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Enterohemorrhagic 'E.Coli' (O157:H7)

INTRODUCTION

The natural habitat of E. coli is the intestinal tract of humans and animals. It is therefore considered an indicator organism for fecal contamination of water and foods. E. coli is the most frequent causative pathogen in human bacterial infections. Extra intestinal infections include urinary tract infections, which occur when the tract is obstructed or spontaneously caused by the pathovar UPEC. The most important other coli infections are cholecystitis, appendicitis, peritonitis, postoperative wound infections, and sepsis. Intestinal infections are caused by the pathovars EPEC, ETEC, EIEC, EHEC, and EAggEC. EPEC and EAggEC frequently cause diarrhea in infants. ETEC produce enterotoxins that cause a choleralike clinical picture. EIEC cause a dysenterylike infection of the large intestine. (1)

EHEC produce verocytotoxins and cause a hemorrhagic colitis as well as the rare hemolytic-uremic syndrome. E. coli bacterial infections are diagnosed by means of pathogen identification. E. coli serotype O157:H7 is a Gram-negative, rod-shaped bacterium. The "O" in the name refers to the cell wall (somatic) antigen number, whereas the "H" refers to the flagella antigen. Other serotypes may cause (usually less severe) illness, but only those with the specific O157:H7 combination is reviewed here. (1, 2)

E. coli is a bacterium that very easily and frequently exchanges genetic information with related bacteria around it, such as Salmonella spp., Shigella spp., and other E. coli strains, through horizontal gene transfer mechanisms. Therefore, E. coli strains may exhibit characteristics that have been acquired from a wide variety of sources. (3) It has been demonstrated that certain E. coli isolates produced a toxin, which was initially called verotoxin because of its distinct effect on vero cells. This family of toxins was subsequently also called Shiga-like toxins (SLT), and more recently Shiga toxins (Stx), because of the close relation to the Stx of Shigella dysenteriae type. Enterohaemorrhagic E. coli (EHEC) is the main group of verotoxigenic strains which has emerged as the leading cause of haemorrhagic colitis and haemolytic uremic syndrome (HUS) in humans. Shigatoxins are categorized into two main groups, stxI and stxII. The majority of Stx genes are bacteriophage borne, which may be important for the spread of shiga toxin-producing E. coli (STEC). EHEC strains are characterized by the ability to form attaching and effacing (A/E) lesions on the surface of epithelial cells in the gastrointestinal tract, and the production of shiga toxins. The first gene to be associated with A/E activity was the intimin gene, eae, and its presence is often used as a marker for the infections caused by EHEC, E. coli O157:H7 was the first serotype associated with haemorrhagic colitis, although more than 100 STEC serotypes have been isolated from different sources, such as food and recreational and drinking water. However, not all pathogenic STEC isolates have been shown to produce intimin. EHEC appears to be transmitted primarily through the ingestion of faecal contaminated foods, particularly undercooked beef. However, a large number of outbreaks of EHEC have also been associated with consumption of contaminated drinking water or contact with recreational water. E. coli O157:H7 infections as a cause of disease have shown a marked increase in many countries. Many serotypes other than O157:H7 are capable of producing Shiga toxins and similar clinical manifestations, but this serotype was the one most commonly isolated in North America and Europe as a cause of illness. (4)

Geographic Distribution

EHEC 0157:H7 infections occur worldwide; infections have been reported on every continent except Antarctica. Other EHEC are probably also widely distributed. The importance of some serotypes may vary with the geographic area. (5)

Transmission

EHEC are transmitted by the fecal–oral route. They can be spread between animals by direct contact or via water troughs, shared feed, contaminated pastures or other environmental sources. Birds and flies are potential vectors. The reservoir hosts and epidemiology may vary with the organism. Ruminants, particularly cattle and sheep, are the most important reservoir hosts for EHEC O157:H7. A small proportion of the cattle in a herd can be responsible for shedding more than 95% of the organisms. These animals, which are called super-shedders, are colonized at the terminal rectum, and can remain infected much longer than other cattle.

Super-shedders might also occur among sheep. Animals that are not normal reservoir hosts for EHEC O157:H7 may serve as secondary reservoirs after contact with ruminants. EHEC O157:H7 is mainly transmitted to humans by the consumption of contaminated food and water, or by contact with animals, feces and contaminated soil. Person-to-person transmission can contribute to disease spread during outbreaks; however, humans do not appear to be a maintenance host for this organism. Most human cases have been linked to direct or indirect contact with cattle, but some have been associated with other species including sheep, goats (unpasteurized goat milk), deer (venison), horses, rabbits and birds.

The infectious dose for humans is estimated to be under 100 organisms, and might be as few as 10. Food borne outbreaks with EHEC O157:H7 are often caused by eating undercooked or unpasteurized animal products, particularly ground beef but also other meats and sausages, and unpasteurized milk and cheese. Other outbreaks have been linked to lettuce, spinach and other contaminated vegetables, as well as unpasteurized cider. Irrigation water contaminated with feces is an important source of EHEC O157:H7 on vegetables. This organism can attach to plants, and survives well on the surface of a variety of fruits, vegetables and fresh culinary herbs. Depending on the environmental conditions, small numbers of bacteria left on washed vegetables may multiply significantly over several days. EHEC O157:H7 can be internalized in the tissues of some plants including lettuce, where it may not be susceptible to washing. Fruit flies can transmit this organism to apples, where it can multiply in wounded tissues. EHEC O157:H7 can remain viable for long periods in many food products. It can survive for at least nine months in ground beef stored at -20°C (-4°F). It is tolerant of acidity, and remains infectious for weeks to months in acidic foods such as mayonnaise, sausage, apple cider and cheddar at refrigeration temperatures. It also resists drying.

Some human cases are caused by exposure to contaminated soil or water. EHEC are usually eliminated by municipal water treatment, but these organisms may occur in private water supplies such as wells. Swimming in contaminated water, especially lakes and streams, has been associated with some infections. Soil contamination has caused outbreaks at campgrounds and other sites, often when the site had been grazed earlier by livestock. The reported survival time for EHEC O157:H7 in contaminated soil varies from a month to more than 7 months. This organism can also survive for 2 months or longer in some freshwater sources, especially at cold temperatures, and it may remain viable for two weeks in marine water. One study indicated that EHEC O157:H7 is inactivated in slurry within two weeks; another suggested that it can survive up to three months. Possible person-to-person transmission has also been reported. (5)

Incubation Period

The incubation period for disease caused by EHEC O157:H7 ranges from one to 16 days. Most infections become apparent after 3-4 days. (5)

Clinical Feature

Humans can be infected asymptomatically or they may develop watery diarrhea, hemorrhagic colitis and/ or hemolytic uremic syndrome. Most symptomatic cases begin with diarrhea. Some cases resolve without treatment in approximately a week; others progress to hemorrhagic colitis within a few days. Hemorrhagic colitis is characterized by diarrhea with profuse, visible blood, accompanied by abdominal tenderness, and in many cases, by severe abdominal cramps. Some patients have a low–grade fever; in others, fever is absent. Nausea and vomiting may be seen, and dehydration is possible. Many cases of hemorrhagic colitis are self–limiting and resolve in approximately a week. Severe colitis may result in intestinal necrosis, perforation or the development of colonic strictures.

Hemolytic uremic syndrome occurs in up to 16% of patients with hemorrhagic colitis. This syndrome is most common in children, the elderly and those who are immunocompromised. It usually develops a week after the diarrhea begins, when the patient is improving. Occasionally, children develop HUS without prodromal diarrhea. HUS is characterized by renal failure, hemolytic anemia and thrombocytopenia. The relative importance of these signs varies. Some patients with HUS have hemolytic anemia and/or thrombocytopenia with little or no renal disease, while others have significant kidney disease but no thrombocytopenia and/or minimal hemolysis. Extra renal signs including CNS involvement with lethargy, irritability and seizures are common. In more severe cases, there may be paresis, stroke, cerebral edema or coma. Respiratory complications can include pleural effusion, fluid overload and adult respiratory distress syndrome. Elevation of pancreatic enzymes or pancreatitis may also be seen. Rhabdomyolysis and myocardial involvement are rare. The form of HUS usually seen in adults, particularly the elderly, is sometimes called thrombotic thrombocytopenic purpura (TTP).

In TTP, there is typically less kidney damage than in children, but neurologic signs including stroke, seizures and CNS deterioration are more common. Death occurs most often in cases with serious extra renal disease such as severe CNS signs. Approximately 65–85% of children recover from HUS without permanent damage; however, long-term renal complications including hypertension, renal insufficiency and end-stage renal failure also occur. Residual extra renal problems such as transient or permanent insulin-dependent diabetes mellitus, pancreatic insufficiency, gastrointestinal complications or neurological defects such as poor fine-motor coordination are possible. (5)

Diagnostic Tests

Because humans do not normally carry EHEC, clinical cases can be diagnosed by finding these organisms in fecal samples. Food and environmental samples may also be tested to determine the source of the infection. EHEC are sometimes difficult to identify. They are a minor population in the fecal flora or food. They also closely resemble commensal E. coli except in verocytotoxin production. However, the verocytotoxin alone does not necessarily identify an organism as EHEC; additional virulence factors must also be present. Many diagnostic laboratories can detect verocytotoxin-producing E. coli (VTEC) and identify EHEC O157:H7.There is no single technique that can be used to isolate all EHEC serotypes.

Selective and differential media have been developed for EHEC O157:H7, based on its lack of β-glucuronidase activity and the inability of most strains to rapidly ferment sorbitol. MacConkey agar containing 1% sorbitol (SMAC), often with cefixime and either rhamnose or potassium tellurite, is frequently used. Hemorrhagic colitis agar can be used to isolate EHEC O157:H7 from foods. Other media, including commercial chromogenic agars (e.g., rainbow agar), are also available. Because other strains of E. coli, as well as other bacteria, can grow on these media, prior enrichment for E. coli O157 aids detection, particularly in samples from food and the environment. For enrichment, samples may be cultured in liquid enrichment medium, or immunomagnetic separation (IMS) can be used to concentrate the members of serogroup O157 before plating. In IMS, magnetic beads coated with an antibody to the O157 antigen are used to bind these organisms.

Colonies suspected to be EHEC O157:H7 are confirmed to be E. coli with biochemical tests, and shown to have the O157 somatic antigen and H7 flagellar antigen with immunoassays. A variety of tests including enzyme-linked immunosorbent assays (ELISAs), agglutination, PCR, immunoblotting or Vero cell assay can be used to detect the verocytotoxin or its genes. Phage typing and pulsed field gel electrophoresis can subtype EHEC O157:H7 for epidemiology; these tests are generally done by reference laboratories. Subtyping is important in finding the source of an outbreak and tracing transmission. The techniques used to identify EHEC O157:H7 can miss atypical strains of this organism, including rare sorbitol-fermenting isolates. They are also ineffective for detecting EHEC O157:H–, which ferments sorbitol and is beta-glucuronidase positive. Identification of EHEC O157:H- is laborious, but it can be done by IMS followed by plating samples onto SMAC and testing individual sorbitol-fermenting colonies to detect the O157 antigen, verocytotoxins or their genes, and/or other virulence factors.

Selective media and isolation techniques have been developed for few non-O157 EHEC. IMS beads are commercially available for concentrating some common EHEC serogroups including O26, O103, O111 and O145. A selective rhamnose MacConkey medium containing cefixime and tellurite (CT-RMAC) is used to isolate and identify EHEC O26. Isolation of most non-O157 EHEC relies on screening colonies for verocytotoxin, the genes that produce this toxin and/or other virulence genes associated with EHEC. MacConkey agar or other media normally used to culture E. coli can be used to grow these organisms. Some prescreening techniques target specific serogroups or serotypes known to be associated with human EHEC disease. Techniques to identify most non-O157 EHEC are very labor-intensive, and these tests are not available at most laboratories. Immunological and nucleic acid-based tests that detect O and H antigens, verocytotoxin or various genes associated with EHEC can be used for presumptive diagnosis. These rapid tests can determine whether potential pathogens are present in samples before isolation. They include dipstick and membrane technologies, agglutination tests, microplate assays, colony immunoblotting, PCR, immunofluorescence and ELISAs. Although verocytotoxin production can aid identification, VTEC are not necessarily EHEC and additional virulence factors must usually be identified. Verocytotoxin-negative derivatives of the EHEC may occasionally be found by the time HUS develops. The results from rapid tests are confirmed by isolating the organism. In humans, EHEC may not be found in feces after one week. Serology is also valuable in humans, particularly later in the course of the disease when EHEC are difficult to find. Indirect ELISAs can detect antibodies to EHEC O157:H7 for months after infection. Cross-reactions with other bacteria can be seen. (5)

Treatment Treatment of hemorrhagic colitis is supportive, and may include fluids and a bland diet. Antibiotics are controversial and are usually avoided: they do not seem to reduce symptoms, prevent complications or decrease shedding, and they may increase the risk of HUS. The use of antimotility (antidiarrheal) agents in hemorrhagic colitis also seems to increase the risk for developing HUS. Patients with complications may require intensive care including dialysis, transfusion and/or platelet infusion. Patients who develop irreversible kidney failure may need a kidney transplant. (5)

Prevention

Frequent hand washing, especially before eating or preparing food, and good hygiene are important in preventing transmission from animals and their environment. Hand washing facilities should be available in petting zoos and other areas where the public may contact livestock, and eating and drinking should be discouraged at these sites. To protect children and other household members, people who work with animals should keep their work clothing, including shoes, away from the main living areas and launder these items separately. Two children apparently became infected with EHEC O157:H7 after contact with bird (rook) feces, possibly via their father’s soiled work shoes or contaminated overalls. After a number of outbreaks associated with camping in the U.K., the Scottish E. coli O157 Task Force has recommended that ruminants not be grazed on land for at least three weeks before camping begins.

Techniques to reduce microbial contamination during slaughter and meat processing can reduce the risk of EHEC from this source. Screening and control programs have been established for EHEC O157:H7 in meat. To prevent cross-contamination during food preparation, consumers should wash their hands, counters, cutting boards, and utensils thoroughly after they have been in contact with raw meat. Meat should be cooked thoroughly to kill E. coli. Unpasteurized milk or other dairy products and unpasteurized juices should be avoided.

Water that may be contaminated should not be used to irrigate vegetable crops, and untreated manure/ effluents should not be used on fruits or vegetables that will be eaten raw. Post-harvest measures include thorough washing of vegetables under running water to reduce bacterial numbers. Vegetables can also be disinfected with a dilute chlorine solution. It is safest to wash vegetables immediately before use; under some environmental conditions, populations of bacteria can build up again after a few days. EHEC carried internally in plant tissues are difficult to destroy except by irradiation or cooking.

Contamination of public water supplies is prevented by standard water treatment procedures. Livestock-should be kept away from private water supplies. Microbiological testing can also be considered. To the extent possible, people should avoid swallowing water when swimming or playing in lakes, ponds and streams.

Good hygiene, careful hand-washing and proper disposal of infectious feces can reduce person-to-person transmission. Thorough hand washing is especially important after changing diapers, after using the toilet, and before eating or preparing food. Bed linens, towels and soiled clothing from patients with hemorrhagic colitis should be washed separately, and toilet seats and flush handles should be cleaned appropriately. In some areas, regulations may prohibit infected children from attending daycare or school until they are no longer shedding organisms. Some authors suggest that isolating infected children from their young siblings or other young household members can significantly decrease the risk of secondary spread.(6)

References 1-	F.H. Kayser, K.A. Bienz, J.Eckert, R.M.zigernagel, 2005, Medical Microbiology, Germany. 2-	See Kauffmann-White-Schema in the German Wikipedia. 3-	cc O antigen at Dorlands Medical Dictionary. 4-	Technical report from European Food Safety Authority and European Center for Disease Prevention and Control 5-	The Center for Food Security and Public Health.Iwoa State University, College of Veterinary Medicine.+Institute for International cooperation in Animal Biologics,Iwoa State University. 6-	- Information sheet, January 2009, Community and Health Services Department.