Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Lipopolysaccharide on the surface of Escherichia coli constitutes the O antigens which are important virulence factors that are targets of both the innate and adaptive immune systems and play a major role in host-pathogen interactions. O antigens are responsible for antigenic specificity of the strain and determine the O serogroup. The designation of O serogroups is important for classifying E. coli strains, for epidemiological studies, in tracing the source of outbreaks of gastrointestinal or other illness, and for linking the source to the infection. For conventional serogroup identification, serotyping by agglutination reactions against antisera developed for each of the O serogroups has been used. In the last decade, many O-antigen gene clusters that encode for the enzymes responsible for the synthesis of the variable oligosaccharide region on the surface of the bacteria have been sequenced and characterized. Unique gene sequences within the O-antigen gene clusters have been targeted for identification and detection of many O groups using the polymerase chain reaction and microarrays. This review summarizes current knowledge on the DNA sequences of the O-antigen gene clusters, genetic-based methods for O-group determination and detection of pathogenic E. coli based on O-antigen and virulence gene detection, and provides perspectives on future developments in the field.

Molecular evidence is limited for the hypothesis that humans, dogs, and cats can become colonized and infected with similar virulent Escherichia coli strains. To further assess this possibility, archived E. coli O6 isolates (n ‫؍‬ 130) from humans (n ‫؍‬ 55), dogs (n ‫؍‬ 59), and cats (n ‫؍‬ 16), representing the three main H (flagellar) types within serogroup O6 (H1, H7, and H31), were analyzed, along with selected reference strains. Isolates underwent PCR-based phylotyping, multilocus sequence typing, PCR-based detection of 55 virulenceassociated genes, and XbaI pulsed-field gel electrophoresis (PFGE) profiling. Three major sequence types (STs), which corresponded closely with H types, accounted for 99% of the 130 O6 isolates. Each ST included human, dog, and cat isolates; two included reference pyelonephritis isolates CFT073 (O6:K2:H1) and 536 (O6:K15:H31). Virulence genotypes overlapped considerably among host species, despite statistically significant differences between human and pet isolates. Several human and dog isolates from ST127 (O6:H31) exhibited identical virulence genotypes and highly similar PFGE profiles, consistent with cross-species exchange of specific E. coli clones. In conclusion, the close similarity in the genomic backbone and virulence genotype between certain human-and animal-source E. coli isolates within serogroup O6 supports the hypothesis of zoonotic potential.Escherichia coli is a major cause of urinary tract infections (UTI) and other extraintestinal infections in humans, dogs, and cats (1,19,25). Several cross-sectional surveys have demonstrated similarities among clinical or fecal E. coli isolates from humans, dogs, and cats with respect to genomic background and virulence-associated accessory traits (virulence factors [VFs]), particularly within serogroups O6 and O4, suggesting possible zoonotic (whether animal-to-human or human-to-animal) transmission (3,5,6,9,11,13,15,16,20,26,(30)(31)(32)(33). Consistent with this possibility, in two longitudinal surveillance studies involving the E. coli flora of human household members and their canine or feline pets, pets were found to be intermittently colonized with the same virulent-appearing E. coli clones that colonized multiple humans and caused acute cystitis in the women (8,22).However, these studies examined only modest numbers of isolates and accessory traits and/or relied on somewhat imprecise phylogenetic methods such as multilocus enzyme electrophoresis, outer membrane protein profiling, or random amplified polymorphic DNA analysis (3,5,6,8,9,11,13,15,16,20,22,26,(30)(31)(32)(33). Accordingly, they leave uncertainty as to the extent of commonality among human-and pet-derived E. coli isolates.To examine more rigorously the question of human-pet commonality with respect to extraintestinal pathogenic E. coli (ExPEC) clones, we used contemporary molecular methods to characterize a large collection of archival E. coli isolates of serotypes O6:H1, O6:H7, and O6:H31 from humans, dogs, and cats, obtained from the E. coli Reference Ce...

O-antigens on the surface of Escherichia coli are important virulence factors that are targets of both the innate and adaptive immune system and play a major role in pathogenicity. O-antigens that are responsible for antigenic specificity of the strain determine the O-serogroup. E. coli O26, O45, O103, O111, O113, O121, O145, and O157 have been the most commonly identified O-serogroups associated with Shiga toxin-producing E. coli (STEC) implicated in outbreaks of human illness all over the world. A multiplex polymerase chain reaction assay was developed to simultaneously detect the eight STEC O-serogroups targeting the wzx (O-antigen-flippase) genes of all O-antigen gene clusters. The sensitivity of the multiplex polymerase chain reaction was found to be 10 colony forming units for each O-group when enriched in broth and 100 colony forming units when enriched in artificially inoculated apple juice diluted with tryptic soy broth for 16 h at 37°C. The method can be used for detecting STEC O-groups simultaneously and may be exploited for improving the safety of food products.

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.Escherichia coli O26 strains, first isolated from cases of infantile diarrhea, have been implicated in causing hemolytic uremic syndrome (HUS) (13) and serious enteric disorders in humans in the United Kingdom (12), Germany (17), Poland (9), Spain (2), and Finland (7). Among the non-O157 Shiga toxin-producing E. coli (STEC) isolates, O26 has been the most common serogroup, composing 18% (1,066 of 5,913) of the total number of STEC isolates reported from 1997 to 1999 (6). E. coli O26 strains have been found to be genetically diverse with unique virulence profiles (19). An eae-negative O113:H21 STEC strain was responsible for an HUS outbreak in South Australia (10). Since traditional E. coli growth and isolation methods show all non-O157 STEC to be phenotypically similar to nonpathogenic E. coli, detection of specific STEC serogroups is problematic. There is no rapid method presently available for detecting specific STEC strains.The O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and consists of many repeats of an oligosaccharide unit (O unit). The O antigen is the major contributor of antigenic variability on the cell surface, and on this basis different O types have been designated. The genes involved in the biosynthesis of O antigens in E. coli are generally clustered and flanked by the galF and gnd genes at the 5Ј and 3Ј ends, respectively. O-antigen gene clusters including O26 and O113 have been cloned and sequenced (4, 11). Analyses of each of the genes in the cluster by National Center for Biotechnology Information genome BLAST and gene alignment software programs showed that the O-unit flippase gene (wzx) and the polymerase gene (wzy) were unique for E. coli O26 as well as for O113 antigens. Therefore, these genes were targeted for developing PCR assays for detecting these serogroups.All E. coli strains used in the study were from the bacterial collection of the Gastroenteric Disease Center at The Pennsylvania State University. Reference standard strains, E. coli O26:HϪ (H31b) and E. coli O113:H21 (6182-50) from the World Health Organization (8), were used for developing the assays. The 179 World Health Organization O reference ...