PCR product we conduct the gel electrophoresis.
After the gel electrophoresis we observed the bands as follows.




















CONCLUSIONS:

The current primer set designed for Shigella sps, Clostridium botulinum, E .coli, Campylobacter jejuni, Listeria monocytogens, showed very good specificity and sensitivity for all the organisms tested. The sensitivity of the test for Shigella sps is 1.6ng/l, for Clostridium botulinum it is 1.54 ng /l, for Listeria monocytogens it is 1.76ng/l, for E. coli the sensitivity is 0.62ng/l, and for Campylobacter jejuni the sensitivity worked for 1.68ng/l. The amplicon sizes vary from 110bp-600bp. The amplicon sizes are as follows for E. coli: 110bp; Listeria monocytogens: 167bp; Campylobacter jejuni: 275bp; Shigella sps: 550bp; Clostridium botulinum: 600bp.Internal standardizations for individual primer are completed and further Multiplex standardization of all the sets to be performed.























REFERENCES

1.Hale TL, Keusch GT (1996). 'Shigella': Structure, Classification, and Antigenic Types. in: Baron's Medical Microbiology (Baron S et al., eds.) (4th ed.). Univ of Texas Medical Branch. ISBN 0-9631172-1-1.
2.World Health Organization. Shigellosis.
3.Hale TL, Keusch GT (1996). Shigella. in: Baron's Medical Microbiology (Baron S et al., eds.) (4th ed.). Univ of Texas Medical Branch. ISBN 0-9631172-1-1.
4.Hill Gaston JS, Lillicrap MS (2003). "Arthritis associated with enteric infection". Best practice & research. Clinical rheumatology 17 (2): 219–39.doi:10.1016/S1521-6942(02)00104-3. PMID 12787523.
5.Yang F et al. (2005). "Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery". Nucleic Acids Res 33 (19): 6445–58.doi:10.1093/nar/gki954. PMID 16275786.
6.Ito H, Kido N, Arakawa Y, Ohta M, Sugiyama T, Kato N (October 1991). "Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp". Appl. Environ. Microbiol. 57 (10): 2912–7. PMID 1746953
7.J. C. Suen, C. L. Hatheway, A. G. Steigerwalt, D. J. Brenner. 1988, Clostridium argentinense sp.nov.: a genetically homogeneous group composed of all strains of Clostridium botulinum type G and some nontoxigenic strains previously identified as Clostridium subterminale or Clostridium hastiforme. Int. J. Sys. Bacteriol. 38: 375–381.
8.Judicial Commission of the International Committee on Systematic Bacteriology (1999) Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes Opinion 69. International Journal of Systematic Bacteriology. 49, 339.
9.L. V. Holdeman, J. B. Brooks. 1970. Variation among strains of Clostridium botulinum and related clostridia. Protocols of the first U.S-Japan conference on Toxic Microorganisms. pp. 278–286
10.L. D. S. Smith, G. Hobbs. 1974. Genus III Clostridium Prazmowski 1880, 23. In R. E. Buchanan, N. E. gibbons (eds.), Bergey’s Manual of Determinative Bacteriology, 8th edition. William & Wilkins, Baltimore. pp. 551–572.
11.Campbell, N. A. and Reese, J. B. (2005). Biology. Pearson.
12.C. L. Hatheway, L. M. McCroskey. 1987. Examination of faeces for diagnosis of infant botulism in 336 patients. J. Clin. Microbiol. 25: 2334–2338.
13.P. Aureli, L. Fenicia, B. P
14.arber, J.M. and P.I. Peterkin (1991). "Listeria monocytogenes, a Food-Borne Pathogen". Micribiol. Rev. 55:476-511. Retrieved January 28, 2009.
15.Todar, K. (2008). "Listeria monocytogenes". Todar's Online Textbook of Bacteriology. Retrieved January 28, 2009.
16.Seafood HACCP Alliance (2007). "Compendium of Fish and Fishery Product Processes, Hazards, and Controls, Chapter 15: Listeria monocytogenes".Seafood Network Information Center. Retrieved January 28, 2009.
17.Dharmarha, Vaishali (December 2008). "A Focus on Listeria Monocytogenes". National Agricultural Library, Food Safety Research Information Office. Retrieved January 28, 2009.
18.Murray, E.G.D., Webb, R.E., Swann, M.B.R. 1926. A disease of rabbits characterized by a large mononuclear leucocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n. sp.). J. Pathol. Bacteriol. 29: 407– 439.
19.Pirie, J.H.H. 1940. Listeria: change of name for a genus of bacteria. Nature. 145:264
20.Potel, J. 1952. Zur Granulomatosis infantiseptica. Zentr. Bakteriol. I. Orig. 158: 329-331
21.Schlech, W.F. III. 2001. Foodborne listeriosis. Clin. Infect. Dis. 31: 770-775.
22.Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S. and Broome, C.V. 1983. Epidemic listeriosis—evidence for transmission by food. New Engl. J. Med. 308:203–206
23."Escherichia coli O157:H7". CDC Division of Bacterial and Mycotic Diseases. Retrieved on 2007-01-25.
24.Vogt RL, Dippold L (2005). "Escherichia coli O157:H7 outbreak associated with consumption of ground beef, June-July 2002". Public Health Rep 120(2): 174–8. PMID 15842119.
25.^ Bentley R, Meganathan R (01 September 1982). "Biosynthesis of vitamin K (menaquinone) in bacteria". Microbiol. Rev. 46 (3): 241–80. PMID 6127606.PMC: 281544.
26.^ a b Hudault S, Guignot J, Servin AL (July 2001). "Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection". Gut 49 (1): 47–55. doi:10.1136/gut.49.1.47. PMID 11413110.
27.^ Reid G, Howard J, Gan BS (September 2001). "Can bacterial interference prevent infection?". Trends Microbiol. 9 (9): 424–8. doi:10.1016/S0966-842X(01)02132-1. PMID 11553454.
28.^ a b c Feng P, Weagant S, Grant, M (2002-09-01). "Enumeration of Escherichia coli and the Coliform Bacteria". Bacteriological Analytical Manual (8th ed.). FDA/Center for Food Safety & Applied Nutrition. Retrieved on 2007-01-25.
29.Nachmankin I; Szymanski CM; Blaser J (editors) (2008). Campylobacter (3rd ed.). ASM Press. pp. 3–25. ISBN 9781555814373.
30.^ a b Ryan KJ; Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 378–80. ISBN 0838585299.
31.^ Moore JE, et al. (2005). "Campylobacter". Vet Res 36 (3): 351–82. doi:10.1051/vetres:2005012. PMID 15845230.
32.^ Sauerwein R, Bisseling J, Horrevorts A (1993). "Septic abortion associated with Campylobacter fetus subspecies fetus infection: case report and review of the literature". Infection 21 (5): 331–3. doi:10.1007/BF01712458. PMID 8300253.
33.^ Fouts DE et al. (2005). "Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species". PLoS Biol 3 (1): e15. doi:10.1371/journal.pbio.0030015. PMID 15660156.

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