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.     

Molecular characterization of Escherichia coli strains isolated from pigs with edema disease.

The present study was conducted to investigate the epidemiological relationship of isolates of Escherichia coli causing edema disease. Classical edema disease has not previously been described in Denmark, but between February 1994 and November 1995 cases appeared in 51 pig herds, among which direct or indirect trading contacts were confirmed for 36 of the herds. A total of 213 isolates from pigs with edema disease in Denmark and other countries and 23 E. coli O139 isolates from pigs with diarrhea or healthy pigs were analyzed to characterize their O serogroups, HindIII ribotypes, and pulsed-field gel electrophoresis (PFGE) types, and 183 of the isolates were also analyzed for their plasmid profiles. The resulting PFGE types of the isolates from pigs with edema disease were examined by cluster analysis. Ten isolates from three herds could not be typed with the available O antisera, whereas all other isolates were of serotype O139. However, all isolates from pigs with edema disease belonged to the same HindIII ribotype, which was not observed among the isolates from pigs with diarrhea or healthy pigs. All isolates from Danish pigs with edema disease except for three isolates originating from two herds belonged to the same or closely related XbaI PFGE types; the other three isolates were assigned to possibly related types. Isolates from pigs with edema disease in different countries belonged to different PFGE types. All isolates from Danish pigs with edema disease grouped together in one cluster, in contrast to isolates from other countries, which did not form any clusters. E. coli strains of serogroup O139 from pigs with diarrhea or isolated from the feces of healthy Danish pigs were very different. Plasmid profiles differed largely among isolates. However, among the isolates from Danish pigs with edema disease, one type predominated within herds. The present study indicated that most, if not all, of the observed cases of edema disease in Denmark were part of the same outbreak. The combination of PFGE typing and ribotyping was useful for studying the possible clonal relationship among strains, whereas plasmid profiling was less informative.     

Occurrence of hlyA and sheA genes in extraintestinal Escherichia coli strains

The association of a hemolytic phenotype with the carriage of the alpha-hemolysin gene (hlyA) and/or the silent hemolysin gene (sheA or clyA) among 540 extraintestinal clinical isolates of Escherichia coli and 110 fecal isolates from healthy individuals was investigated. Though HlyA is an important virulence factor in extraintestinal E. coli infection, the role of SheA is not completely clarified. Two hemolytic sheA+ E. coli strains that lacked hlyA and possessed no other hemolysin genes were identified. No hlyA+ sheA+ strains were identified, suggesting that there is possible incompatibility between hlyA and sheA in the chromosome of E. coli.     

New fimbrial antigen F165 from Escherichia coli serogroup O115 strains isolated from piglets with diarrhea

Sixteen strains of Escherichia coli serogroup O115 isolated from piglets with diarrhea were examined for mannose-sensitive or mannose-resistant hemagglutination (MSHA or MRHA, respectively) for the presence of fimbriae by electron microscopy and for enterotoxigenicity by the ligated gut loop technique in 10-day-old piglets. Four strains demonstrated MRHA of sheep, goat, pig, dog, cat, chicken, and human erythrocytes but no MRHA of calf, horse, guinea pig, and rabbit erythrocytes. They were divided into pattern I (MSHA negative) and pattern II (MSHA positive). The remaining 12 strains were classified as pattern III (MRHA negative, MSHA positive) and pattern IV (hemagglutination negative). An antiserum produced against the MRHA-positive, MSHA-negative strain 4787 and absorbed by the same strain grown at 15 degrees C agglutinated all of the MRHA-positive strains but none of the MRHA-negative strains and completely inhibited the MRHA of these strains. The surface antigen against which this absorbed antiserum was directed was designated "F165." Fimbriae (pili) purified from strain 4787 hemagglutinated erythrocytes in the same mannose-resistant pattern as the strain itself and reacted with the anti-F165 antiserum in an enzyme-linked immunosorbent assay, thus demonstrating the fimbrial nature of the hemagglutinating F165 antigen. The F165 antigen showed no serological relationship with the fimbrial antigens F4, F5, F6, and "F41". A positive correlation between the presence of F165 and the lack of enterotoxigenicity was demonstrated. Thus, we found a new mannose-resistant, hemagglutinating fimbrial antigen, F165, which is produced only by nonenterotoxigenic strains of E. coli serogroup O115. The possible role of F165 as a virulence attribute of E. coli strains causing extraintestinal disease is discussed.     

Virulence genes in Escherichia coli isolated from calves with diarrhoea in Iran

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia coli strains from diarrhoeic calves in Iran. A total of 156 E. coli isolates obtained from 180 diarrhoeic calves were evaluated with two multiplex PCR protocols for the detection of the K99, F41, Sta, Stx1, Stx2 and eaeA genes. Of these 156 isolates, 32 (20.5%) carried at least one virulent gene, 22 (14.1%) possessed enterotoxigenic E. coli (ETEC) virulent genes and 10 (6.4%) possessed shiga toxin genes, virulence factors of shiga toxin-producing E. coli (STEC). None of the isolates carried eaeA gene. All ETEC but one were isolated from 1- to 4-day-old calves (95.4%), and one was isolated from a 6-day-old calf. All STEC were isolated from 7- to 28-day-old calves. All ETEC carried both K99 and F41 fimbriae and possessed Sta enterotoxin gene.     

Occurrence of K99 antigen on Escherichia coli isolated from pigs and colonization of pig ileum by K99+ enterotoxigenic E. coli from calves and pigs.

Several strains of enterotoxigenic Escherichia coli (ETEC) isolated from pigs were found to have an antigen (K99) previously reported only on strains of calf and lamb origin and which facilitates intestinal colonization in the latter two species. Several human ETEC were also tested for K99; however, none were positive. Each of four K99-positive ETEC strains of calf origin and one of pig origin produced K99 in pig ileum in vivo, adhered to villous epithelium in pig ileum, colonized pig ileum, and caused profuse diarrhea in newborn pigs. In contrast to the K99-positive strains above, four K99-negative ETEC from humans and chickens and one K99-positive ETEC from a calf either did not colonize pig ileum or did so inconsistently. When the K99-negative strains did colonize, they had little or no tendency to adhere to intestinal villi. These results are consistent with the hypothesis that K99 facilitates adhesion to and colonization of pig ileum by some ETEC.     

Distribution of serotypes and virulence-associated genes in pathogenic Escherichia coli isolated from ducks

The objective of the present study was to investigate the serotypes and virulence-associated genes of avian pathogenic Escherichia coli (APEC) isolated from duck colibacillosis cases. Two hundred and fifty-four APEC isolates from duck colibacillosis cases were serotyped and amplified for 12 known virulence-associated genes and the betA gene (encoding choline dehydrogenase) by polymerase chain reaction assays. One hundred and forty-three E. coli isolates from cloacal swabs of healthy ducks were also amplified for the same genes. A total of 53 O-serogroups were found in 254 APEC isolates, among which O93, O78 and O92 were predominant serogroups. Polymerase chain reaction results showed that Shiga-toxin-producing E. coli distributed in only 2.4% of ducks compared with 49.2% of the APEC isolates harbouring the irp2 gene, and 44.9% the fyuA gene, respectively. The ibeA gene was only present in 27 APEC isolates and was not found in healthy ducks. The rfaH gene was detected in 20.5% of APEC isolates, whereas 5.6% was found in healthy ducks. A total 79.5% of APEC isolates harboured the betA gene, which was significantly higher than in healthy ducks (16.1%), suggesting that betA may be associated with virulence.     

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.     

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.     

Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.     

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.     

Heat-stable-enterotoxin-producing Escherichia coli strains isolated from dogs.

Five strains of hemolytic Escherichia coli isolated from dogs suffering from diarrhea were shown by radioactive and enzyme-labeled oligonucleotide probes to possess genes coding for heat-stable enterotoxin (STIa). Four of the strains were shown by immunoassay (enzyme-linked immunosorbent assay) and bioassay (infant mouse test) to produce STI in vitro. All five strains, however, were able to induce fluid accumulation in ligated dog intestinal loops. The four STI-producing strains all possessed the K99 fimbrial antigen (F5) and belonged to serotype O42:H37. In these strains, genes encoding STI were located on a 98-megadalton plasmid. In the fifth strain, which produced STI in vitro only after several subcultivations, the STI gene was located on an 80-megadalton plasmid. This strain was nontypable.     

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.     

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.     

The two-faced role of cad genes in the virulence of pathogenic Escherichia coli

In enterobacteria, acid stress induces expression of the cad system which is involved in maintaining intracellular pH at levels compatible with cell survival. Despite its crucial role, the cad operon is silenced in Shigella and in other pathogenic Escherichia coli. In the present review, we will address the question of why and how the cad locus has been sacrificed for the sake of optimal expression of virulence traits.     

Genetic characterization and virulence of enterotoxigenic Escherichia coli mutants which have lost virulence genes in vivo.

Loss of K99 and STaP genes from enterotoxigenic Escherichia coli 431 during infection occurred by either plasmid curing or plasmid deletion. These mutants expressed the F41 adhesion and colonized neonatal pigs, but only those mutants that retained STaP caused diarrhea with significant weight loss.     

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.     

Increased mcr-1 in pathogenic Escherichia coli from diseased swine,Taiwan

PurposeTo investigate the increasing prevalence of colistin resistance in animal Escherichia coli isolates and their mcr-1-carrying plasmids, especially those shared by isolates from human and retail meats.MethodsE. coli from diseased swine and poultry recovered between 2012 and 2016 were studied. Susceptibility was determined using broth microdilution method or Vitek II system. Fifty-eight mcr-1-positive isolates were randomly selected for further testing, including pulsed-field gel electrophoresis (PFGE) for clonality determination, S1- or I-CeuI-PFGE and Southern blotting for localization of mcr-1, and conjugation for transmissibility. Whole genome sequencing (WGS) was performed for the genetic structure of plasmids.ResultsAmong the 1234 E. coli isolates from diseased swine, colistin resistance increased from 14.6% (14/96) in 2012 to 43.8% (63/144) in 2016 with a paralleled increase in mcr-1-positivity from 12.5% (12/96) to 33.3% (48/144) in 2016 (P < 0.001), but no such significant increase was observed in the 489 diseased poultry isolates. The 58 clonally diverse isolates were resistant to multiple antibiotics commonly used in humans. PFGE and Southern blotting revealed one chromosome-located mcr-1 and various mcr-1-borne plasmids, all of which were transferable to E. coli J53. WGS revealed that the prevalent 60-kb and 30-kb plasmid was the same as pHNSHP45 in China and pESTMCR in Estonia, respectively, which were both present in human isolates in Taiwan.ConclusionIncreased colistin resistance and mcr-1-positivity in diseased swine isolates and detection of mcr-1 carried on similar plasmids in isolates from animals and humans stress the need to monitor resistance in both sectors.     

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.