Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

Abstract

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10 6 and 10 8 CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.Strains of Escherichia coli belonging to serogroup O45 have been isolated from animals and humans and classified as both enterotoxigenic E. coli and Shiga toxin-producing enterohemorrhagic E. coli (4,16,25). In addition, E. coli O45 strains isolated from diarrheic dairy calves produced cytotoxic necrotizing factor and were designated as strains of necrotoxigenic E. coli (10). Many E. coli O45 strains isolated from swine have been associated with postweaning diarrhea and demonstrated the attaching and effacing (A/E) phenotype, allowing them to adhere to intestinal epithelial cells (3). Six E. coli serogroups, including O45, were associated with feral pigeons and produced a variant of Shiga toxin 2 called Stx2f (9).E. coli strains belonging to serogroup O55 have been classified as human enteropathogenic strains and are believed to be genetically related to enterohemorrhagic E. coli O157:H7, shown to have been derived from an O55:H7 ancestral clone (15,23). Because of the potential pathogenicity of E. coli isolates belonging to serogroups O45 and O55, rapid and reliable assays for detecting and identifying these serogroups in food, environmental, and clinical samples are needed.Conventionally, antigenic analysis of the ca. 179 different O serogroups in E. coli is performed by agglutination reactions using antisera raised in rabbits against the O standard reference strains. O serotyping is laborious, and cross-reactions between different serogroups often occur, giving equivocal results. Furthermore, E. coli strains occasionally undergo transition from smooth to rough forms as a result of mutations in one or more of the multiple genes controlling synthesis and polymerization of the O antigen. Rough isolates do not produce an O antigen and therefore cannot be typed by using antisera. In view of these facts, there is a need for the development of alternative methods for identifying and typing E. coli serogroups. Lipopolysaccharides (LPS) are essential components of the gram-negative bacterial outer membrane. They are composed of three parts: lipid A, which is composed of sugars and fatty acids and anchors the LPS in the outer membrane through covalent linkages; an oligosacc...

Development of a Serotype-Specific DNA Microarray for Identification of Some Shigella and Pathogenic Escherichia coli Strains

“…The O-serotype-specific genes of each of the S. boydii types 13, 16, and 18 and E. coli O55, O111, O114, and O157 have been reported previously (7,12,13,15,22,33,36). In this study, we identified the specific genes of the other eight serotypes: S. sonnei; S. flexneri type 2a; S. boydii types 7 and 9; S. dysenteriae types 4, 8, and 10; and E. coli O128.…”

“…The diverse forms of O antigen are almost entirely due to genetic variation in the O-antigen gene cluster (29). Due to the relatively low similarity of glycosyltransferase, wzx, and wzy genes among different serotypes, their sequences are normally highly specific to individual O antigens (7,8,11,14,30).…”

A New O-Antigen Gene Cluster Has a Key Role in the Virulence of the Escherichia coli Meningitis Clone O45:K1:H7

“…Numerous O-antigen gene clusters have been sequenced in recent years, and PCR assays targeting the O-antigen unit processing genes, the O-antigen flippase (wzx) and the O-antigen polymerase (wzy), have been developed (5,6,13,16,17,19,44,45). This Ogenotyping approach has several advantages, including low cost, rapidity, and detection of strains with the O antigen of interest in a complex microflora.…”

“…The emergence of the unusual O45 antigen in ExPEC strains prompted us to analyze the O-antigen gene cluster sequence of S88, a strain representative of the O45:K1:H7 clone causing neonatal meningitis, by comparison with the published sequence of strain 96-3285 (O45:H2) (16). We show that the two O-antigen gene clusters are genetically related but not identical, suggesting that S88 expresses a new O antigen.…”

“…In addition, E. coli O45 strains producing cytotoxic necrotizing factor or expressing the "attaching and effacing" phenotype have been isolated from several diarrheic animals (4,32). The importance of O45 antigen in InPEC strains led DebRoy et al to sequence the genomic region involved in O-antigen synthesis in the CDC reference strain 96-3285 (O45:H2) and to develop a specific PCR method to detect E. coli O45 (16). Many PCR tests for E. coli serogroups have recently been developed by targeting the genes involved in O-antigen synthesis and clustered in the so-called O-antigen gene cluster.…”