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.

Diana Radune,Xianghe Yan,Narasimha Hegde,Lauren Brinkac,Robert Li,Pina Fratamico,Jianghong Meng,Magaly Toro,Chitrita DebRoy,Wei Wang,Liliana Losada,David Needleman,Yanhong Liu

doi:10.3390/bios5010051

Abstract

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources.

Highlights

In Escherichia coli and other Gram-negative bacteria, the major component of the outer membrane is lipopolysaccharide, which consists of three components: lipid A embedded in the membrane, an oligosaccharide core, and the lateral polysaccharide O-antigen
DNA sequences obtained from the E. coli O antigen gene clusters of serogroup O62, O68, O131, O140, O142 and O163 contained 9 to 12 ORFs (Figure 1 and Appendix Tables A1–A6), all in the same transcriptional direction from galF to gnd
The O-antigen gene cluster sequences for six E. coli serogroups have been determined, and PCR

Summary

Introduction

In Escherichia coli and other Gram-negative bacteria, the major component of the outer membrane is lipopolysaccharide, which consists of three components: lipid A embedded in the membrane, an oligosaccharide core, and the lateral polysaccharide O-antigen. The O-antigen confers antigenic variability to the bacteria due to differences in the sugar components, the linkages, and the structure of the repeat O-units. Traditional serotyping of E. coli is based on agglutination reactions of the bacteria with antisera raised in rabbits immunized with different O-group reference strains. The E. coli O-antigen is released by heating the bacteria for 2 h at 100 °C, and agglutination or clumping occurs when the O-antigen reacts with its specific antiserum. If the E. coli is capsulated or rough (does not carry O-antigen), agglutination does not occur

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