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.

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.

Since extraintestinal pathogenic Escherichia coli (ExPEC) strains from human and avian hosts encounter similar challenges in establishing infection in extraintestinal locations, they may share similar contents of virulence genes and capacities to cause disease. In the present study, 1,074 ExPEC isolates were classified by phylogenetic group and possession of 67 other traits, including virulence-associated genes and plasmid replicon types. These ExPEC isolates included 452 avian pathogenic E. coli strains from avian colibacillosis, 91 neonatal meningitis E. coli (NMEC) strains causing human neonatal meningitis, and 531 uropathogenic E. coli strains from human urinary tract infections. Cluster analysis of the data revealed that most members of each subpathotype represent a genetically distinct group and have distinguishing characteristics. However, a genotyping cluster containing 108 ExPEC isolates was identified, heavily mixed with regard to subpathotype, in which there was substantial trait overlap. Many of the isolates within this cluster belonged to the O1, O2, or O18 serogroup. Also, 58% belonged to the ST95 multilocus sequence typing group, and over 90% of them were assigned to the B2 phylogenetic group typical of human ExPEC strains. This cluster contained strains with a high number of both chromosome-and plasmid-associated ExPEC genes. Further characterization of this ExPEC subset with zoonotic potential urges future studies exploring the potential for the transmission of certain ExPEC strains between humans and animals. Also, the widespread occurrence of plasmids among NMEC strains and members of the mixed cluster suggests that plasmid-mediated virulence in these pathotypes warrants further attention. Speculation has long existed regarding a food-borne origin for extraintestinal pathogenic Escherichia coli (ExPEC) strains (28,33,42) and has spawned recent work investigating E. coli contaminants of food and the ExPEC strains of food-producing animals (15,18,24,40). Of particular interest in this regard are avian pathogenic E. coli (APEC) strains that cause colibacillosis in poultry (3,9,35,36,38). Although it has been widely assumed that most APEC strains do not possess zoonotic potential, recent reports have suggested otherwise for certain groups of strains (2,9,29,30,35,36), and some researchers have demonstrated that APEC strains and their plasmids may be transmitted to human hosts (27,38). Recently, APEC isolates have been compared to ExPEC isolates from human urinary tract infections (UTIs) and neonatal meningitis, revealing that these "subpathotypes" have some overlap in serogroups, phylogenetic groups, virulence genotypes, and abilities to cause disease in certain animal models (9,30,31,35,36). The validity of these observations was sustained by comparison of the first APEC genome sequence with sequenced ExPEC isolates of humans (25), which revealed that few differences existed between the sequenced APEC strain (APEC O1) and human strains. In fact, results of an in silico multilocus sequence typing ...

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 pappositive 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.

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

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.The virulent strains of Escherichia coli that cause urinary tract infections (UTIs) and other extraintestinal infections in humans (i.e., extraintestinal pathogenic E. coli [ExPEC]) owe their pathogenic potential largely to the presence of specialized virulence factors (VFs) which are absent from commensal members of the species and which allow ExPEC strains to colonize host mucosal surfaces, injure and invade host tissues, foil host defense mechanisms, and incite an injurious host inflammatory response (4,5,9,20). Currently recognized putative VFs (PVFs) of ExPEC include adhesins, siderophores, toxins, protectins, and invasins, some of which are encoded on pathogenicity-associated islands (PAIs) (2,7,8,17,24,26). PAIs are large blocks of established or suspected virulence genes that are inserted into the E. coli genome (often at tRNA loci) and which may provide a mechanisms for coordinate horizontal transfer of virulence genes between lineages within E. coli and even between species (3,7,21,24,25).Two recently described PAI-linked PVF genes of ExPEC, iha and iroN from E. coli (iroN E. coli ), are of interest both in their own right and because of their potential utility as markers for their respective PAIs of origin (23,27). iha, an novel nonhemagglutinating adhesin which in vitro confers HeLa cell adherence capability to (nonadhering) E. coli K-12, was first identified as part of a tellurite resistance-associated PAI (termed TAI, the tellurite resistance-adherence-conferring island) from an E. coli O157:H7 isolate from a patient with hemorrhagic colitis (27). iha exhibits nearly perfect sequence identity with the sequenced portion of an open reading frame (ORF) of unknown function (ORF "R4") from a PAI in archetypal ExPEC strain CFT073 (8,17). In addition to the iha homologue and multiple other ORFs of unknown significance, this PAI from strain CFT073 also contains an hly (hemolysin) operon and one of the strain's two pap operons (named for pilus associated with pyelonephritis; P fimbriae), which includes the F7-2 allele of papA (the P fimbrial structural subunit gene) and allele II of papG (the P fimbrial digalactoside-specific adhesin gene) (8,16,17). Consistent with the hypothesis that iha is a PVF in ExPEC, probes derived from PAI regions immediately adjacent to iha as it occurs in strain CFT073 hybridized significantly more frequent with UTI or bacteremia isolates of E. coli than with commensal E. coli (8).iroN E. coli , a novel catechole siderophore receptor which exhibits increased expression in urine, was recently identified in archetypal ExPEC strain CP9 as part of a PAI which also includes one of this strain's two pap operons (i.e., the pap operon con...

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. (2,33,53,57). This hypothesis is based on several lines of evidence, including (i) the documented similarities between certain canine and human urinary tract infection (UTI) isolates of E. coli with respect to virulence factors (VFs), O antigens, and evolutionary lineage (33,(53)(54)(55)57), (ii) the observation that in dogs with UTI the infecting E. coli strain often derives immediately from the host's own fecal flora (33), and (iii) the high prevalence of UTI-associated VFs among canine fecal E. coli isolates (57). However, doubts regarding the validity of the canine reservoir hypothesis have persisted (2) because of the differences noted in some studies between canine and human ExPEC isolates with respect to adherence phenotypes (8,33,48,55) and surface antigens (48, 56), which presumably reflect clonal relationships. Dogs have been proposed as a possible reservoir of the virulent Escherichia coli strains that cause extraintestinal infections in humans (extraintestinal pathogenic E. coli [ExPEC])The ostensibly atypical agglutination phenotypes of canine UTI isolates were recently shown to be due to expression by canine strains of papG allele III, which encodes a variant of the P-fimbrial adhesin molecule PapG that is now known to be epidemiologically associated with human cystitis (23). The agglutination phenotypes of strains that expressed papG allele III were found to be indistinguishable among canine and human isolates (23). These findings addressed the first major argument against considering canine-derived ExPEC isolates as potential human pathogens. In addition, clonal overlap was documented between human and canine ExPEC isolates...

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.