Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM

Abstract

Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen. Identification of the H7 flagellar antigen is critical for the confirmation of E. coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen. To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E. coli. The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis. Two hundred eighty E. coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed. A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates. Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A. A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern. Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups. The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E. coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E. coli strains.