Clonal and pathotypic analysis of archetypal Escherichia coli cystitis isolate NU14.

Escherichia coli NU14, a cystitis isolate used to study the pathogenesis of cystitis and to develop a FimH (type 1 fimbrial adhesin) vaccine, was assessed for extended virulence genotype, phylogenetic background, and FimH sequence and binding phenotype(s). NU14 exhibited the same virulence genotype and was derived from the same (meningitis- and cystitis-associated) subclone of E. coli O18:K1:H7 as the archetypal neonatal bacterial meningitis (NBM) isolate RS218. NU14 also displayed the same Ser62Ala FimH polymorphism as did NBM isolates RS218 and IHE3034-conferring both collagen binding and a distinct monomannose binding capability (which characterizes uropathogenic but not commensal E. coli and dramatically increases adherence to uroepithelial cells). These findings establish that strain NU14 exhibits numerous urovirulence-associated traits and derives from the single most prevalent clonal group in acute cystitis. They provide further evidence of clonal and pathotypic similarities between cystitis and NBM isolates of E. coli O18:K1:H7.

PCR for specific detection of H7 flagellar variant of fliC among extraintestinal pathogenic Escherichia coli.

A newly developed PCR-based assay for the H7 variant of the Escherichia coli flagellin gene, fliC, was 100% sensitive and specific in comparison with serology and probe hybridization. It revealed broad conservation of the H7 fliC variant among phylogenetically diverse lineages of extraintestinal pathogenic E. coli (ExPEC) and superseded serotyping for certain isolates with ambiguous or non-H7 serotyping results. The H7 primers functioned well when incorporated into a multiplex PCR assay for diverse virulence-associated genes of ExPEC.

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.

Phylogenetic and pathotypic similarities between Escherichia coli isolates from urinary tract infections in dogs and extraintestinal infections in humans.

Seventeen Escherichia coli isolates from dogs with urinary tract infection (UTI) were characterized with respect to phylogenetic background and virulence genotype and were compared with the E. coli reference (ECOR) collection and with human clinical isolates with similar serotypes from patients with diverse extraintestinal infections. Most of the canine urine isolates were from (virulence-associated) E. coli phylogenetic groups B2 or D, expressed papG allele III, and exhibited numerous other putative virulence genes that are characteristic of human extraintestinal pathogenic E. coli (ExPEC). Close phylogenetic and pathotypic correspondence was documented within 5 clonal groups among individual canine and human isolates, including archetypal human ExPEC strains CFT073 (O6:K2:H1), 536 (O6:K15:H31), and J96 (O4:K-:H5). These findings suggest that canine UTI isolates, rather than being dog-specific pathogens, as previously suspected, may pose an infectious threat to humans. Commonality between canine and human ExPEC has potentially important implications for disease prevention, antibiotic resistance avoidance, and studies of pathogenesis.

Canine feces as a reservoir of extraintestinal pathogenic Escherichia coli.

To test the canine reservoir hypothesis of extraintestinal pathogenic Escherichia coli (ExPEC), 63 environmental canine fecal deposits were evaluated for the presence of ExPEC by a combination of selective culturing, extended virulence genotyping, hemagglutination testing, O serotyping, and PCR-based phylotyping. Overall, 30% of canine fecal samples (56% of those that yielded viable E. coli) contained papG-positive E. coli, usually as the predominant E. coli strain and always possessing papG allele III (which encodes variant III of the P-fimbrial adhesin molecule PapG). Multiple other virulence-associated genes typical of human ExPEC were prevalent among the canine fecal isolates. According to serotyping, virulence genotyping, and random amplified polymorphic DNA analysis, over 50% of papG-positive fecal E. coli could be directly correlated with specific human clinical isolates from patients with cystitis, pyelonephritis, bacteremia, or meningitis, including archetypal human ExPEC strains 536, CP9, and RS218. Five canine fecal isolates and (clonally related) archetypal human pyelonephritis isolate 536 were found to share a novel allele of papA (which encodes the P-fimbrial structural subunit PapA). These data confirm that ExPEC representing known virulent clones are highly prevalent in canine feces, which consequently may provide a reservoir of ExPEC for acquisition by humans.

Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli.

The 72 member strains of the Escherichia coli Reference collection were assessed as to genotype for 31 putative extraintestinal virulence factor (VF) genes and DNA sequence for papA, the P fimbrial structural subunit gene. Although most VFs were concentrated in phylogenetic group B2 or jointly in groups B2 and D, others were concentrated primarily in group D, were broadly distributed (without group-specific associations), and/or occurred only outside of group B2. Statistical correlations among VFs suggested linkage on pathogenicity-associated islands or plasmids. Isolates from humans and nonhuman primates had more VFs than did isolates from other animals. Sequence diversity was minimal within each F type-specific papA allele group but was substantial among different papA allele groups. The distribution patterns of papA variants and other VFs suggested multiple horizontal transfer events. These findings provide new insights into the phylogenetic origins of extraintestinal VFs in E. coli.

Molecular comparison of extraintestinal Escherichia coli isolates of the same electrophoretic lineages from humans and domestic animals.

Molecular typing methods were used to characterize 38 Escherichia coli strains that originally were isolated from extraintestinal infections and represented 5 multilocus enzyme electrophoretic types (ETs) recovered from both humans and animals. Within each ET, the human and animal isolates did not consistently segregate by host group, according to individual virulence factors (VFs), composite VF-serotype profiles, or pulsed-field gel electrophoresis profiles. Several close matches with respect to VF-serotype profiles were identified between human and canine isolates from different locales. One canine and 2 human isolates of serogroup O6 closely resembled archetypal human pyelonephritis isolate 536 (O6:K15:H31), according to papA sequence and VF-serotype profile. These findings support the hypothesis that certain pathogenic lineages of E. coli cause disease in both humans and animals and that humans may acquire pathogenic E. coli from domestic pets.

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.

Molecular epidemiological and phylogenetic associations of two novel putative virulence genes, iha and iroN(E. coli), among Escherichia coli isolates from patients with urosepsis.

Two novel putative Escherichia coli virulence genes, iha and iroN from E. coli (iroN(E. coli)), were detected in 55 and 39%, respectively, of 67 E. coli isolates from patients with urosepsis. iha and iroN(E. coli) exhibited divergent associations with other putative virulence genes, phylogenetic markers, host characteristics, and antimicrobial resistance.

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.

Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise.

Among 75 urosepsis isolates of Escherichia coli, 29 virulence factor (VF) genes were detected by use of a novel polymerase chain reaction (PCR) assay. Compared with probe hybridization, the PCR assay's specificity was 100% and sensitivity 97.1%. fyuA (yersiniabactin: overall prevalence, 93%), traT (serum resistance, 68%), and a pathogenicity-associated island marker (71%) occurred in most strains from both compromised and noncompromised hosts. Present in <20% of strains each were sfaS, focG (F1C fimbriae), afa/dra, bmaE (M fimbriae), gafD (G fimbriae), cnf1, cdtB (cytolethal distending toxin), cvaC (colicin V), and ibeA (invasion of brain endothelium). Different VFs were variously confined to virulence-associated phylogenetic group B2 (as defined by multilocus enzyme electrophoresis); concentrated in group B2, but with spread beyond; or concentrated outside of group B2. These findings provide novel insights into the VFs of extraintestinal pathogenic E. coli and demonstrate the new PCR assay's utility for molecular epidemiological studies.