Timothy T. O'Bryan

Phylogenetic Distribution of Virulence-Associated Genes among Escherichia coli Isolates Associated with Neonatal Bacterial Meningitis in The Netherlands

Seventy cerebrospinal fluid Escherichia coli isolates from infants with neonatal bacterial meningitis (NBM), as submitted to the Netherlands Reference Laboratory for Bacterial Meningitis from 1989 through 1997, were assessed for phylogenetic background and extended virulence genotypes, in comparison with the E. coli reference collection, by using molecular methods. Phylogenetic group B2 significantly predominated overall (81%). The 4 major phylogenetic clusters exhibited distinctive virulence genotypes, suggesting diverse evolutionary histories for the individual genes. Many genes not previously studied in NBM, notably diarrhea-associated cdtB (cytolethal distending toxin [46%]) and urinary tract infection-associated ompT (outer membrane protease T [96%]), were as or more prevalent than traditional NBM-associated traits, such as ibeA (invasion of brain endothelium [33%]), sfaS (S fimbriae [59%]), and K1 capsule (81%). These findings provide novel insights into the phylogenetic origins of NBM-associated E. coli and suggest numerous new potential targets for preventive interventions against this dire disease.

Evidence of Commonality between Canine and Human Extraintestinal Pathogenic Escherichia coli Strains That Express papG Allele III

ABSTRACT 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 topapG 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.

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

ABSTRACT 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. 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. 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.

Improved Repetitive-Element PCR Fingerprinting for Resolving Pathogenic and Nonpathogenic Phylogenetic Groups within Escherichia coli

ABSTRACT 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 15E. 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-PCRs 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.

A disseminated multidrug-resistant clonal group of uropathogenic Escherichia coli in pyelonephritis

In acute pyelonephritis, bacterial resistance to co-trimoxazole predicts treatment failure, but the clonal basis of such resistance is undefined. We did molecular and serological analyses of 170 Escherichia coli urine isolates obtained in 1994-96 from women with acute pyelonephritis. 12 (7%) of the pyelonephritis isolates were in clonal group A (CGA; responsible for 38-51% of co-trimoxazole resistance in acute cystitis), including ten (34%) of 29 isolates that were resistant to co-trimoxazole. CGA isolates were obtained from diverse locations across the USA and were related to the O15:K52:H1 clone of the 1986-87 outbreak in London, UK. Thus, CGA is broadly disseminated and contributes to co-trimoxazole resistance in pyelonephritis as well as in cystitis.

Analysis of the F Antigen-Specific papA Alleles of Extraintestinal Pathogenic Escherichia coli Using a Novel Multiplex PCR-Based Assay

ABSTRACT Polymorphisms 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 ofpapA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of thepapA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence thatpapA 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 ofpapA alleles between lineages. Sequencing ofpapA 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.

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.

Phylogenetic and Pathotypic Comparison of Concurrent Urine and Rectal Escherichia coli Isolates from Men with Febrile Urinary Tract Infection

ABSTRACT Among men with febrile urinary tract infection (FUTI), whether the hosts fecal flora is the source for the urine strain (“fecal-urethral” hypothesis), and whether pathogenesis is driven by prevalence versus special pathogenicity, are unknown. Accordingly, pretherapy urine isolates from 65 men with FUTI were compared with concurrent rectal isolates from the same hosts according to serotype, genomic profile, phylogenetic group, and virulence genotype. The hosts multiple rectal colonies included only the urine clone in 25% of subjects, the urine clone plus additional clones in 22%, and only nonurine clones in 54%. Compared with the 67 unique rectal clones, the 65 urine isolates were significantly enriched for phylogenetic group B2, virulence-associated serotypes, and specific virulence genes and contained more virulence genes (median, 10 versus 6: P < 0.001). In multivariable models, phylogenetic group B2, hlyD (hemolysin), cnf1 (cytotoxic necrotizing factor), iroN (siderophore receptor), ompT (outer membrane protease), and malX (pathogenicity island marker) most strongly predicted urine source. These findings challenge the fecal-urethral and prevalence hypotheses for FUTI pathogenesis and instead strongly support the possibility of alternate infection routes in some men and the special pathogenicity hypothesis. They also identify specific bacterial traits as potential targets for anti-FUTI interventions.

Global Molecular Epidemiology of the O15:K52:H1 Extraintestinal Pathogenic Escherichia coli Clonal Group: Evidence of Distribution beyond Europe

ABSTRACT Escherichia coli O15:K52:H1 is a significant extraintestinal pathogen in Europe (G. Prats et al., J. Clin. Microbiol. 38:201-209, 2000). To search for evidence of this clonal group outside of Europe, 75 non-European E. coli isolates of serogroup O15 were compared with five members of the O15:K52:H1 clonal group from Barcelona, Spain, according to genomic background, virulence genotypes, and antimicrobial resistance profiles. Amplification phylotyping showed that 16 (21%) of the 75 non-European O15 isolates corresponded with the O15:K52:H1 clonal group. The 16 non-European O15:K52:H1 clonal group members represented diverse geographic locales. They were isolated almost exclusively from humans with extraintestinal infections and accounted for 50% of all O15 isolates from five human clinical collections studied. Most non-European clonal group members exhibited a consensus virulence factor profile that included the F16 or F7-2 papA alleles (P fimbrial structural subunit), papG allele II (P fimbrial adhesin), iha (putative adhesin siderophore), and iutA (aerobactin receptor). This resembles the virulence profiles of (i) European representatives of the O15:K52:H1 clonal group and (ii) phylogenetically related “clonal group A,” a recently recognized significant contributor to trimethoprim-sulfamethoxazole resistance in the United States (A. R. Manges et al., N. Engl. J. Med. 345:1007-1013, 2001). Antimicrobial resistance profiles were variable, and resistance was inconsistently transferred by conjugation. These findings indicate that the O15:K52:H1 clonal group is broadly distributed beyond Europe, exhibits previously unrecognized phenotypic and genotypic diversity, and contributes significantly to extraintestinal infections in humans.

Host-Pathogen Relationships among Escherichia coli Isolates Recovered from Men with Febrile Urinary Tract Infection

BACKGROUND Host-pathogen relationships in men with febrile urinary tract infection (FUTI) are poorly understood. METHODS Phylogenetic background, extended virulence genotypes, and serotypes were determined for 70 Escherichia coli isolates recovered from urine samples obtained from men with FUTI for comparison with available data for 70 E. coli rectal isolates recovered from uninfected men. Bacterial traits were assessed in relation to underlying host characteristics (age, compromise status, and history of urinary tract infection) and acute manifestations (bacteremia, flank pain, and serum prostate-specific antigen [PSA] level). RESULTS Compared with rectal isolates, FUTI isolates exhibited a significantly higher prevalence of virulence-associated phylogenetic groups, serotypes, and extraintestinal virulence genes. The latter included traditional prostatitis-associated traits (e.g., hemolysin and cytotoxic necrotizing factor), as well as unconventional traits, such as outer membrane protease T. These bacterial traits occurred largely independent of host age, urological compromise status, urinary tract infection history, and acute manifestations. However, certain traits were less prevalent in association with use of urinary tract instrumentation and significantly predicted elevated PSA levels. CONCLUSIONS Considerable virulence capability may be required for an E. coli strain to cause FUTI in men, regardless of whether most compromising conditions are present. Bacterial traits that promote prostatic invasion may be relevant for the pathogenesis of FUTI, even among men without classic manifestations of acute prostatitis.