Identification of Escherichia coli O172 O-antigen gene cluster and development of a serogroup-specific PCR assay

Abstract

Aim: To characterize the locus for O-antigen biosynthesis from Escherichia coli O172 type strain and to develop a rapid, specific and sensitive PCR-based method for identification and detection of E. coli O172. Methods and Results: DNA of O-antigen gene cluster of E. coli O172 was amplified by long-range PCR method using primers based on housekeeping genes galF and gnd Shot gun bank was constructed and high quality sequencing was performed. The putative genes for synthesis of UDP-FucNAc, O-unit flippase, O-antigen polymerase and glycosyltransferases were assigned by the homology search. The evolutionary relationship between O-antigen gene clusters of E. coli O172 and E. coli O26 is shown by sequence comparison. Genes specific to E. coli O172 strains were identified by PCR assays using primers based on genes for O-unit flippase, O-antigen polymerase and glycosyltransferases. The specificity of PCR assays was tested using all E. coli and Shigella O-antigen type strains, as well as 24 clinical E. coli isolates. The sensitivity of PCR assays was determined, and the detection limits were 1 pg ll )1 chromosomal DNA, 0AE2 CFU g )1 pork and 0AE2 CFU ml )1 water. The total time required from beginning to end of the procedure was within 16 h. Conclusion:The O-antigen gene cluster of E. coli O172 was identified and PCR assays based on O-antigen specific genes showed high specificity and sensitivity. Significance and Impact of the Study: An O-antigen gene cluster was identified by sequencing. The specific genes were determined for E. coli O172. The sensitivity of O-antigen specific PCR assay was tested. Although Shiga toxin-producing O172 strains were not yet isolated from clinical specimens, they may emerge as pathogens.

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

“…Primer pairs wl-4397/wl-4398 and wl-4399/wl-4400 from E. coli O174 and wl-4393/wl-4394 and wl-4395/wl-4396 from E. coli O177 were also used to screen for E. coli strains of these two O serogroups in pork and water samples using a method described previously (14).…”

“…PCR methods for the detection of O-antigen-specific genes such as wzx and wzy represent a reliable, rapid, and sensitive alternative to serotyping, particularly for E. coli strains that belong to serogroups that are not covered by the panel of commercially available antisera (5,14,22,27). Moreover, PCR detection of O-antigen-specific genes allows detection of strains which are serologically rough for their O antigens, and last but not least, serological cross-reactions between different O antigens that may cause difficulties in O typing are ruled out.…”

“…More recently, DNA-based typing methods were employed for the detection of E. coli O-antigen-encoding genes, and correspondent PCR protocols for the identification of pathogenic E. coli strains such as O157, O111, O114, and O172 were developed (12,14,28,30). The O antigen, as a part of the lipopolysaccharide moiety of gram-negative bacteria, consists of many repeats of an oligosaccharide unit (O unit) (9).…”

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

“…Traditional serotyping requires the use of a large panel of antisera; moreover, it is subjective and cross-reactive (16). In recent years, PCR assays based on O-serotype-specific genes have been proposed by us and others for molecular typing of many Shigella and E. coli O serotypes (2, 3, 7, 8, 10, 11, 12, 15,18,31,34,36). Molecular typing has many advantages over the traditional method.…”

Molecular analysis ofShigella boydiiO1 O-antigen gene cluster and its PCR typing

“…a Gene size and sequence positions refer to genes in E. coli K-12. b PCR templates were prepared as described (Feng et al 2004c;Guo et al 2004). The PCRs were performed as follows: denaturation at 95°C for 30 s, annealing at the temperature as indicated for 30 s and extension at 72°C for 1 min, for 30 cycles.…”