Large-scale genomic sequencing of extraintestinal pathogenic Escherichia coli strains

Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.With the advent of high-throughput DNA sequencing technologies, it is becoming increasingly tractable to generate whole-genome sequence data from large numbers of clinically relevant bacterial isolates. However, most comparative genome sequencing efforts to date have focused on the biology and molecular epidemiology of organisms involved in disease outbreaks (Chin et al. 2011; Lieberman et al. 2011; Koser et al. 2012; Snitkin et al. 2012; Sanjar et al. 2014). Although illuminating, these studies have shed little light on the agents of bacterial disease that infect an overwhelming majority of patients: commonplace pathogens causing sporadically acquired infections. Outbreaks represent the transmission of a single bacterial clone over a short period of time (Kennedy et al. 2010), providing a necessarily biased sampling that does not encompass the general properties of disease-causing organisms within a larger species. Relatedly, genomic studies of most bacteria are consistent with the distributed genome hypothesis, which proposes that the genetic content of a species is much larger than that of any single strain (Tettelin et al. 2005), necessitating sequencing of large numbers of unrelated clones in order to accurately catalog genetic variation (Rasko et al. 2008).Escherichia coli is among the commonest clinical pathogens and is capable of causing a spectrum of disease both within the intestinal tract (intestinal pathogenic strains) and outside of it (extraintestinal pathogenic E. coli, or ExPEC). The most potentially destructive of these illnesses is bacterial invasion of the bloodstream: E. coli is the most common Gram-negative agent of sepsis, causing ∼30% of all bacteremias and representing the tenth most common cause of death in industrialized nations (Martin et al. 2003; Jaureguy et al. 2008). Far more prevalent are E. coli urinary tract infections, which encompass ∼95% of all community-acquired cases (Lau et al. 2008; Manges et al. 2008). E. coli infections of either type incur significant morbidity and healthcare costs (Sannes et al. 2004; Lau et al. 2008; Ron 2010; Telli et al. 2010); regardless, only a handful of strains causing these diseases have been sequenced, and knowledge of ExPEC E. coli remains incomplete.Here we performed large-scale whole-genome sequencing and analysis of clinical isolates of extraintestinal pathogenic E. coli, obtained from routine diagnostic culture of peripheral blood or urine from patients within a single hospital system. These data enable a robust pan-genome analysis of ExPEC E. coli, high-resolution molecular epidemiological analysis, and genome-wide association studies for identifying antibiotic resistance genes.     

Presence and characterization of extraintestinal pathogenic Escherichia coli virulence genes in F165-positive E. coli strains isolated from diseased calves and pigs

The virulence genotype profile and presence of a pathogenicity island(s) (PAI) were studied in 18 strains of F165-positive Escherichia coli originally isolated from diseased calves or piglets. On the basis of their adhesion phenotypes and genotypes, these extraintestinal pathogenic strains were classified into three groups. The F165 fimbrial complex consists of at least two serologically and genetically distinct fimbriae: F165(1) and F165(2). F165(1) is encoded by the foo operon (pap-like), and F165(2) is encoded by fot (sfa related). Strains in group 1 were foo and fot positive, strains in group 2 were foo and afa positive, and strains in group 3 were foo positive only. The strains were tested for the presence of virulence genes found mainly in extraintestinal pathogenic E. coli (ExPEC) strains. Although all the strains were positive for the papA variant encoding F11 fimbriae incD, traT, and papC, the prevalence of virulence genes commonly found in PAIs associated with ExPEC strains was highly variable, with strains of group 2 harboring most of the virulence genes tested. papG allele III was detected in all strains in group 1 and in one strain in group 3. All other strains were negative for the known alleles encoding PapG adhesins. The association of virulence genes with tRNA genes was characterized in these strains by using pulsed-field gel electrophoresis and DNA hybridization. The insertion site of the foo operon was found at the pheU tRNA locus in 16 of the 18 strains and at the selC tRNA locus in the other 2 strains. Furthermore, 8 of the 18 strains harbored a high-pathogenicity island which was inserted in either the asnT or the asnV/U tRNA locus. These results suggest the presence of one or more PAIs in septicemic strains from animals and the association of the foo operon with at least one of these islands. F165-positive strains share certain virulence traits with ExPEC, and most of them are pathogenic in piglets, as tested in experimental infections.     

Cytotoxic necrotizing factor production by hemolytic strains of Escherichia coli causing extraintestinal infections

Two hundred and nineteen strains of Escherichia coli from extraintestinal infections and feces of healthy subjects were examined for hemolysin (Hly) and cytotoxic necrotizing factor (CNF) production and for mannose-resistant hemagglutination. Of 105 strains from extraintestinal infections, 42 (40.0%) were positive for production of both Hly and CNF, and 21 (20.0%) were positive for Hly alone; on the contrary, only 1 Hly- and CNF-positive strain and 2 Hly-positive strains were found among 114 strains from normal stools. CNF production was not found to occur among the nonhemolytic strains, confirming the close association existing between these toxic factors. Hemolytic strains positive for CNF showed mannose-resistant hemagglutination less frequently than did Hly-positive, CNF-negative strains (25.6 versus 82.6%), suggesting the existence of two distinct classes among hemolytic strains of E. coli.     

Pandemic lineages of extraintestinal pathogenic Escherichia coli

Pathogenic Escherichia coli strains cause a wide variety of intestinal and extraintestinal infections. The widespread geographical clonal dissemination of intestinal pathogenic E. coli strains, such as E. coli O157:H7, is well recognized, and its spread is most often attributed to contaminated food products. On the other hand, the clonal dissemination of extraintestinal pathogenic E. coli (ExPEC) strains is also recognized, but the mechanism of their spread is not well explained. Here, I describe major pandemic clonal lineages of ExPEC based on multilocus sequence typing (MLST), and discuss possible reasons for their global dissemination. These lineages include sequence type (ST) 131, ST393, ST69, ST95, and ST73, which are all associated with both community-onset and healthcare-associated infections, in particular urinary tract infections and bloodstream infections. As with many other types of drug-resistant Gram-negative and Gram-positive bacterial infections, drug-resistant ExPEC infections are recognized to be caused by a limited set of clonal lineages. However, reported observations on these major pandemic lineages suggest that the resistance phenotype is not necessarily the determinant of their clonal dissemination. Both epidemiological factors and their intrinsic biological ‘fitness’ are likely to contribute. An important public health and clinical concern is that pandemicity itself may be a determinant of progressive drug resistance acquisition by clonal lineages. New research is urgently needed to better understand the epidemiological and biological causes of ExPEC pandemicity.     

Prevalence of pathogenicity island IICFT073 genes among extraintestinal clinical isolates of Escherichia coli

Uropathogenic Escherichia coli is the most common cause of urinary tract infection (UTI). Cystitis in women is by far the most common UTI; pyelonephritis in both sexes and prostatitis in men are more severe but are less frequent complaints. The ability of E. coli to cause UTI is associated with specific virulence determinants, some of which are encoded on pathogenicity islands (PAI). One such PAI (PAI IICFT073), of the prototypical uropathogenic E. coli strain CFT073, contains 116 open reading frames, including iron-regulated genes, carbohydrate biosynthetic genes, the serine protease autotransporter picU, a two-partner secretion system, a type I secretion system, mobility genes, and a large number of hypothetical genes. To determine the association of PAI IICFT073 with UTI, PCR was used to examine the prevalence of the five virulence-associated loci among the ECOR collection and a collection of E. coli isolated from patients with cystitis, pyelonephritis, prostatitis, or septicemia. All PAI IICFT073 loci were found to be more prevalent among the B2 phylogenetic group than any other group within the ECOR collection and among invasive prostatitis strains than were cystitis or pyelonephritis strains. These data support the theory that clinical isolates causing prostatitis are more virulent than those producing cystitis or pyelonephritis in women.     

DegS is necessary for virulence and is among extraintestinal Escherichia coli genes induced in murine peritonitis

Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37 degrees C but is impaired at 43 degrees C or in 3% ethanol LB broth at 37 degrees C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for sigma(E) activity, our findings implicate sigma(E) and its regulon in E. coli extraintestinal pathogenesis.     

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.     

Use of deoxyribose by intestinal and extraintestinal pathogenic Escherichia coli strains: a metabolic adaptation involved in competitiveness

We showed that the deoK operon, which confers the ability to use deoxyribose as a carbon source, is more common among pathogenic than commensal Escherichia coli strains. The expression of the deoK operon increases the competitiveness of clinical isolates, suggesting that this biochemical characteristic plays a role in host infectivity.     

Cytolethal distending toxin type I and type IV genes are framed with lambdoid prophage genes in extraintestinal pathogenic Escherichia coli

Five types of cytolethal distending toxin (CDT-I to CDT-V) have been identified in Escherichia coli. In the present study we cloned and sequenced the cdt-IV operon and flanking region from a porcine extraintestinal pathogenic E. coli (ExPEC) strain belonging to serogroup O75. We confirmed that similar to other CDTs, CDT-IV induced phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks, and blocked the HeLa cell cycle at the G(2)-M transition. The cdt-IV genes were framed by lambdoid prophage genes. We cloned and sequenced the cdt-I operon and flanking regions from a human ExPEC O18:K1:H7 strain and observed that cdt-I genes were also flanked by lambdoid prophage genes. PCR studies indicated that a gene coding for a putative protease was always associated with the cdtC-IV gene but was not associated with cdtC genes in strains producing CDT-I, CDT-III, and CDT-V. Our results suggest that the cdt-I and cdt-IV genes might have been acquired from a common ancestor by phage transduction and evolved in their bacterial hosts. The lysogenic bacteriophages have the potential to carry nonessential "cargo" genes or "morons" and therefore play a crucial role in the generation of genetic diversity within ExPEC.     

Virulence genes in verocytotoxigenic Escherichia coli strains isolated from humans and cattle

Verocytotoxigenic Escherichia coli (VTEC) causing diarrhoea, haemorrhagic colitis and haemolytic-uremic syndrome usually have additional traits such as the adhesin intimin and a large plasmid that seems to increase virulence. There are, however, isolates of VTEC causing serious symptoms that do not harbour these traits. In the present study we have used PCR with primers detecting adhesin genes other than eaeA, namely fimA, papC, sfaD/sfaE and daaE. We have also used PCR to detect the genes hlyA and iutA that besides the plasmid-borne gene E-hly possibly support the bacterial access to iron. The aim of the study was to identify and compare the presence of virulence genes in VTEC isolates of human and cattle origin. The main finding was that the absence of E-hly might be compensated for by the gene iutA coding for aerobactin or hlyA coding for alpha-haemolysin as 94% of the human VTEC isolates had at least one of these genes. Interestingly, only 45% of VTEC isolated from cattle had any of these genes. We propose that this might be the reason for the relatively low incidence of symptomatic VTEC infections among humans in relation to the high number of VTEC among cattle.     

Genetic diversity of intimin genes of attaching and effacing Escherichia coli strains

In this study, we determined the sequences of four intimin variant genes detected in attaching and effacing Escherichia coli isolates of human origin. Three of them were novel and were designated eae-eta (eta), eae-iota (iota), and eae-kappa (kappa). The fourth was identical to the recently described eae-zeta (zeta), isolated from a bovine E. coli O84:NM isolate. We compared these sequences with those of published intimin-alpha, intimin-beta, intimin-gamma1, intimin-gamma2, intimin- epsilon, and intimin-theta alleles. Sequence analysis of these 10 intimin alleles confirmed extensive genetic diversity within the intimin gene family in E. coli. The genetic diversity was more prominent in the 3' region (starting at bp 2,112), which encodes the binding domain of intimin. Phylogenetic analyses revealed four groups of closely related intimin genes: alpha and zeta; beta and kappa; gamma1 and gamma2/theta; and epsilon and eta. Calculation of homoplasy ratios of sequences of the 5' region of eae (positions 1 to 2,111) revealed evidence for intragenic recombination. Split decomposition analysis also indicates that recombination events have played a role in the evolutionary history of eae. In conclusion, we recommend an eae nomenclature system based on the Greek alphabet and provide an updated PCR scheme for amplification and typing of E. coli eae.     

Pathogenicity of an Enterotoxigenic Escherichia coli Hemolysin (hlyA) Mutant in Gnotobiotic Piglets

Pigs infected with hemolytic F4⁺ strains of enterotoxigenic Escherichia coli often develop septicemia secondary to intestinal infection. We tested the hypothesis that inactivation of hemolysin would reduce the ability of F4⁺ enterotoxigenic E. coli to cause septicemia in swine following oral inoculation. Inactivation of the hemolysin structural gene (hlyA) did not decrease the incidence of septicemia in the gnotobiotic piglet model.     

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.     

Molecular epidemiology of extraintestinal pathogenic (uropathogenic) Escherichia coli

Molecular epidemiological analyses of extraintestinal pathogenic Escherichia coli (ExPEC), which are also called "uropathogenic E. coli" since they are the principle pathogens in urinary tract infection, involve structured observations of E. coli as they occur in the wild. Careful selection of subjects and use of appropriate methods for genotyping and statistical analysis are required for optimal results. Molecular epidemiological studies have helped to clarify the host-pathogen relationships, phylogenetic background, reservoirs, and transmission pathways of ExPEC, to assess potential vaccine candidates, and to delineate areas for further study. Ongoing discovery of new putative virulence factors (VFs), increasing awareness of the importance of VF expression and molecular variants of VFs, and growing appreciation of transmission as an important contributor to ExPEC infections provide abundant stimulus for future molecular epidemiological studies. Published by Elsevier GmbH.     

Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli.

Virulent and nonvirulent isolates of avian Escherichia coli were tested for the presence of aerobactin genes by colony hybridization with a specific gene probe constructed from plasmid pABN1 (A. Bindereif and J. B. Neilands, J. Bacteriol. 153:1111-1113, 1983). Positive hybridization with the gene probe was highly correlated with virulence, as measured by the 50% lethal dose of the strains for chicks. Evidence for the expression of aerobactin genes in the virulent strains was obtained by demonstrating their susceptibility to cloacin DF13, which binds to the same receptor that binds aerobactin, and their ability to produce aerobactin, as revealed by cross-feeding the E. coli mutant WO987 (aroB fepA iuc iut+), which is unable to synthesize but capable of taking up aerobactin. We suggest that the production of aerobactin is involved in the virulence of avian septicemic E. coli.     

Virulence factors and drug resistance in Escherichia coli isolated from extraintestinal infections

PURPOSE: To determine the virulence factors produced by Escherichia coli isolated from extraintestinal infections, to study the drug resistance pattern in E. coli with special reference to extended spectrum beta -lactamase (ESBL) and to evaluate screening methods for ESBL. METHODS: A total of 152 isolates of E. coli from various extraintestinal infections were screened for virulence factors such as haemolysin, surface hydrophobicity, serum resistance and protease. All the isolates were also studied for antibiotic susceptibility pattern using modified Kirby Bauer disk diffusion method. ESBL production was screened by standard disk diffusion method and confirmed using phenotypic confirmatory method. RESULTS: Among 152 isolates, 36 (23.7%) were haemolytic, 42 (27.6%) were hydrophobic, 132 (86.8%) were serum resistant and only four were positive for protease. Multiple virulence factor were observed in 67 (44%) of isolates. Seventy-nine (51.4%) isolates produced ESBL. ESBL producing isolates showed multidrug resistance. There was a significant association ( P E. coli . CONCLUSIONS: The present study shows the expression of virulence factors and multidrug resistance in E. coli isolated from various extraintestinal infections. The study also shows that appropriate methods of detecting drug resistance and ESBL production are required for the judicious use of antibiotics in managing these infections.     

Antimicrobial resistances of extraintestinal pathogenic Escherichia coli isolates from swine in China

Antibiograms and relevant genotypes of porcine extraintestinal pathogenic Escherichia coli (ExPEC) isolates (n = 315) recovered between 2004 and 2007 in China were assessed. Among the 14 antimicrobials tested, the most prevalent resistance was to ampicillin, trimethoprim, sulfadimidine, tetracycline, neomycin, streptomycin, kanamycin, ciprofloxacin and ofloxacin (ranging from 81.9 to 100%). Forty-six multiresistant patterns were found. For each antimicrobial agent, ampicillin resistance was primarily mediated by bla_TEM, streptomycin resistance by strA and strB, kanamycin/neomycin resistance by aphA1, gentamicin resistance by aac(3)-IV, quinolones resistance by mutations in gyrA, tetracycline resistance by tet(A), tet(B) and tet(G), trimethoprim resistance by dfrA7, dfrA12 and dfrA13, and sulfadimidine resistance by sul1 and sul2. Both bla_TEM-1 and bla_CTX-M-14 were found in two ESBLs-producing isolates. Strains that harbored several genes that conferred resistance to the same antimicrobial agent were often significantly more multiresistant than others. Class 1 integrons were identified in 86 (27.3%) ExPEC isolates, which harbored dfrA14, aadA2, aadA22, dfrA17, aadA5, dfrA17-aadA2, dfrA1-aadA1, dfrA12-aadA2, dfrA17-aadA5 gene cassettes in five major different variable regions, conferring resistance to trimethoprim and aminoglycosides. These results provide novel insights into the epidemiological characteristics of porcine ExPEC strains in China, and suggest the need for the prudent use of antimicrobial agents in food animals.     

Multiplex PCR for detection of three plasmid-borne genes of enteroaggregative Escherichia coli strains

We developed a novel multiplex PCR assay for enteroaggregative Escherichia coli (EAEC) detection, by using three plasmid-borne genes (the aggregative adherence [AA] probe, aap, and aggR). One or more of the loci were detected in 24 (86%) of 28 patient isolates analyzed. The multiplex PCR assay is a fast, convenient, and sensitive molecular test to detect EAEC.