Escherichia coliserogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenicE. coliO2 and O28ac by PCR

Abstract

The O-antigen gene clusters of Escherichia coli serogroups O2 and O28ac were sequenced, and PCR assays were developed to identify strains belonging to these 2 serogroups. Sixteen and 8 open reading frames were mapped to these loci in E. coli O2:H4 U 9-41 and E. coli O28ac:H25 96-3286, respectively. The wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the E. coli O2 and O28ac O-antigen gene clusters were selected as targets for PCR assays for their identification. PCR assays targeting the wzx and wzy genes were specific for these serogroups, with one exception. Escherichia coli serogroup O42 strains gave positive results with wzx and wzy PCR assays targeting E. coli O28ac, and antiserum raised against O42 cross-reacted with serogroup O28ac strains. The O-antigen gene cluster of a strain of E. coli serogroup O42 was sequenced, and there were only 3 nt differences between the O-antigen gene clusters of the O28ac and O42 strains. Multiplex PCR assays targeting the O2 wzx gene, the stx1, stx2, hly, eae, and saa genes, and the O28ac wzx, ial, ipaC, and ipaH genes were developed for detecting Shiga toxin-producing E. coli O2 strains and enteroinvasive E. coli O28ac strains, respectively. The O2 and O28ac wzx and wzy genes can be used as diagnostic markers in PCR assays for rapid identification of these serogroups as an alternative to serotyping, and the multiplex PCR assays targeting serogroup-specific genes in combination with virulence genes can be used to identify and to detect pathogenic serogroup O2 and O28ac strains.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine

“…The O serotype of each isolate was confirmed by agglutination using a single E. coli O-serotypespecific antiserum (Statens Serum Institut, Denmark) and PCR using O-serotype-specific primers described by Fratamico et al (32) and Li et al (33). Assay working stocks were created on the basis of the method described by Burton and Nahm (34).…”

Toll-Like Receptor Stimulation Induces Nondefensin Protein Expression and Reverses Antibiotic-Induced Gut Defense Impairment

“…Since our results showed that antibiotic treatment markedly induced translocation of members of the Enterobacteriaceae to MLNs, the translocation of injected pathogenic K. pneumoniae organisms and the nonvirulent E. coli strain DH10B/DsRed was used to investigate the effect of intestinal microflora depletion on intestinal immunity. The E. coli strain DH10B without the virulence plasmid and with a plasmid carrying the dsRED2 gene fragment was used to generate the nonvirulent DH10B/DsRed strain (25). After antibiotic treatment for 6 days, there was a marked increase in the translocation of pathogenic K. pneumoniae to MLNs, liver, and blood (93-fold, 23-fold, and 41-fold, respectively) but not that of DH10B/DsRed.…”

Comprehensive Approaches to Molecular Biomarker Discovery for Detection and Identification of Cronobacter spp. ( Enterobacter sakazakii ) and Salmonella spp

“…There are 7 genes in this operon, including beta-carotene hydroxylase (crtZ), phytoene synthase (crtB), phytoene dehydrogenase Cronobacter O-antigen gene cluster. Another potential strainor serogroup-specific gene cluster, the O-antigen gene cluster, was investigated to determine its suitability as a biomarker (15,16,38,41). The O-antigen gene cluster contains genes with distinctly different functions, including genes involved in the processing and assembly of the O antigen.…”

“…The O-antigen gene cluster contains genes with distinctly different functions, including genes involved in the processing and assembly of the O antigen. The genes that encode the O antigen flippase (wzx) and the O antigen polymerase (wzy) are extensively used as biomarker genes for Salmonella and E. coli serotyping (15,16) and may be suitable biomarkers for Cronobacter spp. as well (Table 4).…”