Characterisation of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef

Shiga toxin-producing Escherichia coli (STEC) strains isolated in Mangalore, India, were characterised by bead-enzyme-linked immunosorbent assay (bead-ELISA), Vero cell cytotoxicity assay, PCR and colony hybridisation for the detection of stx1 and stx2 genes. Four strains from seafood, six from beef and one from a clinical case of bloody diarrhoea were positive for Shiga toxins Stx1 and Stx2 and also for stx1and stx2 genes. The seafood isolates produced either Stx2 alone or both Stx1 and Stx2, while the beef isolates produced Stx1 alone. The stx1 gene of all the beef STEC was found to be of recently reported stx1c type. All STEC strains and one non-STEC strain isolated from clam harboured EHEC-hlyA. Interestingly, though all STEC strains were negative for eae gene, two STEC strains isolated from seafood and one from a patient with bloody diarrhoea possessed STEC autoagglutinating adhesion (saa) gene, recently identified as a gene encoding a novel autoagglutinating adhesion.     

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus ) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.     

Shiga toxin-producing Escherichia coli, faecal coliforms and coliphage in animal feeds

AIMS: Animal feeds (n = 226), collected from pastures or feeding troughs on UK farms and from feed manufacturers' bulk stores, were analysed for Escherichia coli harbouring shiga-toxin genes (stx), faecal coliforms, coliphages and stx-harbouring bacteriophages. METHODS AND RESULTS: Samples comprised of 79 fresh grasses, 26 silages and 121 dried or heat-processed feeds (DPF). Five of the 79 (6.3%) fresh grass samples contained stx(2)-E. coli. stx-E. coli were not detected in the silages or DPF that were examined. Faecal coliforms were detected in 75/79 (94.9%) of fresh grasses, 19/26 (73.1%) of silages and 36/121 (29.8%) of processed feeds. Coliphages were detected in 63/79 (79.7%) and 18/26 (69.2%) of fresh grasses and silages, respectively. Coliphages were isolated at a significantly lower prevalence of 5% (6/121) from processed feeds. Although stx(2)-phage was isolated from the enrichment of a single grass sample, stx-phages were not detected in any of the silage or processed feeds. We did not detect stx(1)-phage in any of the samples collected. CONCLUSIONS: Pastures have the potential to act as transmission vectors for stx-harbouring E. coli for grazed livestock. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the prevalence of E. coli harbouring stx genes, faecal coliforms, coliphages and stx-harbouring bacteriophages in a range of feedstuffs destined for consumption by UK livestock. This study provides information on the risk of feeds to the spread of stx-phages between livestock and/or the environment.     

A prospective study on Shiga toxin-producing Escherichia coli in children with diarrhoea in Paraná State, Brazil

Aims:  To examine stool specimens from children with diarrhea from Paraná State, southern Brazil, for presence of STEC.
Methods and Results:  A PCR screening assay for stx genes was used to examine a loopful of confluent colonies of 306 stool samples cultures. In six (1.96%) of them, DNA fragments of the expected size were observed, and the presence of stx was confirmed by DNA sequencing. Then up to 100 single colonies from each of the six stool cultures were analyzed using the same PCR protocol. However, stx -positive colonies were found only in two of the cultures. The E. coli strains belonged to serotypes O69:H11 and O178:H19, and presented genotypes stx ₁ eae ehxA and stx ₁ respectively. Shiga toxin production was confirmed using the VTEC Screen Seiken. Except ampicillin, they were susceptible to all the antimicrobials tested.
Conclusions:  These results show that STEC may be an important cause of diarrhea in children of Paraná State, and that they are present in low numbers in stools. The strains belonged to serotypes not commonly found associated with STEC and probably present low virulence.
Significance and Impact of Study:  These results indicate that molecular methods are required to diagnosis of STEC infections.     

Stx genotypes and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli strains isolated from human infections, cattle and foods in Brazil

A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in S?o Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.     

Virulence characteristics of Shiga toxin‐producing Escherichia coli from raw meats and clinical samples

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae‐negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx_2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non‐O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.     

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.     

Screening of food raw materials for the presence of Shiga toxin-producing Escherichia coli O91:H21

Aims:  To provide information on the prevalence and detection, in foods, of Shiga toxin-producing Escherichia coli (STEC) O91:H21.
Methods and Results:  Seven hundred fifteen minced beef meats and 205 raw milk samples were analysed by stx -specific PCR-ELISA. Samples positive for stx were subsequently tested for the presence of wzy -O91, fliC -H21 and the adhesin-encoding gene saa . For minced meat, 16 (2·2%) and 11 (1·5%) samples were found positive for ( stx , wzy -O91, fliC -H21) and ( stx , wzy -O91, fliC -H21, saa ) combinations, respectively. For raw milk, seven (3·4%) samples were found positive for the ( stx , wzy -O91, fliC -H21) combination but none of these contained saa . Two STEC O91:H21 saa -positive strains and three STEC O91 H21– and saa -negative strains were isolated by colony hybridization.
Conclusions:  A low prevalence of potentially pathogenic STEC O91:H21 in food products was found using a combination of PCR assays targeting stx , wzy -O91, fliC -H21 and saa .
Significance and Impact of the Study:  The PCR-based approach described here represents a valuable method for rapid screening of food samples contaminated by STEC O91:H21.     

Animal host associated differences in Shiga toxin-producing Escherichia coli isolated from sheep and cattle on the same farm

AIMS: To investigate if cattle on the same farm as sheep are a possible risk factor for stx in sheep and to determine whether or not sheep and cattle on the same farm share the same stx pool. METHODS AND RESULTS: Faecal samples from sheep and cattle were screened for stx by polymerase chain reaction (PCR). Of these samples, 87.6 and 64.6% were stx positive in sheep and cattle, respectively. There was no difference in stx occurrence in sheep from farms with or without cattle. From stx positive samples, 118 Shiga toxin-producing Escherichia coli (STEC) isolates were recovered by a filter-hybridization method. Serotyping, PCR and pulsed-field gel electrophoresis (PFGE) showed that there was a distinct association between serotypes, stx profiles and animal species. CONCLUSIONS: Keeping animals together in pens, which enhances faecal-oral contact, is suggested as a possible explanation for the differences seen in stx occurrence. Sheep and cattle isolates are distinctly different in serotype and stx profile although isolated from the same farm, and are more related to isolates within the same serotype with the same stx profile than to isolates with different serotype from the same farm. SIGNIFICANCE AND IMPACT OF STUDY: The study supports the animal-host relationship hypothesis suggested in other studies and indicates that the STEC sheep reservoir in Norway may not pose a serious public health risk.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheeses in France

AIMS: To evaluate Shiga toxin-producing Eschericha coli (STEC) prevalence in 1039 French raw milk cheeses including soft, hard, unripened and blue mould cheeses, and to characterize the STEC strains isolated (virulence genes and serotypes). METHODS AND RESULTS: STEC strains were recovered from cheese samples by colony hybridization. These strains were then serotyped and genetically characterized. These strains (32 STEC) were then recovered from 18 of 136 stx-positive samples: 19 strains had stx2 variant genes stx(2vh-a) (n = 2), stx(2NV206) (n = 2), stx(2EDL933) (n = 4) and stx2d (n = 11). Thirty strains had the stx1 gene and one strain, the eae gene. Combinations of stx2 and stx1 genes were present in 17 (81%) of the STEC strains. Nineteen strains belonged to the O6 serogroup and the other strains belonged to the O174, O175, O176, O109, O76, O162 and O22 serogroups in decreasing frequency. CONCLUSIONS: No conclusion can be drawn at the moment concerning the potential risk to consumers because the O6:H1 serotype has already been found associated with the haemolytic uremic syndrome and almost no isolate had the eae gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The large number of STEC strains recovered from the cheese samples evaluated in this study emphasizes the health risks associated with raw milk cheeses. This further emphasizes the immediate need to identify and implement effective pre- and postharvest control methods that decrease STEC carriage by dairy cattle and to eliminate contamination of their cheeses during processing.     

Persistence of Shiga toxin-producing Escherichia coli O26 in cow slurry

AIMS: The main objective of this study was to evaluate the growth and survival of Shiga toxin-producing Escherichia coli (STEC) O26 in cow slurry; this serogroup is regarded as an important cause of STEC-associated diseases. METHODS AND RESULTS: Four STEC were examined by polymerase chain reaction (PCR) to determine whether they harbour key virulence determinants and also by pulsed-field gel electrophoresis (PFGE) to obtain overview fingerprints of their genomes. They were transformed with the pGFPuv plasmid and were separately inoculated at a level of 10(6) CFU ml(-1) in 15 l of cow slurry. All STEC O26 strains could be detected for at least 3 months in cow slurry without any genetic changes. The moisture content of the slurry decreased over time to reach a final value of 75% while the pH increased from 8.5 to 9.5 units during the last 50 days. CONCLUSION: STEC O26 strains were able to survive in cow slurry for an extended period. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term storage of waste slurry should be required to reduce the pathogen load and to limit environmental contamination by STEC O26.     

Detection and genetical characterization of Shiga toxin-producing Escherichia coli from wild deer

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from wild deer in Japan were examined. A total of 43 fecal samples were collected 4 times from 4 different sites around Obihiro City, Hokkaido, Japan, in June and July 1997. Seven STEC strains were isolated by PCR screening, all of them were confirmed by ELISA and Vero cell cytotoxicity assay to be producing only active Stx type 2 (Stx2). Moreover, they seemed to carry the hemolysin and eaeA genes of STEC O157:H7, and some isolates harbored large plasmids which were similar to the 90-kilobase virulence plasmid of STEC O157:H7. Based on their plasmid profiles, antibiotic resistance patterns, PCR-based DNA fingerprinting data obtained by using random amplified polymorphic DNA (RAPD) and the stx2 gene sequences, all isolates were divergent from each other except for 3 isolates from the first and second samplings. A DNA sequence analysis of representative isolates revealed that deer originating STEC strains were closely related to each other, but not to the Stx2-producing STEC strains isolated from a mass outbreak in Obihiro at the same time. A phylogenic analysis of the deduced Stx2 amino acid sequences demonstrated that three distinct clusters existed in the deer originating STEC strains and that the Stx of deer originating STEC was closely associated with that originating from humans, but not those of STEC originating from other animals. These results suggest that STEC contamination of deer carcasses should be considered as a potential source of human infection and adequate sanitary inspection of meat for human consumption is also essential for wild animals.     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli in retail-minced beef using PCR-based techniques, immunoassays and colony hybridization

AIMS: To provide information on detection of Shiga toxin-producing Escherichia coli (STEC) in retail-minced beef using an approach combining (i) PCR-based techniques and automated immunoassay for stx screening and detection of the five major serogroups associated with human infection, and (ii) immunomagnetic separation (IMS) and colony hybridization assays for bacterial strain isolation. METHODS AND RESULTS: Twenty-seven out of 164 minced beef samples were stx-positive by PCR-ELISA, nine of which were also positive by real-time PCR for at least one marker of the five main serogroups tested (O26, O103, O111, O145 and O157). Two E. coli O103 stx-negative strains were isolated from two out of 10 IMS and nine STEC strains that did not belong to the five main serogroups were isolated by colony hybridization. CONCLUSIONS: PCR techniques are applicable for rapid screening of samples containing both an stx gene and an O-group marker of the five main pathogenic STEC serogroups. Isolation of STEC strains belonging to the main non-O157 serogroups remains difficult. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents an evaluation of a multi-faceted approach for the detection of the most frequently reported human pathogenic STEC serogroups. The advantages and limits of this strategy are presented.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.