Development of primers to O-antigen biosynthesis genes for specific detection of Escherichia coli O157 by PCR.

The chemical composition of each O-antigen subunit in gram-negative bacteria is a reflection of the unique DNA sequences within each rfb operon. By characterizing DNA sequences contained with each rfb operon, a diagnostic serotype-specific probe to Escherichia coli O serotypes that are commonly associated with bacterial infections can be generated. Recently, from an E. coli O157:H7 cosmid library, O-antigen-positive cosmids were identified with O157-specific antisera. By using the cosmid DNAs as probes, several DNA fragments which were unique to E. coli O157 serotypes were identified by Southern analysis. Several of these DNA fragments were subcloned from O157-antigen-positive cosmids and served as DNA probes in Southern analysis. One DNA fragment within plasmid pDS306 which was specific for E. coli O157 serotypes was identified by Southern analysis. The DNA sequence for this plasmid revealed homology to two rfb genes, the first of which encodes a GDP-mannose dehydratase. These rfb genes were similar to O-antigen biosynthesis genes in Vibrio cholerae and Yersinia enterocolitica serotype O:8. An oligonucleotide primer pair was designed to amplify a 420-bp DNA fragment from E. coli O157 serotypes. The PCR test was specific for E. coli O157 serotypes. PCR detected as few as 10 cells with the O157-specific rfb oligonucleotide primers. Coupled with current enrichment protocols, O157 serotyping by PCR will provide a rapid, specific, and sensitive method for identifying E. coli O157.     

Detection of Escherichia coli Serogroups O26 and O113 by PCR Amplification of the wzx and wzy Genes

PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as ≤10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.     

Development of an Allele-Specific PCR Assay for Simultaneous Sero-Typing of Avian Pathogenic Escherichia coli Predominant O1, O2, O18 and O78 Strains

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.     

Development of Three Multiplex PCR Assays Targeting the 21 Most Clinically Relevant Serogroups Associated with Shiga Toxin-Producing E. coli Infection in Humans

Escherichia coli serogroups O5, O15, O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145, O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant Shiga toxin-producing E. coli (STEC) serotypes. In this study, three multiplex PCR assays able to specifically detect these 21 serogroups were developed and validated. For this purpose, the O-antigen gene clusters of E. coli O5 and O76 were fully sequenced, their associated genes were identified on the basis of homology, and serogroup-specific primers were designed. After preliminary evaluation, these two primer pairs were proven to be highly specific and suitable for the development of PCR assays for O5 and O76 serogroup identification. Specific primers were also designed for serogroups O15, O45, O55, O91, O104, O113, O118, O123, O128, O146, O157, O165, O172, and O177 based on previously published sequences, and previously published specific primers for serogroups O26, O103, O111, O121, and O145 were also included. These 21 primer pairs were shown to be specific for their target serogroup when tested against E. coli type strains representing 169 known O-antigen forms of E. coli and Shigella and therefore suitable for being used in PCR assays for serogroup identification. In order to validate the three multiplex PCR assays, 22 E. coli strains belonging to the 21 covered serogroups and 18 E. coli strains belonging to other serogroups were screened in a double-blind test and their sensitivity was determined as 1 ng chromosomal DNA. The PCR assays developed in this study could be a faster, simpler, and less expensive strategy for serotyping of the most clinically relevant STEC strains in both clinical microbiology and public health laboratories, and so their development could benefit for clinical diagnosis, epidemiological investigations, surveillance, and control of STEC infections.     

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 Clone Sonnei (Shigella sonnei) Had a Chromosomal O-Antigen Gene Cluster Prior to Gaining Its Current Plasmid-Borne O-Antigen Genes

O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria. The surface-exposed O antigen is subject to selection by the host immune system, which may account for the maintenance of many different O-antigen forms. Characteristically, all genes specific to O-antigen synthesis are clustered in a region close to the his and gnd genes on the chromosome of Escherichia coli and related species. Shigella sonnei, essentially a clone of E. coli (E. coli clone Sonnei), is an important human pathogen and is unusual in that its O-antigen gene cluster is located on a plasmid. Our results suggest that it once had a normal chromosomal O-antigen gene cluster which has been largely deleted. We suggest that the O antigen encoded by the plasmid-borne genes offered a selective advantage in adapting to a new environment and that the chromosomal O-antigen genes were eventually inactivated. We also identified, by PCR and sequencing, a potential ancestor of E. coli Sonnei among the 166 known E. coli serotype strains.     

Lipopolysaccharide with an altered O-antigen produced in Escherichia coli K-12 harbouring mutated, cloned Shigella flexneri rfb genes

Cloning of the rfb genes of Shigella flexneri 2a into Escherichia coli K-12 strain DH1 results in the synthesis of lipopolysaccharides (LPS) with an O-antigen chain having type antigen IV and group antigens 3,4. During genetic studies of these rfb genes in E. coli K-12, we observed that strains harbouring plasmids with certain mutations (inversion and transposon insertions) which should have blocked O-antigen synthesis nevertheless still produced LPS with O-antigen chains. These LPS migrated differently on silver-stained SDS—polyacrylamide gels, compared with the LPS produced by wild-type rfb genes, and the group 3,4 antigens were barely detectable, suggesting that the O-antigen was altered. Investigation of the genetic determinants for production of the altered O-antigen/LPS indicated that: (i) these LPS are produced as a result of mutations which are either polar on rfbF or inactivate rfbF; (ii) the rfbX gene product (or a similar protein in the E. coli K-12 rfb region) is needed for production of the altered O-antigen in the form of LPS; (iii) the rfbG gene product is required for the production of both the parental and altered LPS; (iv) the dTDP-rhamnose biosynthesis genes are required. Additionally, an E. coli K-12 gene product(s) encoded outside the rfb region also contributes to production of the O-antigen of the altered LPS. An antiserum raised to the altered LPS from strain DH1(pPM2217 (rfbX::Tn1725)) was found to cross-react with nearly all S. flexneri serotypes, and with the altered LPS produced by other DH1 strains harbouring plasmids with different rfb mutations, as described above. The reactivity of the altered LPS with a panel of monoclonal antibodies specific for various S. flexneri O-antigen type and group antigens demonstrated that their O-antigen components were closely related to that of S. flexneri serotype 4. The RfbF and RfbG proteins were shown to have similarity to rhamnose transferases, and we identified a motif common to the N-termini of 6-deoxy-hexose nucleotide sugar transferases. We propose that the E. coli K-12 strains harbouring the mutated S. flexneri rfb genes produce LPS with a hybrid O-antigen as a consequence of inactivation of RfbF and complementation by an E. coli K-12 gene product. Analysis of the genetic and immunochemical data suggested a possible structure for the O-antigen component of the altered LPS.     

Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses

Glycoconjugate is one of the most efficacious and safest vaccines against bacterial pathogens. Previous studies of glycoconjugates against pathogen E. coli O157:H7 focused more on the humoral responses they elicited. However, little was known about their cellular responses. In this study, we exploited a novel approach based on bacterial protein N-linked glycosylation system to produce glycoconjugate containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein and examined its humoral and cellular responses in BALB/c mice. The transfer of E. coli O157:H7 O-antigen to MBP was confirmed by western blot and MALDI-TOF MS. Mice injected with glycoconjugate O-Ag-MBP elicited serum bactericidal antibodies including anti-E. coli O157:H7 O-antigen IgG and IgM. Interestingly, O-Ag-MBP also stimulated the secretion of anti-E. coli O157:H7 O-antigen IgA in intestine. In addition, O-Ag-MBP stimulated cellular responses by recruiting Th1-biased CD4⁺ T cells, CD8⁺ T cells. Meanwhile, O-Ag-MBP induced the upregulation of Th1-related IFN-γ and downregulation of Th2-related IL-4, and the upregulation of IFN-γ was stimulated by MBP in a dose-dependent manner. MBP showed TLR4 agonist-like properties to activate Th1 cells as carrier protein of O-Ag-MBP. Thus, glycoconjugate vaccine E. coli O157:H7-specific O-Ag-MBP produced by bacterial protein N-linked glycosylation system was able to elicit both humoral and Th1-biased cellular responses.     

Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes

Background

Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed.

Results

Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157.

Conclusion

Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs.     

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10⁶ and 10⁸ CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.     

Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliC _h7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.     

Detection of Escherichia coli serogroup O103 by real-time polymerase chain reaction

AIMS: The aims of the study were to identify the specific genes of O-antigen gene cluster from Shiga toxin-producing Escherichia coli (STEC) O103 and to provide the basis for a specific real-time PCR test for rapid detection of E. coli O103. METHODS AND RESULTS: The published primers complementary to JUMPstart and gnd gene, the conserved flanking sequences of O-antigen genes clusters in E. coli and related species, were used to amplify the 12-kbp O103 O-antigen biosynthesis locus of STEC O103. A DNA library representative of this cluster allowed two O103-specific probes to be identified in the flippase (wzx) and UDP-galactose-4-epimerase (galE) genes. Two specific O103 serotyping real-time PCR tests based on these two genes were successfully developed. CONCLUSIONS: These results confirm that the O-antigen gene cluster sequences of E. coli allow rapidly a specific O-antigen real-time PCR assay to be designed. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings increase the number of real-time PCR-assays available to replace the classical O-serotyping among E. coli O-antigen.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Mapping of chromosomal loci associated with lipopolysaccharide synthesis and serotype specificity in Vibrio cholerae 01 by transposon mutagenesis using Tn5 and Tn2680

Summary Vibrio cholerae strains of the 01 serotype have been classified into three subclasses, Ogawa, Inaba and Hikojima, which are associated with the O-antigen of the lipopolysaccharide (LPS). The DNA encoding the biosynthesis of the O-antigen, the rfb locus, has been cloned and analysed (Manning et al. 1986; Ward et al. 1987). Transposon mutagenesis of the Inaba and Ogawa strains of V. cholerae, using Tn5 or Tn2680 allowed the isolation of a series of independent mutants in each of these serotypes. Some of the insertions were mapped to the rfb region by Southern hybridization using the cloned rfb DNA as a probe, confirming this location to be responsible for both O-antigen production and serotype specificity. The other insertions allowed a second region to be identified which is involved in V. cholerae LPS biosynthesis.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of the wzx and wzy genes

PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as < or =10 CFU/ml. The O26- and O113-specific PCR assays can potentially be used for typing E. coli O26 and O113 serogroups; these assays will offer an advantage to food and environmental microbiology laboratories in terms of identifying these non-O157 serogroups by replacing antigen-based serotyping.     

Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Avian Hosts in British Columbia,Canada

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study, Escherichia coli isolates (n = 412) from the feces of various avian host sources were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae (enteropathogenic E. coli [EPEC]), est-h, est-p, and elt (encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). None of the isolates were found to be positive for stx₁, while 23% (n = 93) were positive for only stx₂, representing STEC, and 15% (n = 63) were positive for only eae, representing EPEC. In addition, five strains obtained from pheasant were positive for both stx₂ and eae and were confirmed as non-O157 by using an E. coli O157 rfb (rfb_O157) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic E. coli and clustered independently. MANOVA analysis also revealed spatial variation among the E. coli isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health.     

Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coli Groups

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx₁ and stx₂ was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.