Development of PCR Assays Targeting the Genes Involved in Synthesis and Assembly of the New Escherichia coli O174 and O177 O Antigens

ABSTRACT Escherichia coli O174 and O177 are newly described O serogroups which were reported as human pathogens. Identification of these strains by serotyping has been restricted, as the required sera are not commercially available. In this study, a collection of 13 E. coli O174 strains and 12 E. coli O177 strains was studied on the O:H serotypes and virulence markers. The O-antigen gene clusters of E. coli O174 and O177 were sequenced, and associated genes were assigned functions on the basis of homology. Two genes, each specific for E. coli O174 and O177, were identified. PCR assays based on the O-antigen-specific genes were developed and tested on 25 clinical and environmental isolates of those two serogroups as well as 26 isolates of other O serogroups. As little as 1 pg per μl of chromosomal DNA and as few as 0.1 CFU per g of pork and water samples were detected for either strain. The PCR assays established in this study were shown to be highly sensitive and reliable and could be the method of choice for detection of these two human pathogens from clinical, food, and other environmental samples.

[1]  Lei Wang,et al.  Sequence Analysis of the Escherichia coli O15 Antigen Gene Cluster and Development of a PCR Assay for Rapid Detection of Intestinal and Extraintestinal Pathogenic E. coli O15 Strains , 2005, Journal of Clinical Microbiology.

[2]  L. Beutin,et al.  Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48 , 2005, BMC Microbiology.

[3]  J. Blanco,et al.  Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina. , 2004, International microbiology : the official journal of the Spanish Society for Microbiology.

[4]  H. R. Rodríguez,et al.  Shiga toxin-producing Escherichia coli in healthy young beef steers from Argentina: prevalence and virulence properties. , 2004, International journal of food microbiology.

[5]  F. Scheutz,et al.  Designation of O174 and O175 to temporary O groups OX3 and OX7, and six new E. coli O groups that include Verocytotoxin‐producing E. coli (VTEC): O176, O177, O178, O179, O180 and O181 , 2004, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[6]  L. Beutin,et al.  Identification of Escherichia coli O114 O-Antigen Gene Cluster and Development of an O114 Serogroup-Specific PCR Assay , 2004, Journal of Clinical Microbiology.

[7]  L. Feng,et al.  Identification of Escherichia coli O172 O‐antigen gene cluster and development of a serogroup‐specific PCR assay , 2004, Journal of applied microbiology.

[8]  J. Blanco,et al.  Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina. , 2004, Veterinary microbiology.

[9]  L. Beutin,et al.  Serotyping, stx2 Subtyping, and Characterization of the Locus of Enterocyte Effacement Island of Shiga Toxin-Producing Escherichia coli and E. coli O157:H7 Strains Isolated from the Environment in France , 2004, Applied and Environmental Microbiology.

[10]  L. Beutin,et al.  Characterization of Shiga Toxin-Producing Escherichia coli Strains Isolated from Human Patients in Germany over a 3-Year Period , 2004, Journal of Clinical Microbiology.

[11]  J. Blanco,et al.  Serotypes, Virulence Genes, and Intimin Types ofShiga Toxin (Verotoxin)-Producing Escherichia coli Isolatesfrom Cattle in Spain and Identification of a New Intimin VariantGene(eae-ξ) , 2004, Journal of Clinical Microbiology.

[12]  Jinghua Yang,et al.  Structural and Genetic Characterization of the Shigella boydii Type 13 O Antigen , 2004, Journal of bacteriology.

[13]  L. Beutin,et al.  Animal host associated differences in Shiga toxin‐producing Escherichia coli isolated from sheep and cattle on the same farm , 2003, Journal of applied microbiology.

[14]  J. Brisson,et al.  Three Highly Conserved Proteins Catalyze the Conversion of UDP-N-acetyl-d-glucosamine to Precursors for the Biosynthesis of O Antigen in Pseudomonas aeruginosaO11 and Capsule in Staphylococcus aureus Type 5 , 2003, The Journal of Biological Chemistry.

[15]  P. Fach,et al.  Identification of the O‐antigen biosynthesis genes of Escherichia coli O91 and development of a O91 PCR serotyping test , 2002, Journal of applied microbiology.

[16]  P. Reeves,et al.  Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes. , 2002, Gene.

[17]  P. Reeves,et al.  Sequence Analysis of Four Shigella boydii O-Antigen Loci: Implication for Escherichia coli andShigella Relationships , 2001, Infection and Immunity.

[18]  P. Fratamico,et al.  Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes. , 2001, Gene.

[19]  L. Trabulsi,et al.  10.321/eid0805.Typical and Atypical Enteropathogenic Escherichia coli , 2002, Emerging infectious diseases.

[20]  Sequence of the E . coli O 104 antigen gene cluster and identi ® cation of O 104 speci ® c genes , 2001 .

[21]  P. Reeves,et al.  Sequencing of Escherichia coli O111 O-Antigen Gene Cluster and Identification of O111-Specific Genes , 1998, Journal of Clinical Microbiology.

[22]  P. Reeves,et al.  Organization of Escherichia coli O157 O Antigen Gene Cluster and Identification of Its Specific Genes , 1998, Infection and Immunity.

[23]  M. Valvano,et al.  Promoter Region of the Escherichia coli O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence , 1998, Journal of bacteriology.

[24]  L. Beutin,et al.  Rapid detection and isolation of shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay , 1996, Journal of clinical microbiology.

[25]  P. Reeves,et al.  Sequence and analysis of the O antigen gene (rfb) cluster of Escherichia coli O111. , 1995, Gene.

[26]  C. Gyles.  Escherichia coli in Domestic Animals and Humans , 1994 .

[27]  M. Valvano,et al.  Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine , 1994, Journal of bacteriology.

[28]  L. Beutin,et al.  Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids , 1994, Journal of clinical microbiology.

[29]  P. Reeves,et al.  The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters , 1994, Molecular microbiology.

[30]  W. Ewing,et al.  Edwards and Ewing's Identification of Enterobacteriaceae , 1986 .