Strains of Escherichia coli O157:H7 differ primarily by insertions or deletions, not single-nucleotide polymorphisms

Escherichia coli O157:H7 (O157) strains demonstrate varied pulsed-field gel electrophoresis patterns following XbaI digestion, which enable epidemiological surveillance of this important human pathogen. The genetic events underlying PFGE differences between strains, however, are not defined. We investigated the mechanisms for strain variation in O157 by recovering and examining nucleotide sequences flanking each of the XbaI restriction enzyme sites in the genome. Our analysis demonstrated that differences between O157 strains were due to discrete insertions or deletions that contained the XbaI sites polymorphic between strains rather than single-nucleotide polymorphisms in the XbaI sites themselves. These insertions and deletions were found to be uniquely localized within the regions of the genome that are specific to O157 compared to E. coli K-12 (O islands), suggesting that strain-to-strain variation occurs in these O islands. These results may be utilized to devise novel strain-typing tools for this pathogen.     

Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.

Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.     

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin.

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.     

Clonal dissemination of Escherichia coli O157:H7 subtypes among dairy farms in northeast Ohio

To ascertain the extent to which indistinguishable strains of Escherichia coli O157:H7 are shared between farms, molecular characterization was performed on E. coli O157:H7 isolates recovered during a longitudinal study of 20 dairy farms in northeast Ohio. Of the 20 dairy farms sampled, 16 were located in a primary area and 4 were located in two other distant geographical areas. A total of 92 E. coli O157:H7 isolates obtained from bovine fecal samples, water trough sediment samples, free-stall bedding, and wild-bird excreta samples were characterized. Fifty genetic subtypes were observed among the isolates using XbaI and BlnI restriction endonucleases. Most restriction endonuclease digestion profiles (REDPs) were spatially and temporally clustered. However, four REDPs from multiple sources were found to be indistinguishable by pulsed-field gel electrophoresis between four pairs of farms. The geographical distance between farms which shared an indistinguishable E. coli O157:H7 REDP ranged from 9 to 50 km, and the on-farm sources sharing indistinguishable REDPs included cattle and wild bird feces and free-stall bedding. Within the study population, E. coli O157:H7 REDP subtypes were disseminated with considerable frequency among farms in close geographic proximity, and nonbovine sources may contribute to the transmission of this organism between farms.     

Genotypic analyses of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States

Escherichia coli O157:H7 and O157 nonmotile isolates (E. coli O157) previously were recovered from feces, hides, and carcasses at four large Midwestern beef processing plants (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000). The study implied relationships between cattle infection and carcass contamination within single-source lots as well as between preevisceration and postprocessing carcass contamination, based on prevalence. These relationships now have been verified based on identification of isolates by genomic fingerprinting. E. coli O157 isolates from all positive samples were analyzed by pulsed-field gel electrophoresis of genomic DNA after digestion with XbaI. Seventy-seven individual subtypes (fingerprint patterns) grouping into 47 types were discerned among 343 isolates. Comparison of the fingerprint patterns revealed three clusters of isolates, two of which were closely related to each other. Remarkably, isolates carrying both Shiga toxin genes and nonmotile isolates largely fell into specific clusters. Within lots analyzed, 68.2% of the postharvest (carcass) isolates matched preharvest (animal) isolates. For individual carcasses, 65.3 and 66.7% of the isolates recovered postevisceration and in the cooler, respectively, matched those recovered preevisceration. Multiple isolates were analyzed from some carcass samples and were found to include strains with different genotypes. This study suggests that most E. coli O157 carcass contamination originates from animals within the same lot and not from cross-contamination between lots. In addition, the data demonstrate that most carcass contamination occurs very early during processing.     

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.     

Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR,rep-PCR,PFGE, ribotyping and AFLP

A total of 54 isolates were characterized by multiplex-PCR for toxin genes and genotyped using several DNA fingerprinting methods: using repetitive extragenic palindromes (REP) and Box primers (rep-PCR), amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and ribotyping. The known-pathogenic strains tested were from food and clinical samples (34 strains) and included serovars O157:H7, O111:H8, O111:H11, O91:H21 and O55:H7. Two type cultures, Escherichia coli K12 (ATCC 29425) and DUP-101 (ATCC 51739), were included as known non-pathogenic strains and an additional 17 previously unclassified isolates from animal fecal samples. Comparisons of genomic DNA fingerprint patterns using unweighted pair group method with arithmetic averages (UPGMA) cluster analysis of Jaccard similarity indices indicated that all methods tested showed a greater similarity between the E. coli O157:H7 strains than to other isolates. On the basis of these studies, we propose that AFLP, REP-PCR, Box-PCR and ribotyping techniques can all be used for discriminating O157:H7 isolates and are preferred for large-scale screening because of the speed and ease of the methods. The PFGE method is the best to discriminate between subtypes of O157:H7 associated with specific outbreak investigations; however, it is more time consuming and unnecessary if subtyping is not required. There are differences between the dendrograms generated from each method and the relationship between the other strains analyzed. However, the fingerprint profiles of the O157:H7 isolates were virtually identical using REP-PCR and Box-PCR enabling easy distinction of the group. Thus, these typing methods have the potential to aid investigators in identifying the source of an outbreak to prevent or control further spread of E. coli O157:H7.     

Rapid Determination of Escherichia coli O157:H7 Lineage Types and Molecular Subtypes by Using Comparative Genomic Fingerprinting

In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.     

Shedding of Escherichia coli O157:H7 in dairy cattle housed in a confined environment following waterborne inoculation

A study of Escherichia coli O157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration of E. coli O157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers of E. coli O157:H7 isolates shed in these calves varied from 60 to 10(5) CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by using XbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 10(3) to 10(4) CFU of E. coli O157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 10(2) to 10(6) CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 10(6) CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination of E. coli O157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.     

Characteristics of E.coli O157 strains isolated in Poland from clinical material and food samples

E. coli belonging to the O157 serological group are among the organisms isolated most frequently out of all the so called entero-hemorrhagic E. coli strains (EHEC). Since several years they have been isolated also in Poland. The purpose of the present study was determination on selected phenotypic and genotypic properties of E. coli O157 strains isolated in our country from clinical material samples and from food. The serotype of the strains was determined, together with the following properties regarded as pathogenicity markers of verotoxic E. coli strains such as absence of beta-glucuronidase activity and sorbitol fermentation ability, as well as production of verotoxins SLT I and/or SLT II and entero-hemolysin. Besides that, by the PCR method the fragments of the genes coding for verotoxins, intimin and enterohaemolysin were amplified. The products of PCR were analysed by the restriction enzyme analysis (RFLP). All verotoxic E. coli O157 strains isolated in Poland were analysed by the pulsed field gel electrophoresis of genomic DNA (PFGE). The studied group comprised E. coli O157 strains, among them 40 strains were isolated from human faeces and 5 from food. The remaining strains were the reference E. coli O157:H7 EDL 933 and G 5244 strains and strains from NIH collection. The obtained results showed that the tested strains were a very varying population. 21 of them (all isolated from food, 11 from faeces and 5 reference strains) belonged to serotype O157:H7, five were not peritrichous O157:NM and the remaining ones had other ciliary antigen than H7. All strains isolated from food, reference strains and only 3 O157:NM strains isolated from humans were verotoxic. The strains from food and two reference strains produced only SLT II, 2 of 3 strains isolated from humans and one reference strain also produced only SLT II and the other produced both verotoxins. Apart from these 13 verotoxic strains all remaining strains caused sorbitol fermentation.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Competition for proline between indigenous Escherichia coli and E. coli O157:H7 in gnotobiotic mice associated with infant intestinal microbiota and its contribution to the colonization resistance against E. coli O157:H7

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 mice became carriers. It has been reported that the lag time of E. coli O157:H7 growth in 50% GB-3 caecal suspension was extended when compared to GB-4 caecal suspension. In this study, competition for nutrients between intestinal microbiota of GB-3 and GB-4 mice and E. coli O157:H7 was examined. Amino acid concentrations in the caecal contents of GB-3 and GB-4 differed, especially the concentration of proline. The supplementation of proline into GB-3 caecal suspension decreased the lag time of E. coli O157:H7 growth in vitro. When E. coli O157:H7 was cultured with each of the strains used to produce GB-3 mice in vitro, 2 strains of E. coli (proline consumers) out of 5 enterobacteriaceae strains strongly suppressed E. coli O157:H7 growth and the suppression was attenuated by the addition of proline into the medium. These results indicate that competition for proline with indigenous E. coli affected the growth of E. coli O157:H7 in vivo and may contribute to E. coli O157:H7 elimination from the intestine.     

Phylogeny of Shiga toxin-producing Escherichia coli O157 isolated from cattle and clinically ill humans

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor multiple genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor; however, a lack of genome sequence has hindered investigations on the divergence of human- and/or cattle-associated subtypes. Our goals were to 1) identify nucleotide polymorphisms for STEC O157 genetic subtype detection, 2) determine the phylogeny of STEC O157 genetic subtypes using polymorphism-derived genotypes and a phage insertion typing system, and 3) compare polymorphism-derived genotypes identified in this study with pulsed field gel electrophoresis (PFGE), the current gold standard for evaluating STEC O157 diversity. Using 762 nucleotide polymorphisms that were originally identified through whole-genome sequencing of 189 STEC O157 human- and cattle-isolated strains, we genotyped a collection of 426 STEC O157 strains. Concatenated polymorphism alleles defined 175 genotypes that were tagged by a minimal set of 138 polymorphisms. Eight major lineages of STEC O157 were identified, of which cattle are a reservoir for seven. Two lineages regularly harbored by cattle accounted for the majority of human disease in this study, whereas another was rarely represented in humans and may have evolved toward reduced human virulence. Notably, cattle are not a known reservoir for E. coli O55:H7 or STEC O157:H(-) (the first lineage to diverge within the STEC O157 serogroup), which both cause human disease. This result calls into question how cattle may have originally acquired STEC O157. The polymorphism-derived genotypes identified in this study did not surpass PFGE diversity assessed by BlnI and XbaI digestions in a subset of 93 strains. However, our results show that they are highly effective in assessing the evolutionary relatedness of epidemiologically unrelated STEC O157 genetic subtypes, including those associated with the cattle reservoir and human disease.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.     

Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker

The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Dual-Serotype Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and O26:H11 Strains

Escherichia coli O26:H11 strains were able to outgrow O157:H7 companion strains in planktonic and biofilm phases and also to effectively compete with precolonized O157:H7 cells to establish themselves in mixed biofilms. E. coli O157:H7 strains were unable to displace preformed O26:H11 biofilms. Therefore, E. coli O26:H11 remains a potential risk in food safety.