Examination of retail chickens and sausages in Britain for vero cytotoxin-producing Escherichia coli.

Samples from chickens and pork sausages were examined for the presence of Vero cytotoxin-producing Escherichia coli by using DNA probes for the Vero cytotoxin genes. Hybridization was detected in 25% of the 184 sausage samples, but none of the chickens was positive. No E. coli O157:H7 strains were isolated, and serotyping showed that the Vero cytotoxin-producing E. coli strains belonged to eight different O serogroups and that six strains had an unidentifiable O antigen.     

Cefixime–tellurite rhamnose MacConkey agar for isolation of Vero cytotoxin-producing Escherichia coli serogroup O26 from Scottish cattle and sheep faeces

Aims:  To compare rhamnose MacConkey agar supplemented with cefixime and tellurite (CT-RMac) and tryptone bile X-glucuronide (TBX) agars as isolation media for Vero cytotoxin-producing Escherichia coli (VTEC) serogroup O26 from animal faeces.
Methods and Results:  Nine VTEC O26 were isolated from sheep faeces; out of which six were isolated only on CT-RMac and one was isolated only on TBX. One hundred and twelve VTEC O26 were isolated from calf faeces; out of which 97% were from CT-RMac and 52% were from TBX. In a study of E. coli O26 strains, 84% of VT-positive O26 did not ferment rhamnose when compared with 16% of VT-negative O26. VT-positive (19%) and VT-negative (39%) E. coli O26 strains did not grow on CT-RMac agar.
Conclusions:  It is important to consider that VTEC O26 strains either may ferment rhamnose or may be sensitive to the CT supplement of CT-RMac agar.
Significance and Impact of the Study:  This work compares CT-RMac and TBX agars as isolation medium for VTEC O26 from Scottish animal faeces and highlights that VTEC O26 may be missed if only CT-RMac agar is used.     

Isolation of vero cytotoxin-producing Escherichia coli O157 from wild birds

In a survey of wild birds (mainly gulls), 0.9% of the bacterial isolates from faecal samples at an urban landfill site and 2.9% of bacterial isolates from faecal samples on intertidal sediments in Morecambe Bay were Vero cytotoxin-producing Escherichia coli O157. Isolation procedures employing commonly used cultural methods were hindered by the selection of a large number of false positives. The only procedure which resulted in the isolation of E. coli O157 from bird faecal samples was: enrichment (18 h) in a selective tryptone soya broth followed by filtration using hydrophobic grid membranes and growth on Chromagar O157. The majority of isolates selected as potential E. coli O157 by characteristic growth on Chromagar O157 could be eliminated by subsequent growth on CT-SMAC or CR-SMAC. This second identification (characterization) stage reduced the number of potential E. coli O157 requiring further confirmation by typing methods (serotype and Vero cytotoxin) by more than 70%.     

Beta-glucuronidase activity of Vero cytotoxin-producing strains of Escherichia coli, including serogroup 0157, isolated in the United Kingdom

One hundred and twenty Vero cytotoxin-producing (VT⁺) strains of Escherichia coli 0157 (of H type 7 or non-motile) isolated in the UK, failed to ferment sorbitol after 24 h or to hydrolyse 4-methylumbelliferyl-beta- d -glucuronide (MUG). This combination of properties was not found in 167 of 169 other E. coli strains, including VT⁺strains of other serogroups and VT^-strains of serogroup 0157. As the two exceptions were rare VT^-strains of serotype 0157: H7, we conclude that, although biochemical tests can aid the isolation of VT⁺0157 strains, confirmation of VT production is necessary.     

Prevention of drug-resistant Escherichia coli colonization in chickens by treatment with a faecal fluid

This study was performed to prove that intestinal colonization in chickens by resistant Escherichia coli strains present in the environment might be prevented when faeces in which sensitive E. coli strains were dominant was administered to newly hatched chicks. The appearance of resistant E. coli strains was markedly reduced. Escherichia coli O49:H12 was the sensitive E. coli strain which formed the major colonizer in the intestinal tract. In young chickens, this strain persisted as a major component, and even when it was a minor colonizer in the faecal fluid administered, it appeared as a major component soon afterwards. This strain is considered to be a good colonizer in the gut of young chickens.     

Prevention of drug-resistant Escherichia coli colonization in chickens by treatment with a faecal fluid

This study was performed to prove that intestinal colonization in chickens by resistant Escherichia coli strains present in the environment might be prevented when faeces in which sensitive E. coli strains were dominant was administered to newly hatched chicks. The appearance of resistant E. coli strains was markedly reduced. Escherichia coli O49:H12 was the sensitive E. coli strain which formed the major colonizer in the intestinal tract. In young chickens, this strain persisted as a major component, and even when it was a minor colonizer in the faecal fluid administered, it appeared as a major component soon afterwards. This strain is considered to be a good colonizer in the gut of young chickens.     

Escherichia coli serogroup O107/O117 lipopolysaccharide binds and neutralizes Shiga toxin 2

The AB(5) toxin Shiga toxin 2 (Stx2) has been implicated as a major virulence factor of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains in the progression of intestinal disease to more severe systemic complications. Here, we demonstrate that supernatant from a normal E. coli isolate, FI-29, neutralizes the effect of Stx2, but not the related Stx1, on Vero cells. Biochemical characterization of the neutralizing activity identified the lipopolysaccharide (LPS) of FI-29, a serogroup O107/O117 strain, as the toxin-neutralizing component. LPSs from FI-29 as well as from type strains E. coli O107 and E. coli O117 were able bind Stx2 but not Stx1, indicating that the mechanism of toxin neutralization may involve inhibition of the interaction between Stx2 and the Gb(3) receptor on Vero cells.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT-. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.     

Investigation of shiga toxin-producing Escherichia coli in avian species in India

AIMS: To investigate the presence or absence of shiga toxin-producing Escherichia coli (STEC) in avian species in India. METHODS AND RESULTS: Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence of E. coli. A total of 426 (chicken, 401; pigeons, 25) E. coli strains were isolated. Of 426 E. coli strains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection of stx(1), stx(2), eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of stx(1), stx(2) or their variants and saa genes. Overall 11 (2.74%) and seven (1.74%) strains from chickens possessed eaeA and hlyA genes, respectively, while as only six (1.49%) strains from chickens possessed both eaeA and hlyA genes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5.63%), 23 (5.39%), 23 (5.39%) and 20 (4.69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied. CONCLUSIONS: STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings. E. coli strains originating from birds are carrying a low percentage eaeA or hlyA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India.     

Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique

There has been no culture method of choice for detecting non-O157 Shiga toxin-producing Escherichia coli strains (STEC) because of their biochemical diversity The aim of this study was the assessment of verotoxin gene detection (VT1/VT2) within STEC PCR compared with the Vero cells cytotoxicity among O157 and non-O157 STEC serotypes. Stool cultures were performed on Tryptic Soy Broth and sorbitol MacConkey agar with cefixitime and tellurite supplements which were identified as Escherichia coli (E. coli) by BBL crystal. Further identifications were performed including verotoxin production assessment by Vero cells cytotoxicity assay, PCR for specific VT1/VT2 genotyping, and isolates were plated on blood agar and tested for enterohemolysis. Vero cells cytotoxicity assay revealed that 58 of E. coli isolates (71.6%) were STEC. In PCR, 33 (56.9%) of the 58 strains were positive for the VT2 gene, 24 (41.4%) were positive for the VT1 gene and one isolate was positive for both genes. In comparison to Vero cells cytotoxicity, the sensitivity, specificity of PCR were 100%. In comparative study between verotoxin assessment by Vero cells cytotoxicity and enterohemolytic activity, concordance positive results between both were 53 (91.4%). The most common serogroups of STEC were O157 (33%) and O26 (20%). From this study we can conclude that enterohemolysin production can be used as surrogate marker for STEC. The most rapid and promising approach for detection of STEC is by molecular method.     

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.     

Examination of recovery in vitro and in vivo of nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Comparison of Vero cell assay, polymerase chain reaction and an enzyme immunoassay for identification of verocytotoxin-producing Escherichia coli O157:H7

This study evaluated three different analytical methods for identification of Verocytotoxin-producing E. coli O157:H7 (VTEC) strains. A total of 34 E. coli O157:H7 strains isolated from bovine faeces and bovine carcasses were comparatively tested with Vero cell assay (VCA), PCR and the sandwich ELISA "RIDASCREEN Verotoxin" test. The VCA, performed without a neutralization assay, gave a false positive result because a VCA-positive E. coli O157:H7 strain did not possess the VT-coding genes when tested with PCR. The lack of specificity of the VCA could be avoided by testing for neutralization of cytotoxicity. The commercial ELISA system was as sensitive and specific as PCR, with the advantages of being a more rapid and easier procedure which could be employed in all first level diagnostic laboratories.     

Colimetry of marine waters off Fortaleza (Ceará State, Brazil) and detection of enteropathogenic Escherichia coli strains.

Bacteriological analyses of seawater from three main beaches in Fortaleza, Brazil were performed during 1997. Thirty-six samples per beach were collected for a total of 108 samples. For Meireles Beach, 44% of the samples had MPN total coliforms values of at least 1100 or over 2400/100 ml, followed by Formosa and Diários beaches showing lower counts. For fecal coliforms the highest numbers were demonstrated for Formosa, followed by Meireles and Diários beaches in this descending order: 13.0%, 11.1% and 8.3%, respectively. Escherichia coli strains were identified in 76.8% of the 108 samples. Among 295 strains of E. coli, 21 belonged to serogroups O25, O26, O91, O112, O119, O158 and O164. Strains from serogroup O26 were tested using PCR, ELISA and Vero cells to detect Verotoxins VT1 and VT2 and all strains were negative. No LT and ST, as determined by ELISA and suckling mice assays, were detected among the 295 strains. All strains of E. coli were sensitive to ampicillin, cephalothin, gentamicin, tetracycline, sulfametox-trimethoprim, chloramphenicol and ciprofloxacin. Although the E. coli strains were not toxigenic, their presence in high numbers could be of public health significance.     

Examination of raw beef products for the presence of Vero cytotoxin producing Escherichia coli, particularly those of serogroup O157

Fifty-four of 310 (17%) samples of raw beef products contained Vero cytotoxin (VT)-producing Escherichia coli (VTEC) detected by DNA probes for the VT genes. VTEC strains examined in detail from a selection of the positive samples belonged to several O serogroups, some of which have been associated with human diarrhoea or haemolytic uraemic syndrome. Some of the strains possessed properties that may contribute to virulence in man. None of the food samples contained VT-producing E. coli O157 when tested by a combination of VT probe tests and colony immunoblotting with commercially available anti-O157 serum. Quantification of the immunoblotting technique indicated that O157 VTEC could be recovered from artificially-inoculated meat samples at a level of less than one organism per gram. Five of the food samples carried E. coli O157 strains that did not produce VT and differed in other properties from O157 VTEC.     

Lactate dehydrogenase release assay from Vero cells to distinguish verotoxin producing Escherichia coli from non-verotoxin producing strains

The Vero cell assay presently used for virulence testing of verotoxigenic Escherichia coli (VTEC) requires at least 48-96 h where cytotoxicity effects are examined under a microscope. Here, a complimentary rapid assay was developed that measures endogenous lactate dehydrogenase (LDH) release from Vero or HEp-2 cells as an indicator of cytotoxicity. Toxin preparations from 24 VTEC strains induced 36-89% LDH from Vero cells and 15-62% LDH from HEp-2 cells in 12-16 h. A verotoxin-positive but enterohemolysin negative strain also showed a similar cytotoxicity effect. In contrast, three VT-negative strains caused only 13-16% LDH from Vero cells and 1-7% LDH from HEp-2 cells. Five presumptive E. coli isolates from naturally contaminated food and clinical sources did not induce significant LDH release from either cell lines. PCR analysis confirmed the presence of vt1 or vt2 genes in E. coli showing positive LDH values. Similarly, RiboPrinter analysis confirmed and identified the test strains as E. coli except for two meat isolates, which were identified as Hafnia alvei. Cytopathic effects of toxin preparations from VTEC revealed severe lysis, vacuole formation and death in Vero cells and multiple vacuoles and cell elongation in HEp-2 cells. The colorimetric cytotoxicity assay described here can provide quantitative data for determining the virulence potential of verotoxigenic E. coli in 12-16 h.     

禽源大肠杆菌的分离及其毒力因子的检测

从临床疑似大肠杆菌感染的病禽组织中分离到69株细菌(其中鹅源29株,鸡源40株);通过常规形态学、培养特性和生化特征的研究,确定为大肠杆菌。PCR检测表明,其中46株(66.7%)为F1 大肠杆菌,10株(14.5%)为F1 HPI 大肠杆菌,2株(2.9%)为HPI 大肠杆菌;通过比较还发现,F1菌毛和HPI在鹅源和鸡源大肠杆菌中以及不同脏器来源的菌株中具有相似的分子流行病学。O抗原鉴定结果表明鹅源大肠杆菌的O抗原型主要有O26、O78、O18、O117,鸡源大肠杆菌的O抗原型主要有O109、O24、O18、O139、O78。药敏试验表明,其中绝大多数菌株对先锋霉素V、呋喃妥因、庆大霉素敏感,对环丙沙星因菌株差异而不同,林可霉素、四环素、多粘菌素多不敏感。     

Cloning of genes determining the production of vero cytotoxin by Escherichia coli

Sequences encoding the production of a cytotoxin (VT) active on Vero cells were cloned in Escherichia coli K12 from a VT-determining phage that originated in E. coli strain H19 of serotype O26.H11. Subcloning resulted in the identification of a 2.5 kb fragment that still coded for VT production. Mutagenesis with transposon Tn1000 was used to map VT sequences and a 0.75 kb probe was developed. In colony hybridization tests with strains isolated from patients with haemolytic uraemic syndrome or diarrhoea, this probe derived from the H19 VT genes detected only some of the VT+ strains belonging to serogroup 0157. A VT+ strain, E32511, serotype 0157.H-, which was negative in colony hybridization was the source of another VT-determining phage from which VT sequences were cloned. Southern hybridization of the VT genes from E32511 with the H19 probe was negative under stringent conditions but there was weak homology under conditions of low stringency. These results indicate that there are differences in the VT genes of pathogenic E. coli.     

Production of cytotoxin VT in enteropathogenic and non-enteropathogenic Eschericha coli strains of porcine origin

Abstract We have investigated the production of verotoxin (VT) in 55 enteropathogenic and 48 non-enteropathogenic Escherichia coli strains of porcine origin, belonging to 48 serotypes. VT cytotoxin was only produced by seven out of the eight enteropathogenic strains with serotypes O138:K81, O139:K82, O141:K85ab and O141:K85ac. Five haemolytic non-enteropathogenic E. coli , four with serotypes O75:K-, O75:K95 and O2:K2, and one not typable, synthesized a new thermolabile product not related with enterotoxin (LT) or VT, and which induced the formation of multinucleate cells in Vero monolayers.