Widespread distribution of urinary tract infections caused by a multidrug-resistant Escherichia coli clonal group.

BACKGROUND: The management of urinary tract infections is complicated by the increasing prevalence of antibiotic-resistant strains of Escherichia coli. We studied the clonal composition of E. coli isolates that were resistant to trimethoprim-sulfamethoxazole from women with community-acquired urinary tract infections. METHODS: Prospectively collected E. coli isolates from women with urinary tract infections in a university community in California were evaluated for antibiotic susceptibility, O:H serotype, DNA fingerprinting, pulsed-field gel electrophoretic pattern, and virulence factors. The prevalence and characteristics of an antibiotic-resistant clone were evaluated in this group of isolates and in those from comparison cohorts in Michigan and Minnesota. RESULTS: Fifty-five of the 255 E. coli isolates (22 percent) from the California cohort were resistant to trimethoprim-sulfamethoxazole as well as other antibiotics. There was a common pattern of DNA fingerprinting, suggesting that the isolates belonged to the same clonal group (clonal group A), in 28 of 55 isolates with trimethoprim-sulfamethoxazole resistance (51 percent) and in 2 of 50 randomly selected isolates that were susceptible to trimethoprim-sulfamethoxazole (4 percent, P<0.001). In addition, 11 of 29 resistant isolates (38 percent) from the Michigan cohort and 7 of 18 (39 percent) from the Minnesota cohort belonged to clonal group A. Most of the clonal group A isolates were serotype O11:H(nt) or O77:H(nt), with similar patterns of virulence factors, antibiotic susceptibility, and electrophoretic features. CONCLUSIONS: In three geographically diverse communities, a single clonal group accounted for nearly half of community-acquired urinary tract infections in women that were caused by E. coli strains with resistance to trimethoprim-sulfamethoxazole. The widespread distribution and high prevalence of E. coli clonal group A has major public health implications.

Ongoing horizontal and vertical transmission of virulence genes and papA alleles among Escherichia coli blood isolates from patients with diverse-source bacteremia.

The phylogenetic distributions of multiple putative virulence factors (VFs) and papA (P fimbrial structural subunit) alleles among 182 Escherichia coli blood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coli Reference (ECOR) collection, and other collections of extraintestinal pathogenic E. coli (ExPEC) was assessed. Although among the 182 bacteremia isolates phylogenetic group B2 predominated, exhibited the greatest concentration of individual VFs, and contained the largest number of familiar virulent clones, other phylogenetic groups exhibited greater concentrations of certain VFs than did group B2 and included several additional virulent clones. Certain of the newly detected VF genes, e.g., fyuA (yersiniabactin; 76%) and focG (F1C fimbriae; 25%), were as prevalent or more prevalent than their more familiar traditional counterparts, e.g., iut (aerobactin; 57%) and sfaS (S fimbriae; 14%), thus possibly offering additional useful targets for preventive interventions. Considerable diversity of VF profiles was observed at every level within the phylogenetic tree, including even within individual lineages. This suggested that many different pathways can lead to extraintestinal virulence in E. coli and that the evolution of ExPEC, which involves extensive horizontal transmission of VFs and continuous remodeling of pathogenicity-associated islands, is a highly active, ongoing process.

Clonal relationships and extended virulence genotypes among Escherichia coli isolates from women with a first or recurrent episode of cystitis.

To identify bacterial predictors of recurrence and/or persistence in acute cystitis, extended virulence genotypes were compared with clonal background and epidemiologic status among 74 Escherichia coli urine isolates from women with first or recurrent episodes of urinary tract infection (UTI). Sequential isolates from patients with recurrent UTI were classified, using macrorestriction analysis, as having caused an isolated recurrence versus a single or multiple same-strain recurrences. papA, papG allele II, iha, and iutA predicted multiple same-strain recurrences, whereas nfaE and the absence of sfaS or fyuA predicted isolated recurrences. Phylogenetic group B2 accounted for 70% of isolates and for most of the putative virulence factors (VFs) studied. The meningitis-associated O18:K1:H7 clonal group comprised 18% of isolates, exhibited multiple VFs, and caused "once-only" recurrences less commonly than did other strains. These findings identify specific VFs and clonal groups against which preventive interventions might be beneficial and illustrate the importance of delineating pathogenetically relevant subgroups within the "recurrent cystitis" population.

Novel molecular variants of allele I of the Escherichia coli P fimbrial adhesin gene papG.

P fimbriae of extraintestinal pathogenic Escherichia coli mediate digalactoside-specific adherence via the tip adhesin molecule PapG, which occurs in three known variants (I to III), which are encoded by the corresponding three alleles of papG. In the present study, newly discovered variants of papG allele I and the respective wild-type source strains were characterized. One of the new papG allele I variants conferred a unique agglutination phenotype that combined the phenotypes associated with papG alleles I, II, and III. Comparative hydrophilicity analysis of predicted PapG peptides revealed regions that might explain the observed phenotypic similarities and differences between the PapG variants. The new papG allele I variants occurred either as the sole papG allele or together with both papG alleles II and III, rather than with only papG allele III, as in archetypal strains J96 and CP9. They also occurred in the absence of the usual F13 papA allele. One of the new papG allele I variants occurred in a serogroup O6 strain that, according to random amplified polymorphic DNA analysis, was phylogenetically distant from the "J96-like" clonal group of E. coli O4:H5, which includes all previously identified examples of papG allele I. Cluster analysis of nucleotide and predicted peptide sequences suggested that papG allele I represents the earliest evolutionary branch from a common papG ancestor. These results demonstrate unexpected diversity within papG allele I and, together with previous findings, suggest that the J96-like clonal group of E. coli O4:H5 may represent the original source of papG within the species.

Escherichia coli O18:K1:H7 isolates from patients with acute cystitis and neonatal meningitis exhibit common phylogenetic origins and virulence factor profiles.

Escherichia coli isolates of serotype O18:K1:H7, taken from women with acute cystitis, healthy control patients, and infants with neonatal bacterial meningitis (NBM), were analyzed and were compared with phylogenetically diverse control strains from the E. coli Reference collection. Clonal relationships were defined by amplification phylotyping, nicotinamide auxotrophy, and outer membrane protein patterns (OMPs). Virulence factor profiles were determined by multiplex polymerase chain reaction, probe hybridization, and hemagglutination testing. The O18:K1:H7 cystitis, fecal, and NBM isolates were clonally derived. The cystitis isolates and archetypal NBM isolates RS218 and C5 were from the OMP6 subclone of E. coli O18:K1:H7 and exhibited a consensus virulence genotype that included papG allele III (cystitis-associated P fimbrial adhesin), sfaS (S fimbrial adhesin), hlyA (hemolysin), cnf1 (cytotoxic necrotizing factor), iroN (putative siderophore), and ibeA (invasion of brain endothelium). The demonstrated commonality between O18:K1:H7 isolates from cystitis and NBM suggests common pathogenetic mechanisms and the possibility of new approaches to prevention.

Evidence of commonality between canine and human extraintestinal pathogenic Escherichia coli strains that express papG allele III.

Although dogs have been proposed as carriers of extraintestinal pathogenic Escherichia coli (ExPEC) with infectious potential for humans, presumed host species-specific differences between canine and human ExPEC strains have cast doubt on this hypothesis. The recent discovery that allele III of papG (the P fimbrial adhesin gene) predominates among human cystitis isolates and confers an adherence phenotype resembling that of canine ExPEC prompted the present reevaluation of the canine-human ExPEC connection. Sixteen paired pap-positive urine and rectal E. coli isolates from dogs with urinary tract infection were studied. papG (adhesin) and papA (pilin) allele type, agglutination phenotypes, virulence factor genotypes, and randomly amplified polymorphic DNA and pulsed-field gel electrophoresis fingerprints were analyzed and compared with those of human ExPEC controls. The 16 canine strains contained predominantly papG allele III. Agglutination phenotypes segregated strictly according to papG allele status and were homogeneous among strains with the same papG allele profile irrespective of their human versus canine origin. Canine and human PapG variant III peptide sequences were highly homologous, without host species-specific differences. The most prevalent canine papA allele was F48, a novel variant recently identified among human urosepsis isolates. In addition to pap, human ExPEC-associated virulence genes detected among the canine strains included sfa/focDE, sfaS, fyuA, hlyA, cnf1, cdtB, kpsMT-II and -III, rfc, traT, ompT, and a marker for a pathogenicity-associated island from archetypal human ExPEC strain CFT073. Molecular fingerprinting confirmed the fecal origin of all but one canine urine isolate and showed one pair of O6 canine urine and fecal isolates to be extremely similar to an O6 human urosepsis isolate with which they shared all other genotypic and phenotypic characteristics analyzed. These data demonstrate that canine ExPEC strains are similar to, and in some instances essentially indistinguishable from, human ExPEC strains, which implicates dogs and their feces as potential reservoirs of E. coli with infectious potential for humans.

Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli.

Repetitive-element PCR (rep-PCR) fingerprinting is a promising molecular typing tool for Escherichia coli, including for discriminating between pathogenic and nonpathogenic clones, but is plagued by irreproducibility. Using the ERIC2 and BOXA1R primers and 15 E. coli strains from the ECOR reference collection (three from each phylogenetic group, as defined by multilocus enzyme electrophoresis [MLEE], including virulence-associated group B2), we rigorously assessed the effect of extremely elevated annealing temperatures on rep-PCR's reproducibility, discriminating power, and ability to reveal MLEE-defined phylogenetic relationships. Modified cycling conditions significantly improved assay reproducibility and discriminating power, allowing fingerprints from different cyclers to be analyzed together with minimal loss of resolution. The correspondence of rep-PCR with MLEE with respect to tree structure and regression analysis of distances was substantially better with modified than with standard cycling conditions. Nonetheless, rep-PCR was only a fair surrogate for MLEE, and when fingerprints from different days were compared, it failed to distinguish between different clones within all-important phylogenetic group B2. These findings indicate that although the performance and phylogenetic fidelity of rep-PCR fingerprinting can be improved substantially with modified assay conditions, even when so improved rep-PCR cannot fully substitute for MLEE as a phylogenetic typing method for pathogenic E. coli.

Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay.

Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia 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 of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA 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 of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. 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 of papA alleles available to any laboratory with PCR capability.