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.     

Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.     

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.     

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.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.     

Shiga toxin-producing Escherichia coli in the animal reservoir and food in Brazil

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathotype associated with human gastrointestinal disease that may progress to severe complications. Ruminants, especially cattle, are the main reservoirs of STEC contaminating the environment and foods of animal or vegetable origin. Besides Shiga toxin, other virulence factors are involved in STEC virulence. O157:H7 remains the most frequent serotype associated with disease. In Brazil, the prevalence of STEC reaches values as high as 90% in cattle and 20% in meat products which may impact the Brazilian food export trade. However, only few reports are related to human disease. The stx1 gene prevails in cattle, whereas the stx2 gene is more frequent in food. Several STEC serotypes have been isolated from cattle and food in Brazil, including the O157:H7, O111:NT, NT:H19 as well as O26 and O103 serogroups. O113: H21 STEC strains are frequent in ruminants and foods but with no report in human disease. The virulence profile of Brazilian STEC strains from cattle and food suggests a pathogenic potential to humans, although some differences with clinical strains have been detected. Further studies, employing recent and more discriminative techniques are in need to better clarify their virulence potential.     

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.     

Variants of eae and stx genes of atypical enteropathogenic Escherichia coli and non-O157 Shiga toxin-producing Escherichia coli from calves

AIMS: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. METHODS AND RESULTS: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx(1) exhibited stx(1c) in 13 (31.70%) isolates, while that of stx(2) revealed stx(2c) in eight (24.24%) and stx(2d) in two (6.06%) isolates. Subtyping of eae gene showed the presence of eae-beta, eae-eta and eae-zeta in two, three and four isolates respectively. None of the E. coli isolates possessed stx(2e), stx(2f), eae-alpha, eae-delta, eae-epsilon and eae-xi. All EPEC isolates were atypical. CONCLUSIONS: stx(1), stx(1c), stx(2), stx(2c), stx(2d), eae-beta, eae-eta and eae-zeta subtypes are prevalent in STEC and EPEC isolates in India. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first subtype analysis of stx(2) and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans.     

Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China

Background

Shiga toxin-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Swine plays an important role as a carrier of this pathogen. In this study we determined the prevalence and characteristics of STEC from healthy swine collected between May 2011 and August 2012 from 3 cities/provinces in China.

Results

A total of 1003 samples, including 326 fecal, 351 small intestinal contents and 326 colon contents samples, was analyzed. Two hundred and fifty five samples were stx-positive by PCR and 93 STEC isolates were recovered from 62 stx-positive samples. Twelve O serogroups and 19 O:H serotypes including 6 serotypes (O100:H20/[H20], O143:H38/[H38], O87:H10, O172:H30/[H30], O159:H16, O9:H30/[H30]) rarely found in swine and ruminants were identified. All 93 STEC isolates harbored stx ₂ only, all of which were stx _2e subtype including 1 isolate being a new variant of stx _2e. 53.76%, 15.05% and 2.15% STEC isolates carried astA, hlyA and ehxA respectively. Four STEC isolates harbored the high-pathogenicity island. Of the 15 adherence-associated genes tested, 13 (eae, efa1, iha, lpfA _O113, lpfA _O157/OI-154, lpfA _O157/OI-141, toxB, saa, F4, F5, F6, F17 or F41) were all absent while 2 (paa and F18) were present in 7 and 4 STEC isolates respectively. The majority of the isolates were resistant to tetracycline (79.57%), nalidixic acid (78.49%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin (55.91%). The STEC isolates were divided into 63 pulsed-field gel electrophoresis patterns and 21 sequence types (STs). Isolates of the same STs generally showed the same or similar drug resistance patterns. A higher proportion of STEC isolates from Chongqing showed multidrug resistance with one ST (ST3628) resistant to 14 antimicrobials.

Conclusions

Our results indicate that swine is a significant reservoir of STEC strains in China. Based on comparison by serotypes and sequence types with human strains and presence of virulence genes, the swine STEC may have a low potential to cause human disease.     

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.     

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.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India. METHODS AND RESULTS: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples. CONCLUSIONS: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.     

Virulence properties and serotypes of Shiga toxin-producing Escherichia coli from healthy Australian cattle

The virulence properties and serotypes of complex Shiga toxin-producing Escherichia coli (cSTEC) were determined in two studies of healthy cattle in eastern Australia. In the first, a snapshot study, 84 cSTEC isolates were recovered from 37 of 1,692 (2.2%) fecal samples collected from slaughter-age cattle from 72 commercial properties. The second, a longitudinal study of three feedlots and five pasture beef properties, resulted in the recovery of 118 cSTEC isolates from 104 animals. Of the 70 serotypes identified, 38 had not previously been reported.     

Serotypes and virulence factors of Shiga toxin-producing Escherichia coli isolated from healthy Norwegian sheep

AIMS: To characterize a number of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep and to discuss the potential of these isolates as human pathogens. METHODS AND RESULTS: Twelve different O-groups and seven different H-types were identified by standard serotyping methods. The most common serotypes were O5:NM, O6:H10, O91:NM and O128:NM. Polymerase chain reaction (PCR) was used for the detection of virulence factor genes. Of 102 isolates, 86.3% carried stx1 and 83% of these were also positive in the stx1OX3-specific PCR. stx2 was carried by 55.9% of the isolates and 77.2% of these were also positive in the stx2d-specific PCR. The Vero cell assay showed high toxin production in 70.6% of the isolates. None of the isolates carried eae. CONCLUSIONS: The study supports the animal-host relationship suggested in other studies with STEC serogroups O5, O91 and O128 strongly associated with sheep. Most sheep STEC carry stx1OX3 (except O91) and the dominating stx2 variant is stx2d. One stx profile clearly dominates within a serotype. SIGNIFICANCE AND IMPACT OF THE STUDY: In spite of the predominance of certain sheep-associated STEC, sheep cannot be excluded as carriers of human pathogenic STEC.     

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.     

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.