Open AccessAnalysis of the F Antigen-Specific papA Alleles of Extraintestinal Pathogenic Escherichia coli Using a Novel Multiplex PCR-Based Assay
Author(s) -
James R. Johnson,
Adam L. Stell,
Flemming Scheutz,
Timothy T. O’Bryan,
Thomas A. Russo,
Ulrike B. Carlino,
Claudine E. Fasching,
Justine A. Kavle,
Linda van Dijk,
Wim Gaastra
Publication year - 2000
Publication title -
infection and immunity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.508
H-Index - 220
eISSN - 1070-6313
pISSN - 0019-9567
DOI - 10.1128/iai.68.3.1587-1599.2000
Subject(s) - biology , serotype , genetics , multiplex polymerase chain reaction , phylogenetic tree , escherichia coli , allele , multiplex , polymerase chain reaction , virology , gene
Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenicEscherichia coli , are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants ofpapA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapA alleles among 75E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies ofpapA , of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctionalpap fragments. Among the urosepsis isolates, the assay revealed considerable segregation ofpapA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer ofpapA alleles between lineages. Sequencing ofpapA from two strains that werepapA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novelpapA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the knownpapA alleles. These findings provide novel insights into thepapA alleles of extraintestinal pathogenicE. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection ofpapA alleles available to any laboratory with PCR capability.