MOLECULAR CHARACTERIZATION OF PATHOGENS AND THEIR RESPONSES TO ENVIRONMENTAL FACTORS
Location: Molecular Characterization of Foodborne Pathogens
Title: DNA SEQUENCING, ANALYSIS, AND IDENTIFICATION OF SEROGROUP-SPECIFIC GENES IN THE ESCHERICHIA COLI O28AC AND O118 O ANTIGEN GENE CLUSTERS
Submitted to: Foodborne Pathogens and Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 1, 2008
Publication Date: August 1, 2008
Citation: Liu, Y., Fratamico, P.M., Debroy, C., Bumbaugh, A., Allen, J. 2008. DNA SEQUENCING, ANALYSIS, AND IDENTIFICATION OF SEROGROUP-SPECIFIC GENES IN THE ESCHERICHIA COLI O28AC AND O118 O ANTIGEN GENE CLUSTERS. Foodborne Pathogens and Disease. 5(4):449-457.
Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and non-harmful E. coli types, referred to as serogroups, also exist. Traditionally, a procedure called serotyping is used to distinguish among the ca. 170 different E. coli serogroups. This procedure, which relies on the use of antibodies raised in rabbits against different surface polysaccharides of the bacteria, can only be performed in specialized laboratories, is labor intensive and may require several days to complete, and one antiserum can react with multiple E. coli serogroups, rendering identification of the specific strain of E. coli difficult. Thus, due to the lack of simple, rapid, and reliable methods for detection and identification of harmful and non-harmful E. coli types, the incidence of disease caused by harmful strains of E. coli may be underestimated, and epidemiological studies are difficult to perform. To develop a more rapid and simple method for detection and typing of important pathogenic E. coli belonging to serogroups O28ac and O118, the DNA sequence of a cluster of genes involved in the production of specific surface polysaccharides of these bacteria was determined. Genes required for biosynthesis of the surface polysaccharides (O antigen) in each of the serogroups were identified, and polymerase chain reaction (PCR) assays were developed targeting unique DNA sequences of two genes present in each of these serogroups. The PCR assays were serogroup specific when tested against a variety of different bacterial strains. Thus, the use of the E. coli O28ac- and O118-specific PCR assays facilitates the ability to identify, detect, and type these bacteria, and potentially can eliminate the use of the labor-intensive serotyping procedure. While testing for the specificity of the PCR assays targeting E. coli O118, however, cross reactivity against E. coli serogroup O151 occurred. The DNA sequence of the O antigen gene cluster of E. coli O151 was determined, and it was very similar to that of E. coli O118. The similarity in the genes encoding the O antigens of E. coli O151 and O118 indicate that these serotypes shared a common ancestor and that E. coli O151 may be a subgroup of E. coli O118.
The DNA sequence of the O antigen gene clusters of Escherichia coli serogroups O28ac and O118 was determined, and 7 and 13 ORFs were identified, respectively, encoding genes required for O antigen sugar biosynthesis, transfer, and processing. Analysis of the DNA sequence revealed that the wzx (O antigen flippase) and wzy (O antigen polymerase) genes in the O antigen gene clusters of E. coli O28ac and O118 shared little homology with similar genes in other E. coli; therefore PCR assays targeting these genes were designed for identification of these serogroups. Specificity testing using strains belonging to E. coli O28ac and O118 isolated from various sources, representative strains of 167 different E. coli O serogroups, and non-E. coli bacteria revealed that the PCR assays were specific for E. coli O28ac and O118. Thus, the PCR assays can be used for rapid identification of these serogroups as an alterative to typing using antisera. However, the PCR assays targeting the E. coli O118 wzx and wzy genes were positive using E. coli serogroup O151 DNA. Therefore, the sequence of the O antigen gene cluster of E. coli O151 was determined, and it was almost identical to that of E. coli O118, with only 9 nucleotide differences. Six and 3 of the differences resulted in synonomous and non-synonomous substitutions, respectively. Analysis of the sequences using the Jameson-Wolf antigenic index predicted differences in the antigenic determinants near the sites of residue replacement. Multilocus sequencing typing of E. coli O118 and O151 strains revealed minor variation at the nucleotide level; over a total of 3753 bp, 61 sites (1.6%) differed among the isolates examined. Since E. coli O118 strains are more frequently isolated from humans, animals, and the environment than E. coli O151, serogroup O151 may likely be a minor variant of E. coli O118. Further studies are needed to elucidate this possibility