Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.Escherichia coli O157:H7 is a food- and waterborne zoonotic pathogen with serious effects on public health. E. coli O157:H7 causes diseases in humans ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (30). Typically, enterohemorrhagic E. coli (EHEC) strains express two groups of important virulence factors: one or more Shiga toxins (Stx; also called verotoxins), encoded by lambda-like bacteriophages, and a pathogenicity island called the locus of enterocyte effacement (LEE) encoding all the proteins necessary for attaching and effacing lesions of epithelial cells (41). Comparative genomic studies of E. coli O157:H7 strains revealed extensive genomic diversity related to the structures, positions, and genetic contents of bacteriophages and the variability of putative virulence genes encoding non-LEE effector proteins (29, 43).Ruminants and, in particular, healthy cattle are the major reservoir of E. coli O157:H7, although the prevalence of O157:H7 strains in cattle may vary widely, as reviewed by Caprioli et al. (12). E. coli O157:H7 has been found to persist and remain infective in the environment for a long time, e.g., for at least 6 months in water trough sediments, which may be an important environmental niche.In Hungary, infections with E. coli O157 and other Shiga toxin-producing E. coli (STEC) strains in humans in cases of “enteritidis infectiosa” have been notifiable since 1998 on a case report basis. Up to now, the disease has been sporadic, and fewer than 100 (n = 83) cases of STEC infection among 2,700 suspect cases have been reported since 2001. However, until the present study, no systematic, representative survey of possible animal sources had been performed.In this study, our aim was to investigate healthy cattle in Hungary for the presence of strains of E. coli O157 and the genes encoding Shiga toxins (stx₁ and stx₂) and intimin (eae) and a wide range of putative virulence genes found in these strains. In addition, the phage type (PT) was determined, and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to further compare the strains at the molecular level. Shiga toxin and cytolethal distending toxin (CDT) production was also examined, and phage induction experiments were conducted. The high incidence of enteropathogenic E. coli (EPEC; eae-positive) O157:H7 strains and atypical (eae- and stx-negative) O157 strains indicates that cattle are a major reservoir of not only EHEC O157 but also EPEC O157 and atypical E. coli O157 strains. These atypical, non-sorbitol-fermenting O157 strains frequently produced CDT-V and may represent a novel O157 clade as demonstrated by MLST and PFGE.     

Chromosomal Dynamism in Progeny of Outbreak-Related Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx₂) in the outbreak strain or with the loss of this gene. The two stx₂ alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx₂ genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.     

Chromosomal dynamism in progeny of outbreak-related sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) is a unique clone that causes outbreaks of hemorrhagic colitis and hemolytic-uremic syndrome. In well-defined clusters of cases, we have observed significant variability in pulsed-field gel electrophoresis (PFGE) patterns which could indicate coinfection by different strains. An analysis of randomly selected progeny colonies of an outbreak strain after subcultivation demonstrated that they displayed either the cognate PFGE outbreak pattern or one of four additional patterns and were <89% similar. These profound alterations were associated with changes in the genomic position of one of two Shiga toxin 2-encoding genes (stx2) in the outbreak strain or with the loss of this gene. The two stx2 alleles in the outbreak strain were identical but were flanked with phage-related sequences with only 77% sequence identity. Neither of these phages produced plaques, but one lysogenized E. coli K-12 and integrated in yecE in the lysogens and the wild-type strain. The presence of two stx2 genes which correlated with increased production of Stx2 in vitro but not with the clinical outcome of infection was also found in 14 (21%) of 67 SF EHEC O157:NM isolates from sporadic cases of human disease. The variability of PFGE patterns for the progeny of a single colony must be considered when interpreting PFGE patterns in SF EHEC O157-associated outbreaks.     

Genotypic characterization to identify markers associated with putative hypervirulence in Swedish Escherichia coli O157:H7 cattle strains

Aims: To establish whether investigated subtyping methods could identify any specific characteristics that distinguish Swedish VTEC O157:H7 strains isolated from cattle farms associated with human enterohaemorrhagic Escherichia coli (EHEC) cases from cattle strains isolated in prevalence studies. Methods and Results: Strains (n = 32) isolated in a dairy herd prevalence study and strains isolated from farms associated with human cases (n = 13) were subjected to typing. Partial sequencing of the vtx₂ genes could not identify any unique variants of vtx₂ or vtx_2c in strains associated with human cases. A specific variant of VTEC O157:H7, which was overrepresented among farms associated with human cases (P = 0·01), was by two different single‐nucleotide‐polymorphism (SNP) assays identified as clade 8, a subgroup of VTEC O157:H7 strains considered to be putatively hypervirulent. Multi‐locus variable number tandem repeat analysis (MLVA) typing of all strains produced similar results as pulsed‐field gel electrophoresis (PFGE) typing regarding clustering of the strains, but MLVA distinguished slightly better among strains than PFGE. Conclusion: In Sweden, VTEC O157:H7 strains from the putatively hypervirulent clade 8 are overrepresented among isolates from cattle farms associated with human cases compared with VTEC O157:H7 strains isolated in prevalence studies. Significance and Impact of the Study: Real‐time PCR SNP typing for clade 8 can be used to identify cattle farms that are at higher risk of causing EHEC infections in humans.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

Prevention of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 infection in gnotobiotic mice associated with <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains

Previous reports have shown that Escherichia coli O157:H7 infection is strongly modified by intestinal microbes. In this paper, we examined whether bifidobacteria protect against E. coli O157:H7 infections using gnotobiotic mice di-associated with Bifidobacterium strains (6 species, 9 strains) and E. coli O157:H7. Seven days after oral administration of each Bifidobacterium strain, the mice were orally infected with E. coli O157:H7 and their mortality was examined. Bifidobacterium longum subsp. infantis 157F-4-1 (B. infantis 157F) and B. longum subsp. longum NCC2705 (B. longum NS) protected against the lethal infection, while mice associated with all other Bifidobacterium strains, including type strains of B. longum subsp. infantis and B. longum subsp. longum, died. There were no significant differences in the numbers of E. coli O157:H7 in the faeces among the Bifidobacterium-associated mouse groups. However, the Shiga toxin concentrations in the cecal contents and sera of the GB mice associated with B. infantis 157F and B. longum NS were significantly lower than those of the other groups. However, there were no significant differences in the volatile fatty acid concentrations and histopathological lesions between these two groups. These data suggest that some strains of B. longum subsp. longum/infantis can protect against the lethal infections of E. coli O157:H7 by preventing Shiga toxin production in the cecum and/or Shiga toxin transfer from the intestinal lumen to the bloodstream.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Protection from Hemolytic Uremic Syndrome by Eyedrop Vaccination with Modified Enterohemorrhagic E. coli Outer Membrane Vesicles

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.     

出血性大肠杆菌O157基因缺失疫苗株的构建及其免疫

出血性大肠杆菌O157感染是重要的新发食物源性传染病,主要致病特征之一是能引起人肠上皮细胞特征性的A/E损伤,A/E损伤主要是由LEE致病岛所编码的毒力因子所引起,ler是LEE致病岛毒力基因群的中心调节基因,对LEE致病岛所编码的毒力因子有正调控作用。O157:H7另一个毒力因子是由整合到染色体上的原噬菌体编码的Stx毒素。以O157:H786-24为始发菌株,利用自杀性质粒pCVD442和同源重组的原理构建了O157:H7的ler基因缺失突变菌株(缺失了ler基因中第73-351位的碱基,共279bp),并利用噬菌体消除技术筛选到消除了编码Stx的原噬菌体DNA的菌株,构建出了O157:H7ler/stx基因缺失突变弱毒菌株,并对该菌株的Vero细胞毒性、小鼠模型的安全性以及乳鼠的被动免疫保护作用进行了研究。结果表明,O157:H7ler/stx基因缺失突变菌株丧失了对Vero细胞的毒性作用,并丧失了对实验小鼠的致病性,具有良好的安全性。乳鼠被动免疫保护性实验表明,用该菌株免疫母鼠后,乳鼠通过吸吮母乳可以获得良好的被动免疫保护作用。因此本研究所构建的O157:H7ler/stx基因缺失突变弱毒菌株可作为预防EHEC O157:H7感染的疫苗候选株,为最终研究制出O157的基因工程菌苗奠定基础。     

Functional analysis of the type III secretion system in enteropathogenic Escherichia coli O157:H45

A mass outbreak of Escherichia coli O157:H45 was first reported in Japan in 1998. This pathogen was classified as an enteropathogenic E. coli (EPEC) O157 because it was characterized by the Shiga toxin gene (stx)-negative and bundle-forming pilus (bfp) gene-positive genotypes. In this study, we investigated the type III secretion system in EPEC O157. Although no type III secreted proteins, Esps (E. colisecreted proteins), in EPEC O157:H45 were detectable in culture supernatant, secreted proteins were induced by the introduction of an EPEC plasmid-encoded regulator, per. In further contrast to EHEC O157:H7, EPEC O157:H45 triggered the accumulation of tyrosine phosphorylated proteins beneath the adherent bacteria. These results suggest that regulation of the type III secretion apparatus and host signal transduction events between E. coli O157:H45 and O157:H7 are completely different.     

Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.     

Evaluation of the Anti-Terminator Q933 Gene as a Marker for Escherichia coli O157:H7 with High Shiga Toxin Production

The anti-terminator Q933 gene of the bacteriophage 933W was evaluated as a marker for Escherichia coli O157:H7 strains with high Shiga toxin production. In total, 262 environmental strains of E. coli O157:H7 isolated from feces of beef cattle and the digestive tract of houseflies were screened for the Q933 and Q21 (anti-terminator Q21 of bacteriophage 21) genes by polymerase chain reaction. Nine (3.4%) isolates tested positive for Q933 alone, 161 (61.5%) were positive for the Q21 gene alone, and 92 (35.1%) isolates carried both Q alleles. Results from the enzyme-linked immunosorbent assay show that the isolates with Q933 alone produced significantly more Shiga toxin than the remaining isolates. The difference was even greater after the induction of the toxin production by a short exposure of cells to ultraviolet light. These data suggest that Q933 is a promising indicator for environmental E. coli O157:H7 with high production of Shiga toxins and, therefore, for potentially clinically relevant strains.     

Heparin blocks the adhesion of E. coli O157:H7 to human colonic epithelial cells

Adhesion of Shiga toxin-producing Enterohemorrhagic Escherichia coli (EHEC) O157:H7 to human colonic epithelium is a critical step for infection by this type of bacteria. Here, we demonstrate that adherence of EHEC O157:H7 to cultured human colonic T84 epithelial monolayers can be blocked by heparin and heparan sulfate in a dose-dependent fashion. In doing this, heparin and heparan sulfate also prevent dysfunction of the T84 barrier and disorganization of epithelial tight junction protein ZO-1 caused by EHEC O157:H7. This inhibition by heparin and heparan sulfate seems to result from a block in the binding interactions of bacteria intimin with epithelial β₁ integrins. This study provides evidence, for the first time, that heparin and heparan sulfate can serve as novel effective blockers in preventing EHEC O157:H7 infection.     

Characterization of Escherichia coli O157:H7 Strains Isolated from Supershedding Cattle

Previous reports have indicated that a small proportion of cattle shedding high levels of Escherichia coli O157:H7 is the main source for transmission of this organism between animals. Cattle achieving a fecal shedding status of 10⁴ CFU of E. coli O157:H7/gram or greater are now referred to as supershedders. The aim of this study was to investigate the contribution of E. coli O157:H7 strain type to supershedding and to determine if supershedding was restricted to a specific set of E. coli O157:H7 strains. Fecal swabs (n = 5,086) were collected from cattle at feedlots or during harvest. Supershedders constituted 2.0% of the bovine population tested. Supershedder isolates were characterized by pulsed-field gel electrophoresis (PFGE), phage typing, lineage-specific polymorphism assay (LSPA), Stx-associated bacteriophage insertion (SBI) site determination, and variant analysis of Shiga toxin, tir, and antiterminator Q genes. Isolates representing 52 unique PFGE patterns, 19 phage types, and 12 SBI clusters were obtained from supershedding cattle, indicating that there is no clustering to E. coli O157:H7 genotypes responsible for supershedding. While being isolated directly from cattle, this strain set tended to have higher frequencies of traits associated with human clinical isolates than previously collected bovine isolates with respect to lineage and tir allele, but not for SBI cluster and Q type. We conclude that no exclusive genotype was identified that was common to all supershedder isolates.     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.