Isolation of Escherichia coli O157:H7 strains that do not produce Shiga toxin from bovine, avian and environmental sources

AIMS: To determine the potential for naturally occurring Shiga toxin-negative Escherichia coli O157 to acquire stx(2) genes. METHODS AND RESULTS: Multiple E. coli O157:H7 isolates positive for eae and ehxA, but not for stx genes, were isolated from cattle, water trough sediment, animal bedding and wild bird sources on several Ohio dairy farms. These isolates were experimentally lysogenized by stx(2)-converting bacteriophage. CONCLUSIONS: Shiga toxin-negative strains of E. coli O157 are present in multiple animal and environmental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: Shiga toxin-negative strains of E. coli O157 present in the food production environment are able to acquire the stx genes, demonstrating their potential to emerge as new Shiga toxin-producing E. coli strains.     

Complete DNA sequence analysis of enterohemorrhagic Escherichia coli plasmid pO157_2 in β-glucuronidase-positive E. coli O157:H7 reveals a novel evolutionary path

Strains of enterohemorragic Escherichia coli (EHEC) O157:H7 that are non-sorbitol fermenting (NSF) and β-glucuronidase negative (GUD(-)) carry a large virulence plasmid, pO157 (>90,000 bp), whereas closely related sorbitol-fermenting (SF) E. coli O157:H(-) strains carry plasmid pSFO157 (>120,000 bp). GUD(+) NSF O157:H7 strains are presumed to be precursors of GUD(-) NSF O157:H7 strains that also carry pO157. In this study, we report the complete sequence of a novel virulence plasmid, pO157-2 (89,762 bp), isolated from GUD(+) NSF O157:H7 strain G5101. PCR analysis confirmed the presence of pO157-2 in six other strains of GUD(+) NSF O157:H7. pO157-2 carries genes associated with virulence (e.g., hemolysin genes) and conjugation (tra and trb genes) but lacks katP and espP present in pO157. Comparative analysis of the three EHEC plasmids shows that pO157-2 is highly related to pO157 and pSFO157 but not ancestral to pO157. These results indicated that GUD(+) NSF O157:H7 strains might not be direct precursors to GUD(-) NSF O157:H7 as previously proposed but rather have evolved independently from a common ancestor.     

Characterization of inducible stx2-positive Escherichia coli O157:H7/H7- strains isolated from cattle in France

Aims:  To quantify the variability of the Shiga toxin 2 (Stx2) production by a panel of stx2 -positive Escherichia coli O157:H7/H7- isolates from healthy cattle before and after induction with enrofloxacin.
Methods and Results:  ProSpecT^® ELISA was used to quantify the Stx2 production by stx2 -positive E. coli O157:H7/H7- isolates in native conditions (basal level) or after induction with enrofloxacin. Whereas only 15·2% of the E. coli O157:H7/H7- strains studied displayed significant amounts of detectable Stx2 without induction, most of them were shown to be inducible, and at various levels, in presence of subinhibitory concentrations of enrofloxacin.
Conclusions:  We demonstrated the capability of a highly elevated proportion of stx2 -positive, but constitutively Stx2 -negative, E. coli O157:H7/H7- isolates from healthy cattle to produce significant levels of Shiga toxin Stx2 in presence of subtherapeutic concentrations of enrofloxacin, an antibiotic of the fluoroquinolones family only licensed for veterinary use.
Significance and Impact of the Study:  This study documents the risk that bovine-associated Shiga toxin producing E. coli isolates may become more frequently pathogenic to humans as a side-effect of the increasing use of veterinary fluoroquinolones in the oral treatment of food animals like cattle or poultry.     

Draft genome sequences of six Escherichia coli isolates from the stepwise model of emergence of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has been implicated in food-borne illnesses worldwide. An evolutionary model was proposed in which the highly pathogenic EHEC O157:H7 serotype arose from its ancestor, enteropathogenic E. coli (EPEC) O55:H7 (sorbitol fermenting [SOR(+)] and β-glucuronidase positive [GUD(+)]), through sequential gain of virulence, phenotypic traits, and serotype change. Here we report six draft genomes of strains belonging to this evolutionary model: two EPEC O55:H7 (SOR(+) GUD(+)) strains, two nonmotile EHEC O157:H(-) strains (SOR(+) GUD(+)) containing plasmid pSFO157, one EHEC O157:H7 (SOR(-) GUD(+)) strain, and one O157:H7 strain containing plasmid pSFO157 (SOR(+) GUD(+)).     

Introduction to the food safety concerns of verotoxin-producing Escherichia coli

Verotoxin-producing Escherichia coli (VTEC) have emerged in the past two decades as food-borne pathogens that can cause major outbreaks of human illnesses worldwide. The number of outbreaks has increased in recent years due to changes in food production and processing systems, eating habits, microbial adaptation, and methods of VTEC transmission. The human illnesses range from mild diarrhea to hemolytic uremic syndrome (HUS) that can lead to death. The VTEC outbreaks have been attributed to O157:H7 and non-O157:H7 serotypes of E. coli. These E. coli serotypes include motile (e.g., O26:H11 and O104:H21) and nonmotile (e.g., O111:H-, O145:H-, and O157:H-) strains. In the United States, E. coli O157:H7 has been the major cause of VTEC outbreaks. Worldwide, however, non-O157:H7 VTEC (e.g., members of the O26, O103, O111, O118, O145, and O166 serogroups) have caused approximately 30% of the HUS cases in the past decade. Because large numbers of the VTEC outbreaks have been attributed to consumption of ruminant products (e.g., ground beef), cattle and sheep are considered reservoirs of these food-borne pathogens. Because of the food safety concern of VTEC, a global perspective on this problem is addressed (Exp Biol Med Vol. 228, No. 4). The first objective was to evaluate the known non-O157:H7 VTEC strains and the limitations associated with their detection and characterization. The second objective was to identify the VTEC serotypes associated with outbreaks of human illnesses and to provide critical evaluation of their virulence. The third objective was to determine the rumen effect on survival of E. coli O157:H7 as a VTEC model. The fourth objective was to explore the role of intimins in promoting attaching and effacing lesions in humans. Finally, the ability of VTEC to cause persistent infections in cattle was evaluated.     

Occurrence, virulence characteristics and antimicrobial resistance of Escherichia coli O157 in slaughtered cattle and diarrhoeic calves in West Bengal, India

AIMS: (i) To study the occurrence of Escherichia coli serotype O157 in cattle stool in West Bengal, India, and (ii) the virulence properties and antimicrobial resistance of the E. coli isolates. METHODS AND RESULTS: Following enrichment in modified EC broth and plating onto HiCrome MS.O157 agar, a total of 14 strains of E. coli serotype O157 was isolated from faecal samples from two (2.04%) slaughtered cattle and six (7.59%) diarrhoeic calves. By multiplex PCR, Shiga toxin genes were detected in all the isolates. The enterohaemolysin phenotype was found in all, but one strain. Among 14 strains, ten were resistant to at least one of the antimicrobial agents tested. Multiple antibiotic resistance was frequent. CONCLUSIONS: The study showed that occurrence of Shiga toxin-producing and multiple antibiotic-resistant E. coli O157 among cattle population in this region of India is significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering routine human contacts with cattle, a large human population in this region may be at risk for exposure to Shiga toxin-producing E. coli O157.     

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

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

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.     

Isolation and characteristics of sorbitol-fermenting Escherichia coli O157 strains from cattle

Cattle can be a reservoir of sorbitol-fermenting Escherichia coli O157 (SF E. coli O157) and a source of human diseases. In this study, six strains of SF E. coli O157 were isolated and characterized from cattle using an immunomagnetic separation procedure. PCR analysis of the SF E. coli O157 virulence markers showed that all six isolates tested positive for sfpA, rfbE and eaeA, and negative for terA, ureA, katP and espP. Two of the isolates contained the stx genes. Four isolates tested positive for enterohemorrhagic E. coli hlyA (EhlyA) by PCR but were nonhemolytic on the blood agar. Five isolates tested positive for the cdtA gene. The possession of these virulence factors was an indication of their pathogenic potential. The random amplified polymorphic DNA patterns, which were generated by the arbitrarily primed PCR of the SF E. coli O157 isolates from the cattle, were significantly different from those of the non-sorbitol-fermenting E. coli O157 (NSF E. coli O157) strains originating from cattle or humans. GelCompar analysis showed that the SF E. coli O157 isolates had only a 57% genetic similarity with the NSF E. coli strains. The minimal inhibitory concentration assay showed that imipenem inhibited the growth of the six isolates at a concentration of <4 microg/ml.     

Diversity,frequency, and persistence of Escherichia coli O157 strains from range cattle environments

Genetic diversity, isolation frequency, and persistence were determined for Escherichia coli O157 strains from range cattle production environments. Over the 11-month study, analysis of 9,122 cattle fecal samples, 4,083 water source samples, and 521 wildlife fecal samples resulted in 263 isolates from 107 samples presumptively considered E. coli O157 as determined by culture and latex agglutination. Most isolates (90.1%) were confirmed to be E. coli O157 by PCR detection of intimin and Shiga toxin genes. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested preparations revealed 79 unique patterns (XbaI-PFGE subtypes) from 235 typeable isolates confirmed to be E. coli O157. By analyzing up to three isolates per positive sample, we detected an average of 1.80 XbaI-PFGE subtypes per sample. Most XbaI-PFGE subtypes (54 subtypes) were identified only once, yet the seven most frequently isolated subtypes represented over one-half of the E. coli O157 isolates (124 of 235 isolates). Recurring XbaI-PFGE subtypes were recovered from samples on up to 10 sampling occasions and up to 10 months apart. Seven XbaI-PFGE subtypes were isolated from both cattle feces and water sources, and one of these also was isolated from the feces of a wild opossum (Didelphis sp.). The number of XbaI-PFGE subtypes, the variable frequency and persistence of subtypes, and the presence of identical subtypes in cattle feces, free-flowing water sources, and wildlife feces indicate that the complex molecular epidemiology of E. coli O157 previously described for confined cattle operations is also evident in extensively managed range cattle environments.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli

AIMS: The verotoxigenic Escherichia coli (VTEC) serotype most commonly associated with verotoxin (VT) production is O157:H7, but other serotypes have also been implicated in food-borne illness. These serotypes exhibit much greater genetic and biochemical diversity than E. coli O157:H7, making screening for all VTEC difficult. Here we describe development and testing of novel multi-analyte antibody-based dipstick methods for presumptive detection of VTEC cells and VTs, including non-O157 serotypes. METHODS AND RESULTS: The dipsticks are formatted as paddle-style and lateral flow devices. Test materials included raw milk, minced beef, apple juice and salami, spiked with VTEC. Prototype paddle dipsticks gave 47 of 48 E. coli O157-positive samples correct, and, simultaneously, 27 of 31 O26-positive samples correct, across the four food types. Prototype lateral flow dipsticks gave 12 of 12 E. coli O157-positive milk samples correct and, simultaneously, 28 of 28 positive VT samples correct. CONCLUSIONS: This work demonstrates that simple and rapid detection of more than one VTEC characteristic (toxin production and type, serogroup) is possible in a single dipstick test device, directly from a food enrichment culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of simple easy-to-use rapid methods for simultaneous detection and preliminary characterization of VTEC will enable the risk presented by all VTEC to be more thoroughly assessed (e.g. in surveillance studies, outbreak investigations).     

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

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

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

The characterization of Shiga toxin-non-producing Escherichia coli serotype O157:H7 isolated from carcasses of cattle at a slaughter house.

A bacteriological investigation of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 was performed on 298 carcasses of cattle at slaughter houses between July 1996 and January 1997 in Gifu Prefecture, Japan. As a result, four Stx-non-producing Escherichia coli O157:H7 strains were isolated from two slaughtered carcasses of cattle. The purpose of this study was to examine the characterization of isolates. Isolates possessed the E. coli attaching and effacing gene (eaeA), and hemolysin gene (hlyA), and harbored 3.0-MDa and 60-MDa plasmids. The Xba I pulsed-field gel electrophoresis (PFGE) pattern showed three similar patterns. Consequently, a closely related genotype of Stx-non-producing E. coli O157:H7 may widely exist in cattle.     

Verotoxin-producing Escherichia coli in sheep grazing an irrigated pasture or arid rangeland forages

Worldwide, verotoxin-producing Escherichia coli (VTEC) have been recognized as the cause of many sporadic cases or major outbreaks of human illnesses involving consumption of contaminated meat, especially beef. Although sheep products have not been linked to reported human illnesses, their role as a food safety risk factor should not be ignored. The objective of this study was to assess VTEC prevalence in two groups of ewes (20 each) grazing an irrigated pasture or arid range in a western United States environment (Nevada) over 1 year (summer of 1999 to summer of 2000). A random sample (n = 504) of potential VTEC isolates were tested for verotoxicity and were screened for the presence (polymerase chain reaction [PCR]) and expression (VTEC-reversed passive latex agglutination assay) of the toxin genes (i.e., VT1 and VT2). Forty-one VTEC isolates (16 having only the VT1 gene and 25 having both VT1 And VT2 genes) were detected in both groups of ewes. Except for seven isolates, the genotype and phenotype data matched. All the isolates (nonmotile [H-]) were non-O157:H7 VTEC (i.e., O91:H- [n = 25], O128:H- [n = 9], and untypeable ones [n = 7]). More infected ewes (nine versus three) and different VTEC strains were found in the irrigated pasture than in the arid range. Because our ewes were shedding two VTEC serotypes known to cause human illnesses, it is beneficial to identify VTEC-positive sheep before slaughter as an initial control point before entering the food chain.     

Shiga toxin-producing Escherichia coli in sheep and pre-slaughter lambs in eastern Australia

Sheep and lambs from 14 farms in southern Queensland and one from central New South Wales were surveyed to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC). STEC, isolated from 45% of 144 sheep faeces collected on the farms and 36% of 72 lamb faeces from abattoir yards, were tested for the presence of genes encoding virulence factors. Most (64%) of the 117 STEC isolates contained Shiga toxin 1 and 2 genes, 22% contained those encoding Shiga toxin 1, and 14% contained genes encoding Shiga toxin 2. The genes encoding the E. coli attaching and effacing factor were present in 2.6% of STEC and 26% contained the enterohaemolysin gene. The isolates that contained the E. coli attaching and effacing gene were serotype O157:H. This study has shown that STEC are widely distributed in eastern Australian sheep and lambs and are shed in their faeces prior to slaughter. Thus, there is potential for contamination of carcasses and entry of STEC into the human food chain.     

Genetic Characterization of Escherichia coli O157:H7 Strains Isolated from the One-Humped Camel (Camelus dromedarius) by Using Microarray DNA Technology

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.