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ABSTRACTExtraintestinal Escherichia coli(ExPEC), a heterogeneous group of pathogens, encompasses avian, neonatal meningitis, and uropathogenic E. colistrains. While several virulence factors are associated with ExPEC, there is no core set of virulence factors that can be used to definitively differentiate these pathotypes. Here we describe a multiplex of four virulence factor-encoding genes, yfcV, vat, fyuA, and chuA, highly associated with uropathogenic E. colistrains that can distinguish three groups of E. coli: diarrheagenic and animal-associated E. colistrains, human commensal and avian pathogenic E. colistrains, and uropathogenic and neonatal meningitis E. colistrains. Furthermore, human intestinal isolates that encode all four predictor genes express them during exponential growth in human urine and colonize the bladder in the mouse model of ascending urinary tract infection in higher numbers than human commensal strains that do not encode the four predictor genes (P= 0.02), suggesting that the presence of the predictors correlates with uropathogenic potential.

ABSTRACTEscherichia colisequence type ST131 (O25b:H4) has emerged over the past decade as a globally disseminated, multidrug-resistant pathogen. Unlike traditional antimicrobial-resistant E. coli, ST131 derives from virulence-associated phylogenetic group B2 and exhibits extraintestinal virulence factors. This, plus preliminary evidence of virulence in experimental animals, has suggested that ST131's epidemic emergence may be due to high virulence potential, compared with other E. colitypes. To test this hypothesis, we compared a large number of matched ST131 and non-ST131 E. coliclinical isolates, both fluoroquinolone resistant and susceptible, plus isolates from classic extraintestinal pathogenic E. coli(ExPEC) sequence types (STs) and case report ST131 household transmission isolates, for virulence in a mouse subcutaneous sepsis model. Overall, in mice, the study isolates produced a wide range of lethality and clinical illness. However, neither ST131 status nor fluoroquinolone phenotype correlated with this diversity of illness severity, which occurred within each of the 6 study groups. In contrast, multiple known or suspected ExPEC virulence genes, including pap(P fimbriae), vat(vacuolating toxin), kpsMII (group 2 capsule), ibeA(invasion of brain endothelium), and clbB/N(colibactin synthesis), plus molecularly defined ExPEC status, were significantly associated with virulence. These findings point away from ST131 isolates as having higher virulence potential compared with other E. colitypes in causing invasive extraintestinal infections and suggest instead that ST131's epidemiological success may reflect enhanced fitness for upstream steps in pathogenesis or in colonization and transmission. Additionally, the extensive within-ST virulence diversity suggests an opportunity to compare closely related strains to identify the responsible genetic determinants.

ABSTRACTEscherichia coli, a cause of ∼90% of urinary tract infections (UTI), utilizes fimbrial adhesins to colonize the uroepithelium. Pyelonephritis isolate E. coliCFT073 carries 12 fimbrial operons, 5 of which have never been studied. Using multiplex PCR, the prevalence of these 12 and 3 additional fimbrial types was determined for a collection of 303 E. coliisolates (57 human commensal, 32 animal commensal, 54 asymptomatic bacteriuria, 45 complicated UTI, 38 uncomplicated cystitis, and 77 pyelonephritis). The number of fimbrial types per E. coliisolate was distributed bimodally: those with low (3.2 ± 1.1) and those with high (8.3 ± 1.3) numbers of fimbrial types (means ± standard errors of the means). The fimbrial genes ygiL, yadN, yfcV, and c2395were significantly more prevalent among urine isolates than human commensal isolates. The effect of deletion of Ygi and Yad fimbrial operons on growth, motility, biofilm formation, adherence to immortalized human epithelial cells, and pathogenesis in the mouse model of UTI was examined. Yad fimbriae were necessary for wild-type levels of adherence to a bladder epithelial cell line and for biofilm formation. Deletion of these fimbrial genes increased motility. Ygi fimbriae were necessary for wild-type levels of adherence to a human embryonic kidney cell line, biofilm formation, and in vivofitness in the urine and kidneys. Complementation of each fimbrial mutant restored wild-type levels of motility, biofilm formation, adherence and, for ygi, in vivofitness. A double deletion strain, Δygi Δyad, was attenuated in the urine, bladder, and kidneys in the mouse model, demonstrating that these fimbriae contribute to uropathogenesis.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a debilitating syndrome of unknown etiology often postulated, but not proven, to be associated with microbial infection of the prostate gland. We hypothesized that infection of the prostate by clinically relevant uropathogenic Escherichia coli (UPEC) can initiate and establish chronic pain. We utilized an E. coli strain newly isolated from a patient with CP/CPPS (strain CP1) and examined its molecular pathogenesis in cell culture and in a murine model of bacterial prostatitis. We found that CP1 is an atypical isolate distinct from most UPEC in its phylotype and virulence factor profile. CP1 adhered to, invaded, and proliferated within prostate epithelia and colonized the prostate and bladder of NOD and C57BL/6J mice. Using behavioral measures of pelvic pain, we showed that CP1 induced and sustained chronic pelvic pain in NOD mice, an attribute not exhibited by a clinical cystitis strain. Furthermore, pain was observed to persist even after bacterial clearance from genitourinary tissues. CP1 induced pelvic pain behavior exclusively in NOD mice and not in C57BL/6J mice, despite comparable levels of colonization and inflammation. Microbial infections can thus serve as initiating agents for chronic pelvic pain through mechanisms that are dependent on both the virulence of the bacterial strain and the genetic background of the host.

ABSTRACTChronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a debilitating syndrome of unknown etiology often postulated, but not proven, to be associated with microbial infection of the prostate gland. We hypothesized that infection of the prostate by clinically relevant uropathogenic Escherichia coli(UPEC) can initiate and establish chronic pain. We utilized an E. colistrain newly isolated from a patient with CP/CPPS (strain CP1) and examined its molecular pathogenesis in cell culture and in a murine model of bacterial prostatitis. We found that CP1 is an atypical isolate distinct from most UPEC in its phylotype and virulence factor profile. CP1 adhered to, invaded, and proliferated within prostate epithelia and colonized the prostate and bladder of NOD and C57BL/6J mice. Using behavioral measures of pelvic pain, we showed that CP1 induced and sustained chronic pelvic pain in NOD mice, an attribute not exhibited by a clinical cystitis strain. Furthermore, pain was observed to persist even after bacterial clearance from genitourinary tissues. CP1 induced pelvic pain behavior exclusively in NOD mice and not in C57BL/6J mice, despite comparable levels of colonization and inflammation. Microbial infections can thus serve as initiating agents for chronic pelvic pain through mechanisms that are dependent on both the virulence of the bacterial strain and the genetic background of the host.

Uropathogenic Escherichia coli (UPEC), the most frequent cause of urinary tract infection (UTI), is associated with an inflammatory response which includes the induction of cytokine/chemokine secretion by urothelial cells and neutrophil recruitment to the bladder. Recent studies indicate, however, that UPEC can evade the early activation of urothelial innate immune response in vitro. In this study, we report that infection with the prototypic UPEC strain NU14 suppresses tumor necrosis factor alpha (TNF-)-mediated interleukin-8 (CXCL-8) and interleukin-6 (CXCL-6) secretion from urothelial cell cultures compared to infection with a type 1 piliated E. coli K-12 strain. Furthermore, examination of a panel of clinical E. coli isolates revealed that 15 of 17 strains also possessed the ability to suppress cytokine secretion. In a murine model of UTI, NU14 infection resulted in diminished levels of mRNAs encoding keratinocyte-derived chemokine, macrophage inflammatory peptide 2, and CXCL-6 in the bladder relative to infection with an E. coli K-12 strain. Furthermore, reduced stimulation of inflammatory chemokine production during NU14 infection correlated with decreased levels of bladder and urine myeloperoxidase and increased bacterial colonization. These data indicate that a broad phylogenetic range of clinical E. coli isolates, including UPEC, may evade the activation of innate immune response in the urinary tract, thereby providing a pathogenic advantage.

ABSTRACTWe have found an avian pathogenic Escherichia coli(APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli(UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. colistrain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coliand their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.

ABSTRACTVirulence factors of pathogenic Escherichia colibelonging to a recently emerged and disseminated clonal group associated with urinary tract infection (UTI), provisionally designated clonal group A (CGA), have not been experimentally investigated. We used a mouse model of ascending UTI with CGA member strain UCB34 in order to identify genes of CGA that contribute to UTI. ihawas identified to be expressed by strain UCB34 in the mouse kidney using selective capture of transcribed sequences. ihafrom strain UCB34 demonstrated a siderophore receptor phenotype when cloned in a catecholate siderophore receptor-negative E. coliK-12 strain, as shown by growth promotion experiments and uptake of 55Fe complexed to enterobactin or its linear 2, 3-dihydroxybenzoylserine (DHBS) siderophore derivatives. Siderophore-mediated growth promotion by Iha was TonB dependent. Growth and iron uptake were more marked with linear DHBS derivatives than with purified enterobactin. The reported phenotype of adherence to epithelial cells conferred by expressing ihafrom a multicopy cloning vector in a poorly adherent E. coliK-12 host strain was confirmed to be specific to iha, in comparison with other siderophore receptor genes. ihaexpression was regulated by the ferric uptake regulator Fur and by iron availability, as shown by real-time reverse transcriptase PCR. In a competitive infection experiment using the mouse UTI model, wild-type strain UCB34 significantly outcompeted an isogenic ihanull mutant. Iha thus represents a Fur-regulated catecholate siderophore receptor that, uniquely, exhibits an adherence-enhancing phenotype and is the first described urovirulence factor identified in a CGA strain.

ABSTRACTTo survive within the host urinary tract, Escherichia colistrains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of Escherichia colias a possible virulence factor in UTI by determining the impact of oxyRinactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47phox−/−) mice. The oxyRand oxySgenes of wild-type E. colistrain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an oxyRSmutant. During in vitro growth in broth or human urine, the oxyRSmutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and oxyRSmutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H2O2, which, like the altered growth kinetics observed with oxyRSinactivation, were reversed by restoration of oxyRon a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its oxyRSmutant (by >1 log10) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an oxyR-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47phox−/−C57BL mice were similarly susceptible, and the oxyRmutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47phoxgenotype, as in CBA/J mice. Within the E. coliReference collection, 94% of strains were positive for oxyR. These findings fulfill the second and third of Koch's molecular postulates for oxyRas a candidate virulence-facilitating factor in E. coliand indicate that oxyRis a broadly prevalent potential target for future preventive interventions against UTI due to E. coli. They also suggest that neutrophil phagocyte oxidase is not critical for defense against E. coliUTI and that the major oxidative stresses against which OxyR protects E. coliwithin the host milieu are not phagocyte derived.

ABSTRACTThe role of the Escherichia coliiron-regulated gene homologue adhesin (Iha) in the pathogenesis of urinary tract infections (UTIs) is unknown. We performed a series of complementary analyses to confirm or refute the hypothesis that Iha is a virulence factor in uropathogenic E. coli. Fecal E. coliisolates exhibited significantly lower prevalences of iha(range, 14 to 22%) than did clinical isolates from cases of pediatric cystitis or pyelonephritis, adult pyelonephritis or urosepsis, or bacteremia (range, 38 to 74%). Recombinant Iha from E. colipyelonephritis isolate CFT073 conferred upon nonadherent E. coliORN172 the ability to adhere to cultured T-24 human uroepithelial cells. In a well-established mouse model of ascending UTI, CFT073 and its derivative UPEC76 (a pap[P fimbriae] mutant version of strain CFT073) each significantly outcompeted their respective ihadeletion mutants in CBA/J mice 48 h after bladder challenge (P< 0.03 for urine, both kidneys, and bladders of both constructs, except for bladders of mice challenged with UPEC76 and its deletion mutant, where P= 0.11). These data suggest that IhaCFT073is a virulence factor and might be a target for anti-UTI interventions.

ABSTRACTIsogenic Enterococcus faecalisstrains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS+, constitutive EBS+) failed to outcompete isogenic derivative INY3000 (AS−EBS−) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS+, EBS+) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by E. faecalisand that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.

ABSTRACTOur laboratory is studying an extraintestinal pathogenic isolate ofEscherichia coli(CP9) as a model pathogen. We have been using human urine, ascites, and blood ex vivo to identify genes with increased expression in these media relative to expression in Luria-Bertani (LB) broth. Such genes may represent new or unrecognized virulence traits. In this study, we report the identification of a new gene, ireA(iron-responsive element). This gene has an open reading frame of 2,049 nucleotides, and its peptide has a molecular mass of 75.3 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its expression is increased a mean of 3.6-fold in human urine, 16.2-fold in human ascites, and 6.6-fold in human blood relative to expression in LB medium, and it is Fe repressible. IreA also exhibits peptide similarities (48 to 56%) to previously identified proteins that function as siderophore receptors, suggesting that IreA is involved in iron acquisition. PCR-based analysis ofireA's phylogenetic distribution detectedireAin none (0%) of 14 fecal isolates that represented probable commensal strains, but in 13 (26%) of 50 random urine and blood clinical isolates (P= 0.05) and in 5 (100%) of 5 representatives of the J96-like, clonal group of which CP9 is a member (P< 0.001). In a mouse urinary tract infection model, the presence of ireAcontributed significantly to CP9's ability to colonize the bladder (P< 0.02), evidence that IreA is a urovirulence factor. Taken together, these findings demonstrate thatireAencodes a new virulence factor, which is likely involved in Fe acquisition.

ABSTRACTThe phylogenetic distributions of multiple putative virulence factors (VFs) and papA(P fimbrial structural subunit) alleles among 182 Escherichia coliblood isolates from patients with diverse-source bacteremia were defined. Phylogenetic correspondence among these strains, the E. coliReference (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. coliand 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.

ABSTRACTP fimbriae of extraintestinal pathogenic Escherichia colimediate 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 papGallele I and the respective wild-type source strains were characterized. One of the new papGallele I variants conferred a unique agglutination phenotype that combined the phenotypes associated withpapGalleles 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 papGallele I variants occurred either as the sole papGallele or together with both papGalleles II and III, rather than with only papGallele III, as in archetypal strains J96 and CP9. They also occurred in the absence of the usual F13 papAallele. One of the newpapGallele 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 ofE. coliO4:H5, which includes all previously identified examples of papGallele I. Cluster analysis of nucleotide and predicted peptide sequences suggested that papGallele I represents the earliest evolutionary branch from a commonpapGancestor. These results demonstrate unexpected diversity within papGallele I and, together with previous findings, suggest that the J96-like clonal group of E. coliO4:H5 may represent the original source of papGwithin the species.

ABSTRACTAlthough 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. coliisolates 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 papGallele III. Agglutination phenotypes segregated strictly according topapGallele status and were homogeneous among strains with the same papGallele 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 papAallele 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. coliwith infectious potential for humans.

ABSTRACTTwo novel putative Escherichia colivirulence genes,ihaand iroNfrom E. coli(iroNE. coli), were detected in 55 and 39%, respectively, of 67 E. coliisolates from patients with urosepsis. ihaand iroNE. coliexhibited divergent associations with other putative virulence genes, phylogenetic markers, host characteristics, and antimicrobial resistance.

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.

ABSTRACTAlleles I, II, and III of the P adhesin gene papGwere sought by PCR among 75 Escherichia coliblood isolates from adults with urosepsis, and the papGgenotype was compared with associated bacterial characteristics and host compromise status. Allele II predominated over allele III in the total population (71% of the strains versus 7% for allele III; P< 0.01). Allele I was not encountered. In comparison with allele II, allele III was significantly associated with the presence of hlyand the absence of iuc(which encode hemolysin and aerobactin biosynthesis), nonagglutination of digalactoside-coated beads, absence of aerobactin production, membership in serogroups O6 and O18, and host compromise, particularly cancer and upper urinary tract abnormalities.