The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Abstract

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroupspecific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx 1 and stx 2 genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx 1 and stx 2 genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples.

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping

“…Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Long PCR assays were performed to amplify the O-antigen gene clusters using the Expand Long Template PCR system (Roche Applied Science, Mannheim, Germany) and the JUMPSTART (named for J ust U pstream of M any P olysaccharide-associated gene STARTs) and GND (6-phosphogluconate dehydrogenase gene) primer set targeting sequences that flank the E. coli O-antigen gene clusters as described previously [ 12 ]. However, some modifications were made to the JUMPSTART and GND primer sequences, and they are the following: JUMPSTART primer 5 ' -CATGGTAGCTGTAAAGCCAGGGGCGGTAGCGTG-3 ' ; GND primer 5 ' -CATGCTGCCATACCGACGACGCCGATCTGTTGCTTKGACA-3 ' (Integrated DNA Technologies, Coralville, IA, USA).…”

“…Lin et al [ 11 ] performed PCR assays targeting the wzx and wzy genes of ten Shiga toxin-producing E. coli (STEC) serogroups, and then used the Luminex system to identify the ten serogroups through binding of the PCR products to fluorescent microspheres conjugated to specific DNA probes for each of the ten serogroups. Furthermore, multiplex assays can be designed to detect specific pathogenic E. coli serogroups targeting O-antigen gene cluster sequences and virulence genes [ 7 , 12 ]. Use of the Luminex system (Luminex, Austin, TX, USA) employing monoclonal antibodies coated to carboxylated magnetic microbeads to simultaneously detect Shiga toxin serogroup O157, as well as Shiga toxin 1 and Shiga toxin 2 has also been reported [ 13 ].…”

Detection by Multiplex Real-Time Polymerase Chain Reaction Assays and Isolation of Shiga Toxin–ProducingEscherichia coliSerogroups O26, O45, O103, O111, O121, and O145 in Ground Beef

“…Two internal amplification controls were evaluated with each assay. The first consisted of the use of primers and probe targeting the 16 rRNA gene of gamma proteobacteria (99-bp product) (Fratamico et al, 2009a). The other internal system evaluated employed linearized pUC19 and primers and probe targeting the pMB1 rep gene carried on the plasmid (118-bp product).…”

Detection of Escherichia coli O157:H7 in Food Using Real-Time Multiplex PCR Assays Targeting the stx 1, stx 2, wzy O157, and the fliC h7 or eae Genes

“…The optimal concentration of linearized pUC19 was 10 4 copies/PCR (Ct about 25-27), and the size of the PCR product was 118 bp. The third internal control system targeted the 16S rRNA gene (gamma-proteobacteria) as previously described by Fratamico et al (2009); however, a probe labeled with Cy5 was used ( Table 1). The primers were used at a concentration of 0.1 μM and the probe at 0.03 μM.…”