E.coli O157:h7

Introduction

According to the Latin proverb, we are born between the urine and faeces.Thus from birth, we acquire the faecal flora of our mothers.

Over a century ago,Escherich described the bacteria that he isolated from the faeces of human neonates as Bacterium colicommune (Bettelheim,1986; Escherich,1988).He demonstrated that the organisms now known as Escherichia coli were present in the faeces and intestinal contents of humans and were considered as commensal organisms.

 Most E.coli strains are harmless commensals, however, some strains are pathogenic and cause diarrhoeal disease. E.coli strains that cause diarrhoeal illness are categorized into specific groups based on virulence properties, mechanisms of pathogenicity, clinical syndromes and distinct O: H serogroups. These categories include enteropathogenic E.coli strains (ETEC), enteroinvasive E.coli strains (EIEC), diffuse adhering E.coli strains (DAEC), enteroaggregative E.coli strains (EAggEC) and enterohaemorrhagic E.coli strains (EHEC).

 EHEC were first identified as human pathogens in 1982, when E.coli of serotype O 157:H7 was associated with two outbreaks of haemorrhagic colitis. All EHEC produce factors cytotoxic to African green monkey kidney (VERO) cells, which have been described as verotoxins (VTs).E.coli O157:H7 and many serotypes of E.coli have subsequently shown to produce VTs; hence they have named VT-producing E.coli (VTEC).

 E.coli O157:H7

 The first confirmed isolation of E.coli O157:H7 in the United States was in 1975 from a California woman with bloody diarrhoea.The first reported isolation of E.coli O157:H7 from cattle was from a less than 3 week old calf with colibacillosis in Argentina in 1977. The bacterium was first identified as a human pathogen in 1982, when it was associated with two food borne outbreaks of Hemorrhagic colitis.

 Since then O157 VTEC have been identified in many outbreaks and in sporadic cases of bloody diarrhoea in North America and Great Britain and a close association has been established between VTEC and haemorrhagic uremic syndrome (HUS).

 Characteristics of E.coli O157:H7

 Most strains of E.coli O157:H7 possess several characteristics uncommon to most other E.coli.

 i) Acid tolerance: Unlike most food borne pathogens, E.coli O157:H7 uniquely tolerant to acidic environments. Acid tolerance is a complex phenomenon, both growth phase dependant and inducible.E.coli cells in stationary phase of growth are substantially more acid tolerant than cells in the exponential phase. This increased tolerance is associated with expression of genes regulated by the rpoS sigma factor operon(Cheville et al.1996;Rowbury,1998; Small et al.1994) examined three mechanisms of acid resistance, that is,oxidative-arginine dependent and glutamate dependent, and found that all three contribute to the microorganisms overall acid tolerance. Induction of acid tolerance in E.coli can enhance its survival in acidic foods (Cheville et al, 1996; Layer et al, 1995).

 (ii) Antibiotic resistance: Sufficient evidence indicates that the bacterium is resistant to most antibiotics.

 (iii) Thermal inactivation: Studies on the thermal sensitivity of E.coli O157:H7 in ground beef have revealed that the pathogen has no unusual resistance to heat, with D values at 57.2°C,60°C, 62.8°C and 64.3°C of 270,45,24 and 9.6 s respectively. Pasteurization of milk(72°C,16.2s) has also been determined to be an effective treatment that will kill more than 104 E.coli O157:H7 cells per ml(D’Aoust et al,1988).Proper heating of foods of animal origin (63°C) is an important critical control point to ensure activation of E.coli O157:H7.

 (iv) Inability to grow well: It is important to note that many VTEC strains do not grow well at 44°C, if at all, above 44°C.The minimum growth temperature for E.coli O157:H7 under otherwise optimal condition is approximately 8 to 10°C (Buchanan&Bagi, 1994).

 (v) Inability to ferment sorbitol within 24 hours: Most but not all O157 VTEC strains do not ferment sorbitol.

 (vi) Inability to produce ß-glucoronidase: Most of the O157 VTEC strains will not hydrolyze 4-methyl umbelliferyl-D-glucoronide.

 (vii) Inability to produce gas and indole at 44°C: Hence such methods would probably fail to detect VTEC.

 (viii) Possession of an attaching and effacing (eae) gene: This property contributes to VTEC to establish its pathogenicity.

 (ix) Carriage of a 60-MDa plasmid: E.coli O157:H7 isolates associated with human illness harbour a plasmid (pO157) of approximately 60 MD and that contains DNA sequences common to plasmids present in other serotypes of VTEC isolated from patients with Haemorrhagic colitis. It was hypothesized that the plasmid is believed to play a role in the pathogenicity of disease, but its function is unclear.

 (x) Expression of an uncommon 5,000 to 8,000 molecular weight outer membrane protein.

 Reservoirs and sources of E.coli O157:H7

 Several reservoirs and sources of E.coli O157:H7 have been identified. The association of E.coli O157:H7 with undercooked ground beef and raw rice led to investigations of the role of cattle as a reservoir of the pathogens. Several surveys of faecal shedding of E.coli O157:H7 produced the following general observations.

Young animals tend to carry E.coli O157:H7 more frequently than adults(Zhao et al.,1995)

Prevalence of faecal excretion varies substantially among positive herds (Zhao etal.,1995).

 E.coli O157:H7 levels in calf faeces range from less than 10 2 CFU/g to 10 5 CFU/g (Zhao et al.,1995)

 Faecal shedding of E.coli O157:H7 frequently is intermittent and of short duration, i.e. several weeks to months (Brown et al.,1997; Cray & Moon,1995)

 More than one strain of E.coli O157:H7 can be isolated from faeces of the same animal or different animals within the same herd (Faith et al., 1996; Ming et al.,1995).

 Calves have been experimentally infected with E.coli O157:H7 (Brown et al.,1997; Cray and Moon,1995).The results revealed that,

 1. E.coli O157:H7 is not pathogenic to calves. Inoculation with 1010 CFU did not induce significant clinical disease.

 2. E.coli O157:H7 is continued to the gastrointestinal tract, with the fore stomachs (rumen, reticulum and omasum) and distal sites (distal ileum, proximal caecum, spiral colon and descending colon) being the principal sites of localization.

 3. E.coli O157:H7 did not form attaching and effacing lesions and did not colonize mucosal surfaces.

 The prevalence of E.coli O157 in cattle has been reported to range from 0.1 to 16%.The organism has also been isolated from the faeces of geese, sheep, horses, dogs, seagulls, goats and deer. The organism has also been isolated from environmental sources such as cattle, water troughs and soil.

 Sources of E.coli O157:H7 for cattle have not been clearly identified. Possible sources include contaminated feedstuffs or water,colonized animals in herds,infected wildlife and humans or contaminated facilities and equipment surfaces from contact with faeces.

 Transmission of E.coli O157:H7

 Although a variety of foods have been implicated in E.coli O157:H7 associated illness, most outbreaks have been associated with consumption of raw or undercooked foods of bovine origin. E.coli O157:H7 infections also were associated with eating other foods, including vegetables, apple cider, cantaloupe, mayonnaise-containing salad dressing and salami. Contact of foods with E.coli O157:H7 containing meat or faeces (human or bovine) is a likely source of cross-contamination. Person-to-person transmission (13.2%) and waterborne (4.4%) outbreaks have been documented.

The mechanism of transmission in food chain is not fully understood, but contamination of meat from intestinal contents at slaughter is probably an important factor.

 Detection of E.coli O157:H7

 i) meat: It is not easy to detect VTEC in raw meats and foods where low levels of E.coli may be swamped by high numbers of other bacteria. So far, there are no widely recommended methods for routine food examination. Currently traditional isolation methods for foods involve enrichment in a selective broth followed by plating on to sorbitol Mac conkey agar with additives. This agar is only suitable for 0157:H7 strains (most but not all 0157 VTEC strains do not ferment sorbitol).The composition of the enrichment broth and plating agar is important if VTEC is to be isolated from contaminated materials and several researches are going on determining the optimum combination of selective agents.

 (ii) Stool: Methods used in medical laboratories to detect the organism from stools are more successful, probably the number of VTEC cells present in the stools of someone made ill by the organism is relatively high in comparison to the background flora. For E.coli O157 (but not all VTEC serotypes) commercial kits are available for isolation and identification (ELISA methods) and for confirmation of suspect colonies (latex agglutination).Recipes for several effective broths and agars have been published but there is no consensus yet on which is the best. Another technique used to enhance isolation of VTEC from enrichment broths is the use of commercially available immunomagnetic beads coated with specific O157 antiserum. An immunoblotting technique is available for rapid identification of O157 colonies on agar plates.

 For epidemiological purpose/surveillance O157 VTEC can be distinguished by phage typing (Ahmed et al., 1987) which can be combined with typing of the VT genes to give added discrimination.

 Other methods such as plasmid profile analysis, pulsed field gel electrophoresis and multilocus enzyme electrophoresis may further differentiate O157 VTEC and can be applied to VTEC of other serogroups. Restriction fragment length polymorphism analysis of genomic DNA probed with phage ë or the DNA of a VT encoding phage have also been used to differentiate O157 VTEC strains. Thus for surveillance, isolation of E.coli O157 should be attempted using readily available methods (e.g.: cefixime tellurite sorbitol Mac conkey Agar) and testing of non-forming sorbitol colonies with 0157 antiserum. Biochemical and serological confirmation tests may also be needed to exclude false positives.VTEC strains other than 0157 do not have biochemical markers that assist in their identification.

 The general methods which rely on enrichment followed by plating onto selective agar and biochemical confirmation all at 37°C would be more likely to isolate VTEC. Method developed using cultural and rapid techniques are progressing fast.

 Characteristics of the disease

 Fortunately infection from E.coli 0157 is relatively rare. Its principal symptom, diarrhoea, is also a symptom of other gastrointestinal infections. This has meant that the relatively few E.coli O157 have led to be found from among many more infections with this routine symptom.

 E.coli O157 infections are associated with a range of illness in humans, although a proportion may be asymptomatic. Where symptoms do occur, the incubation period is 2 to 10 days, with most cases occurring in 3 days.

 The range of clinical disease includes:

 Mild diarrhoea, fever, abdominal pain, vomiting

 Hemorrhagic colitis (HC), which consists of inflammation of the large bowel, with severe blood.

 Haemolytic Uraemic syndrome (HUS), a combination of anaemia, acute kidney failure and low platelet count which may be accompanied by fever.

 Thrombotic thrombocytopenic purport (TTP) characterized by fever, via skin and central nervous system involvement, resulting from aggregation of platelets in various

 organs.

 HUS largely affects children and TTP largely affects adults.TTP is a rare syndrome of E.coli O157:H7 infection.

 Immunity to E.coli O157:H7

 An infected patient’s serological response against surface epitopes of E.coli O157:H7 can last from weeks to months. It can be useful for epidemiological studies to determine the serological responses of patients suspected of E.coli O157:H7 infection when stool cultures are negative for recovery of E.coli O157:H7. However, such studies are limited by the fact that all patients who were E.coli O157:H7 culture positive have a demonstrable antibody response. Oral inoculation of calves and steers with 1010 E.coli O157:H7 induced prompt and sustained increases in serum antibodies to the O157 antigenic LPS and to a lesser extent to Stx 1(Johnson et al., 1996).The serological responses, however, do not correlate with elimination of carriage by cattle or protection of calves against reinfection by the same strain. The ability of E.coli O157:H7 to persist in and reinfect cattle that have a strong immune response is likely to contribute to the introduction and persistence of infection in herds.

Prevention and control methods

The prevention of infection requires control measures at all stages of the food chain from agricultural production on the farm, to processing, manufacturing and preparation of foods in both commercial establishments and the domestic environment.

 There are insufficient data to recommend specific intervention methods on the farm in order to reduce the incidence of E.coli O157:H7 in cattle and other ruminants.

 Farms: An important component of Hazard Analysis Critical Control Point (HACCP) application in animal production is reducing the carriage of E.coli O157:H7 by animals. Two approaches that have potential are competitive exclusion and vaccination. Competitive exclusion involves the use of microbial cultures that out compete pathogens from colonizing specific niches. This approach uses defined bacterial cultures that can greatly reduce colonization of campylobacter jejuni in poultry (Schoeni and Doyle,1992).

 Vaccination: Traditional vaccination approaches are not likely to be successful with E.coli O157:H7.Recent observations showed that E.coli O157:H7 does not form attaching or effacing lesions or colonize mucosal surfaces of the gastrointestinal tract (Brown et al., 1997; Cray and Moon, 1998) and cattle exposed to E.coli O157:H7 are not protected from reinfection (Johnson et al., 1996).Hence innovative approaches will be needed for vaccines to be effective.

 Slaughterhouse: Like other E.coli, it is assumed that the ultimate source of E.coli O157:H7 in carcasses is faecal contamination during animal production and slaughter operations. Faecal contamination is associated primarily with contamination of the carcass during hide removal and spreading of contamination to other carcasses by equipment and workers hands (Dickson & Anderson, 1992).

 Quality assurance programmes in slaughter houses should stress the need to minimize the faecal contamination of carcasses and to chill meat rapidly. Screening of raw meats for VTEC is not an effective control mechanism because isolation rates from raw beef are low and the organism has been found in the faeces of a small proportion of healthy cattle, so currently it is unlikely that it can be eliminated at source. Simple and reliable methods suitable for routine VTEC detection in foods are not widely available. Similarly, because of the low contamination rate of VTEC in other foods, routine screening specifically for this organism is unlikely to be worthwhile or successful.

 As screening can never detect all contaminated loss, it is a poor control procedure. Adequate cooking of meat is the only sure way of eliminating the danger of VTEC infection from this source.

 For most food manufacturers, surveillance of raw and in-process materials, finished products and the manufacturing environment should be based on needs identified by HACCP evaluation, and the end product specification. Monitoring trends of indicator organisms and standard plate count indicate deviations from quality standards.

 Food processing: E.coli O157:H7 can be controlled readily through traditional thermal processing techniques.

 Recommendations to reduce the risk of acquiring an E.coli O157:H7 infection.

 1. Cook ground beef thoroughly (minimum 160°F) before eating.

 2. Drink only pasteurized milk and apple juice.

 3. Wash fresh fruits and vegetables thoroughly before eating.

 4. Wash hands thoroughly after handling animals, particularly cattle,deer,goats or dogs.

 5. Wash hands thoroughly after changing diapers or after providing care to children or adults suffering from a diarrhoeal disease.

 6. Do not use fresh manure from ruminants to fertilize vegetables or fruits.

 7. Avoid swimming in lakes or ponds used by cattle and drinking surface water that has not been properly treated to eliminate pathogens.

 Conclusion and recommendations

 The serious nature of the symptoms of haemorrhagic colitis and HUS and the apparent low infectious dose (<100 cells) of E.coli O157:H7 places this food borne pathogen a most serious of known food borne pathogens.

 Persuasive evidence suggests that healthy cattle are a reservoir of O157 and they can enter the food chain to provide a source of exposure for humans. A possible route of transmission of O157 VTEC may involve infections initially in calves that shed their organism into faecal slurry that may be used on grazing grass. This provides potential for infection of other animals from which the organism may contaminate milk or carcasses at slaughter. Possible sources of VTEC in healthy animals other than cattle and a wider range of foodstuffs require further investigation.

 Many features of E.coli O157:H7 strains remain poorly understood. It includes:

 (i) Role of virulent genes in the animal,

 (ii) Mechanism of evolution of the organism,

 (iii) The progress of individual cases of E.coli O157:H7 infection, and

 (iv) The difference in incidence of infection in different geographical areas.

 References

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 2. Bettelheim,K.A., (1986): Zentralbl Bacteriology I Abstract Original A 261:255.

 3. Brown, C.A., Harmon, B.G., Zhao, T., and Doyle, M.P. (1997): Applied Environmental Microbiology 63:27-32.

 4. Brunton,J.,(1994): Molecular biology and role in disease of the verotoxins(Shiga-like toxins) of Escherichia coli,pgs:391-404.In V.L.Miller, J.B.Kapper,D.A.Portnoy and R.R.Isberg (ed.) Molecular Genetics of BacterialPathogenesis, American soc. for Micro.Washington,D.C.

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 8. D’ Aoust, J.K., Park, C.E., Szabo, R.A., Todd, E.C.D., Emmons, D.B and Mckellar, R.C., (1988): Journal of Dairy science 71: 3230-3236.

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 13. Leyer, G.J., Wang, L.L and Johnson, E.A. (1995): Applied Environmental Microbiology 61:3752- 3755.

 14. Ming, J., Zhao,S., Zhao,T. and Doyle,M.P.(!995): Journal of Medical Microbiology 42:258-263

 15. Rowbury, R.I. (1995): Letters in Applied Microbiology 20:333-337.

 16. Small,P., Blankenhom,D., Welly,D., Zinner,E. and Sloncreweki, J.L. (1994): Journal of Bacteriology 176:1729-1737.

 17. Zhao,T.,Doyle,M.P.,Share,J.,and Garber,C. (1995): Applied Environmental Microbiology 61:1290-1293.

Source by Dr.Anjum Sherasiya

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