Analysis of the F Antigen-Specific papAAlleles of Extraintestinal Pathogenic Escherichia coliUsing a Novel Multiplex PCR-Based Assay

ABSTRACTPolymorphisms 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 ofpapAand then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapAalleles among 75 E. coliisolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapAsequences 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 of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional papfragments. Among the urosepsis isolates, the assay revealed considerable segregation of papAalleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer ofpapAalleles between lineages. Sequencing ofpapAfrom two strains that were papApositive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papAvariants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papAalleles. These findings provide novel insights into the papAalleles of extraintestinal pathogenic E. coliand indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papAalleles available to any laboratory with PCR capability.