Verotoxigenic Escherichia Coli Research Articles

Behaviour of E. coli 0157:H7 strains in model system and in raw meat by hpp: Microbial and technological aspects

Verotoxigenic Escherichia coli (VTEC) is an important pathogen which can cause serious illnesses in humans. It is of particular importance to the meat industry to be able to completely destroy E. coli. A technique used to destroy E. coli could be the high pressure process. For this reason a mixture of 8 strains of E. coli 0157:H7 was inoculated in raw minced meat and in broth. These samples were treated at pressures up to 700 MPa at WC, causing up to 5 decimal reductions. HP treatment is also effective in decreasing microbial contamination and increasing the shelf-life of raw minced meat under refrigerated conditions. The texture of raw minced meat improved with HP level, while the colour was less characteristic in HP-treated samples; a combined HP-treatment caused less colour change than an individual HP-treatment. No difference in colour of cooked minced meat was observed in untreated and HP-treated samples. The texture of cooked minced meat improved in HP-treated samples.

Application of a DNA hybridization-hydrophobic-grid membrane filter method for detection and isolation of verotoxigenic escherichia coli.

Verotoxigenic Escherichia coli (VTEC) strains were isolated from food and animal fecal samples by using PCR to screen for the presence of VTEC after broth enrichment and then filtering VTEC-positive cultures through hydrophobic-grid membrane filters (HGMFs) which were incubated on MacConkey agar. The filters were probed with a digoxigenin-labeled PCR product generated by amplification of a conserved verotoxin gene sequence. Replication of the growth on filters allowed probe-positive colonies to be picked. When ground beef samples were inoculated with VTEC strains, 100% of the strains were recovered, and the detection limit was 0.1 CFU per g. Similar results were obtained with seven types of artificially contaminated vegetables. A survey of 32 packages of vegetables and 23 samples of apple cider obtained at the retail level did not reveal the presence of VTEC. However, the intestinal fecal contents of a moose, 1 of 35 wild mammals and birds examined, contained E. coli O157:H7. The DNA hybridization-HGMF method was also used in a prevalence survey of 327 raw and 744 ready-to-eat products; VTEC strains were recovered from 4.9% of the raw products and 0.7% of the ready-to-eat products. No serotype O157:H7 strains were detected. This method is particularly suited for surveys in which low numbers of VTEC-positive samples are expected and isolates are required.

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC.

Certain strains of verotoxigenic Escherichia coli (VTEC), and in particular those belonging to serogroup O157, cause attaching and effacing (AE) lesions of the host gut mucosa during pathogenesis. The mechanisms involved with bacterial attachment and the destruction of microvilli are determined by a cluster of genes within the LEE region, which also encode five secreted proteins. Sera from patients with antibodies to the lipopolysaccharide (LPS) of E. coli O157 and other VTEC were tested for antibodies to these secreted proteins. Twenty-one of 34 (62%) sera with antibodies to the lipopolysaccharide (LPS) of E. coli O157 also contained antibodies to one or more of the secreted proteins. Five of 12 sera containing antibodies to the LPS of a range of other VTEC serogroups also contained antibodies to 1 or more of the 5 secreted proteins, as did 16 of 70 (23%) sera from patients with haemolytic uraemic syndrome (HUS), haemorrhagic colitis (HC) or diarrhoea, but without bacteriological evidence of infection with VTEC and which did not contain antibodies to VTEC serogroups O5, O115, O145, O153 or O157. The detection of serum antibodies to secreted proteins may provide additional information for interpreting the results of established lipopolysaccharide-based VTEC serology.

Survey of Ontario Bulk Tank Raw Milk for Food-Borne Pathogens

Raw (unpasteurized) milk can be a source of food-borne pathogens. Raw milk consumption results in sporadic disease outbreaks. Pasteurization is designed to destroy all bacterial pathogens common to raw milk, excluding spore-forming bacteria and possibly Mycobacterium paratuberculosis, but some people continue to drink raw milk, believing it to be safe. Current methods for assessing the bacteriological quality of raw milk, such as aerobic plate counts, are not usually designed to detect specific pathogens. The objective of this study was to estimate the proportion of pick-ups (loads of raw milk from a single farm bulk tank) from Ontario farm bulk tanks that contained Listeria monocytogenes. Salmonella spp., Campylobacter spp., and/or verotoxigenic Escherichia coli (VTEC). Samples from 1,720 pick-ups of raw milk were tested for the presence of these pathogens, and 47 L. monocytogenes, three Salmonella spp., eight Campylobacter spp., and 15 VTEC isolates were detected, representing 2.73, 0.17, 0.47, and 0.87% of milk samples, respectively. Estimates of the proportion of theoretical tanker truck loads of pooled raw milk contaminated with pathogens ranged from a low of 0.51 % of tankers containing raw milk from 3 bulk tanks being contaminated with Salmonella spp. to a high of 34.41 % of tankers containing raw milk from 10 bulk tanks being contaminated with at least one of the pathogens. Associations between the presence of pathogens and raw milk sample characteristics were investigated. The mean somatic cell count was higher among VTEC- or L. monocytogenes-positive samples, and the mean aerobic plate count was found to be higher among L. monocytogenes-positive samples. These results confirm the presence of bacterial food pathogens in raw milk and emphasize the importance of continued diligence in the application of hygiene programs within dairies and the separation of raw milk from pasteurized milk and milk products.

Hemolysin phenotypes and genotypes of eaeA-positive and eaeA-negative bovine verotoxigenic Escherichia coli.

Intimin or EaeA protein has been implicated in the attaching/effacing lesion caused by entero-hemorrhagic Escherichia coli (EHEC) in the intestine but it is not produced by all EHEC and is therefore not adequate as a marker for EHEC. Hemolysins are produced by a high percentage of verotoxigenic E. coli (VTEC) and could be a marker for EHEC, but their distribution and relation to virulence are not known. We used PCR amplification to determine the presence or absence of eaeA sequences in 281 VTEC isolates from the feces of healthy cattle. There were 101 eaeA-positive isolates, which belonged to O groups 5, 26, 69, 80, 84, 98, 103, 111, 119, 145, 157 and 108 eaeA-negative isolates, which belonged to O groups 8, 22, 38, 113, 119, 116, 132, 153, 156 or untypable. All isolates were tested for hemolysis on horse blood agar and on washed sheep blood agar. PCR amplification was used to test for EHEC hemolysin, Ehly1, Ehly2 and alpha-hemolysin D sequences. Among eaeA-positive isolates 98% were positive for EHEC hemolysin sequences and were hemolytic on washed sheep red blood cell agar; the corresponding percentage for eaeA-negative isolates was 36%. Ehly1 and Ehly2 sequences were present in only 11 isolates (O groups 26, 84, 119 and 132). None of the eaeA-positive and 13 of the eaeA-negative isolates (including all 11 isolates of O group 132) were positive for alpha-hemolysin D gene sequences by PCR and alpha-hemolysin production on horse blood agar. We conclude that since Ehly1, Ehly2 and alpha-hemolysin occur at low frequency among bovine VTEC and serotypes implicated in human disease they are unlikely to be significant virulence factors. In contrast, EHEC hemolysin was present in almost all eaeA-positive VTEC isolates and in approximately one-third the eaeA-negative ones; it may be both a virulence marker and virulence factor but further testing is required. The study identified isolates with all combinations of eaeA and EHEC hly, which may be useful for further testing.

Influence of substrate and low temperature on growth and survival of verotoxigenic Escherichia coli

Growth and survival of verotoxigenic Escherichia coli(VTEC) were studied in tryptic soy broth (TSB), brain–heart infusion (BHI), and whole milk at 12, 9.5, 8.5, 7.5, 6.5, and 5.5°C (±0.2°C). Colony forming units (cfu) were enumerated by surface plating on eosin methylene blue (EMB) agar plates and incubating at 37°C for 24 h. Growth curves were plotted and lag and generation times were determined. Results indicated that the average generation times for each strain was similar in each medium (4.6±0.9 h) at 12°C. However, at 9.5°C the average generation time differed significantly among the media ( P<0.05). The average generation time was longer in BHI compared to TSB and whole milk at 9.5°C (14 h in BHI vs 11 h in milk and TSB). Also, the generation time of individual strains in BHI varied from 9.5–22 h at 9.5°C. Decrease of temperature to 8.5°C resulted in lack of growth of two strains in BHI. At 7.5°C none of the strains grew in BHI, one grew in TSB, whereas all grew in milk. A further decrease of temperature to 6.5°C did not allow growth of any of the strains in TSB or BHI whereas all grew in milk. Addition of 5% lactose to TSB and BHI enabled survival and growth of all strains at 6.5°C. The results of this study demonstrate the influence of growth medium composition on the minimum growth temperature of verotoxigenic E. coli.

Prevalence and characteristics of human and bovine verotoxigenic Escherichia coli strains isolated in Galicia (north-western Spain).

An epidemiological study was carried out to determine the incidence and the serotypes of verotoxigenic Escherichia coli (VTEC) that cause infections in Galicia (north-western Spain). Although, VTEC strains were isolated from 55 (14%) of the 387 calves sampled and the majority of bovine VTEC strains belonged to serotypes (026:H11 or H-, 091:H21, O103:H2, 0105:H18, O111:H-O113:H21, O126:H-, O128:H- and O157:H7 or H-) previously associated with human haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) in other countries, VTEC are not a common cause of human infections in Spain. Thus, VTEC (O26:H11 and O86:H10) were isolated from only 3 (0.6%) of the 482 children with diarrhoea investigated. We examined the 69 (3 humans and 66 bovines) VTEC strains that were initially isolated as E. coli producing a toxin cytotoxic to Vero and HeLa cells by polymerase chain reaction (PCR) using specific primers for VT1, VT2 and eae genes. PCR showed that 38 (55%) of VTEC strains carried VT1 genes. 18 (26%) possessed VT2 genes, and 10 (14%) carried both VT1 and VT2 genes. Three (one human and two bovine) strains which were formerly VTEC had lost the ability to produce verotoxins upon subculture and became negative for VT1 and VT2 by PCR. In total 35 (51%) of 69 VTEC strains, including the two human VT1+ strains of serotype O26:H11, were positive for eae sequences when tested by PCR. Presence of the eae gene was significantly more frequent (100%; 21/21) among VTEC strains with serotypes (O26:H11, O111:H-, O157:H-and O157:H7) considered as enterohaemorrhagic E. coli (EHEC) than among VTEC strains with non-EHEC serotypes (29%; 14/48) (p < 0.001). Results obtained in this study indicate that cattle may be an important source of VTEC involved in human disease. However, severe clinical syndromes caused by VTEC, such as HC and HUS, are uncommon in Spain, in comparison with North America and the UK. In any case, VTEC disease can appear on the scene very suddenly, as occurred in the UK and North America in the 1980s.

Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario.

This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA gene showed that 151 (35.2%) strains were positive for the eaeA gene. All isolates (9-21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7-34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA-negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle.

Virulence factors and phenotypical traits of verotoxigenic strains of Escherichia coli isolated from human patients in Germany.

Fecal isolates of Escherichia coli which were collected from human patients in different parts of Germany between 1985 and 1992 were examined for production of verotoxins (VT). Among 2165 isolates 54 (2.5%) verotoxigenic E. coli (VTEC) were found. The 54 VTEC belonged to 13 different serotypes, 46 (85.2%) of these were enterohemorrhagic E. coli (EHEC) types as O157:H7, O157:H-, O145:H-, O111:[H8] and O26:[H11]. Of the 54 VTEC 50 (92.6%) hybridized with one or both of the DNA probes specific for VT1 and VT2. The 4 VTEC strains which were negative for VT1 and VT2 differed from all other VTEC by many phenotypical trains such as serotype, production of alpha-hemolysin and absence of EHEC-plasmid and "attaching and effacing" (eae)-specific DNA sequences. In contrast, VTEC which were positive for VT1, VT2 or both were frequently positive for eae sequences (92.0%), EHEC-plasmids (90.0%) and for production of enterohemolysin (88.0%). With enterohemolysin as an epidemiological marker more VTEC strains (81.5%) could be identified than with others such as the absence of beta-glucuronidase activity (61.1%) or non-fermentation of sorbitol (48.1%). Case reports were available for 42 of the 54 VTEC strains. The clinical presentation of 42 cases with VTEC ranged from uncomplicated diarrhea to severe diseases as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). However, bloody diarrhea, HC and HUS were more associated with the O157 group than with other VTEC groups.

Verotoxigenic (enterohaemorrhagic) Escherichia coli in infants and toddlers in Czechoslovakia

The results of the investigation indicate that verotoxigenic Escherichia coli (VTEC) belonging to enteropathogenic and other serogroups including Escherichia coli O157:H7 or H- are important enteropathogens in infants and toddlers in Czechoslovakia. As to enteropathogenic serotypes, verotoxin (VT) production was proved most frequently in strains of serogroup O26, and also O111 and O128. Diseases caused by them were as a rule manifested by febrile watery diarrhoea with mucus in the stool. In two of five infants with Escherichia coli O26 :H11 with VT1 production in titres of greater than or equal to 1:512 (blood was present) in the stool and one suffered from marked abdominal pain. In one infant haemorrhagic colitis due to Escherichia coli O157:H- was found. Haemolytic uraemic syndrome associated with VTEC of serogroups O157, O26, O18, O5 and O1 with VT1 and/or VT2 was observed in six children including five who contracted the disease during an outbreak in a small town, and the source of infection was probably contaminated water. Five children recovered and one died; the postmortem examination revealed haemorrhagic colitis and necrosis of the renal cortex. Haemorrhagic colitis caused by Escherichia coli O157 in infants and toddlers differed from the course hitherto described in older subjects by fever and the presence of mucus in the stools.