Hemolytic porcine intestinal Escherichia coli without virulence-associated genes typical of intestinal pathogenic E. coli

Testing 1,666 fecal or intestinal samples from healthy and diarrheic pigs, we obtained hemolytic Escherichia coli isolates from 593 samples. Focusing on hemolytic E. coli isolates without virulence-associated genes (VAGs) typical for enteropathogens, we found that such isolates carried a broad variety of VAGs typical for extraintestinal pathogenic E. coli.     

Virulence factor gene profiles of Escherichia coli isolates from clinically healthy pigs

Nonpathogenic, intestinal Escherichia coli (commensal E. coli) supports the physiological intestinal balance of the host, whereas pathogenic E. coli with typical virulence factor gene profiles can cause severe outbreaks of diarrhea. In many reports, E. coli isolates from diarrheic animals were classified as putative pathogens. Here we describe a broad variety of virulence gene-positive E. coli isolates from swine with no clinical signs of intestinal disease. The isolation of E. coli from 34 pigs from the same population and the testing of 331 isolates for genes encoding heat-stable enterotoxins I and II, heat-labile enterotoxin I, Shiga toxin 2e, and F4, F5, F6, F18, and F41 fimbriae revealed that 68.6% of the isolates were positive for at least one virulence gene, with a total of 24 different virulence factor gene profiles, implying high rates of horizontal gene transfer in this E. coli population. Additionally, we traced the occurrence of hemolytic E. coli over a period of 1 year in this same pig population. Hemolytic isolates were differentiated into seven clones; only three were found to harbor virulence genes. Hemolytic E. coli isolates without virulence genes or with only the fedA gene were found to be nontypeable by slide agglutination tests with OK antisera intended for screening live cultures against common pathogenic E. coli serogroups. The results appear to indicate that virulence gene-carrying E. coli strains are a normal part of intestinal bacterial populations and that high numbers of E. coli cells harboring virulence genes and/or with hemolytic activity do not necessarily correlate with disease.     

Comparison of extraintestinal pathogenic Escherichia coli strains from human and avian sources reveals a mixed subset representing potential zoonotic pathogens

Since extraintestinal pathogenic Escherichia coli (ExPEC) strains from human and avian hosts encounter similar challenges in establishing infection in extraintestinal locations, they may share similar contents of virulence genes and capacities to cause disease. In the present study, 1,074 ExPEC isolates were classified by phylogenetic group and possession of 67 other traits, including virulence-associated genes and plasmid replicon types. These ExPEC isolates included 452 avian pathogenic E. coli strains from avian colibacillosis, 91 neonatal meningitis E. coli (NMEC) strains causing human neonatal meningitis, and 531 uropathogenic E. coli strains from human urinary tract infections. Cluster analysis of the data revealed that most members of each subpathotype represent a genetically distinct group and have distinguishing characteristics. However, a genotyping cluster containing 108 ExPEC isolates was identified, heavily mixed with regard to subpathotype, in which there was substantial trait overlap. Many of the isolates within this cluster belonged to the O1, O2, or O18 serogroup. Also, 58% belonged to the ST95 multilocus sequence typing group, and over 90% of them were assigned to the B2 phylogenetic group typical of human ExPEC strains. This cluster contained strains with a high number of both chromosome- and plasmid-associated ExPEC genes. Further characterization of this ExPEC subset with zoonotic potential urges future studies exploring the potential for the transmission of certain ExPEC strains between humans and animals. Also, the widespread occurrence of plasmids among NMEC strains and members of the mixed cluster suggests that plasmid-mediated virulence in these pathotypes warrants further attention.     

Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic E. coli

Extraintestinal pathogenic Escherichia coli (ExPEC) are an important cause of urinary tract infections, neonatal meningitis and septicaemia in humans. Animals are recognized as a reservoir for human intestinal pathogenic E. coli, but whether animals are a source for human ExPEC is still a matter of debate. Pathologies caused by ExPEC are reported for many farm animals, especially for poultry, in which colibacillosis is responsible for huge losses within broiler chickens. Cases are also reported for companion animals. Commensal E. coli strains potentially carrying virulence factors involved in the development of human pathologies also colonize the intestinal tract of animals. This review focuses on the recent evidence of the zoonotic potential of ExPEC from animal origin and their potential direct or indirect transmission from animals to humans. As antimicrobials are commonly used for livestock production, infections due to antimicrobial-resistant ExPEC transferred from animals to humans could be even more difficult to treat. These findings, combined with the economic impact of ExPEC in the animal production industry, demonstrate the need for adapted measures to limit the prevalence of ExPEC in animal reservoirs while reducing the use of antimicrobials as much as possible.     

HlyA knock out yields a safer Escherichia coli A0 34/86 variant with unaffected colonization capacity in piglets

Escherichia coli A0 34/86 (O83:K24:H31) has been successfully used for prophylactic and therapeutic intestinal colonization of premature and newborn infants, with the aim of preventing nosocomial infections. Although E. coli A0 34/86 was described as a nonpathogenic commensal, partial sequencing revealed that its genome harbours gene clusters highly homologous to virulence determinants of different types of E. coli, including closely linked genes of the alpha-haemolysin operon (hlyCABD) and for the cytotoxic necrotizing factor (cnf1). A haemolysin-deficient mutant (Delta hlyA) of E. coli A0 34/86 was generated and its colonization capacity was determined. The results show that a single dose of the A0 34/86 wild-type or Delta hlyA strains resulted in efficient intestinal colonization of newborn conventional piglets, and that this was still considerable after several weeks. No difference was observed between the wild-type and the mutant strains, showing that haemolysin expression does not contribute to intestinal colonization capacity of E. coli A0 34/86. Safety experiments revealed that survival of colostrum-deprived gnotobiotic newborn piglets was substantially higher upon colonization by the nonhaemolytic strain than following inoculation by its wild-type ancestor. We suggest that the E. coli A0 34/86 Delta hlyA mutant may represent a safer prophylactic and/or immunomodulatory tool with unaffected colonization capacity.     

Adherent-invasive Escherichia coli phenotype displayed by intestinal pathogenic E. coli strains from cats, dogs, and swine

The adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohn's disease, shows similar traits to human and animal extraintestinal pathogenic E. coli (ExPEC) with respect to their phylogenetic origin and virulence gene profiles. Here, we demonstrate that animal ExPEC strains generally do not share the AIEC phenotype. In contrast, this phenotype is very frequent among animal intestinal pathogenic E. coli (InPEC) strains, particularly of feline and canine origin, that genetically resemble ExPEC. These results strengthen the particular identity and disease specificity of the AIEC pathotype and the putative role animals might play in the transmission of AIEC-like strains to humans.     

Escherichia coli Nissle 1917 for probiotic use in piglets: evidence for intestinal colonization

Aims:  This study was prompted to investigate the intestinal localization and colonization of orally administered Escherichia coli Nissle 1917 (EcN) in piglets.
Methods and Results:  EcN was fed to ten EcN-negative piglets (3 months) over seven consecutive days. Faecal samples were collected repeatedly and tested for EcN-DNA by a combined culture/PCR assay and for viable EcN by culture methods, respectively. EcN-DNA was detectable in faeces of all piglets within the first 24 h after it was added to the feed. After the administration of EcN had been stopped, the presence of EcN-DNA in faecal samples indicated that all piglets shedded EcN with their faeces intermittently through up to 33 days. In addition, E. coli strains indistinguishable from EcN by all markers tested (rdar colony morphotype, multiplex PCR and GEI II-PCR analyses, Xba I-pattern, K5 phage susceptibility) were isolated from faecal samples and from mucosal swabs taken at euthanasia at the end of the experiment.
Conclusions:  EcN colonizes the intestine and persists in conventionally reared piglets for at least 4 weeks upon oral administration.
Significance and Impact of the Study:  Results of this study have implications for efficacy and safety assessments of EcN as a probiotic strain for use in pigs.     

Adhesion of Human and Animal Escherichia coli Strains in Association with Their Virulence-Associated Genes and Phylogenetic Origins

Intestinal colonization is influenced by the ability of the bacterium to inhabit a niche, which is based on the expression of colonization factors. Escherichia coli carries a broad range of virulence-associated genes (VAGs) which contribute to intestinal (inVAGs) and extraintestinal (exVAGs) infection. Moreover, initial evidence indicates that inVAGs and exVAGs support intestinal colonization. We developed new screening tools to genotypically and phenotypically characterize E. coli isolates originating in humans, domestic pigs, and 17 wild mammal and avian species. We analyzed 317 isolates for the occurrence of 44 VAGs using a novel multiplex PCR microbead assay (MPMA) and for adhesion to four epithelial cell lines using a new adhesion assay. We correlated data for the definition of new adhesion genes. inVAGs were identified only sporadically, particularly in roe deer (Capreolus capreolus) and the European hedgehog ( Erinaceus europaeus). The prevalence of exVAGs depended on isolation from a specific host. Human uropathogenic E. coli isolates carried exVAGs with the highest prevalence, followed by badger (Meles meles) and roe deer isolates. Adhesion was found to be very diverse. Adhesion was specific to cells, host, and tissue, though it was also unspecific. Occurrence of the following VAGs was associated with a higher rate of adhesion to one or more cell lines: afa-dra, daaD, tsh, vat, ibeA, fyuA, mat, sfa-foc, malX, pic, irp2, and papC. In summary, we established new screening methods which enabled us to characterize large numbers of E. coli isolates. We defined reservoirs for potential pathogenic E. coli. We also identified a very broad range of colonization strategies and defined potential new adhesion genes.     

Occurrence of virulence and antimicrobial resistance genes in Escherichia coli isolates from different aquatic ecosystems within the St. Clair River and Detroit River areas

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.     

Transcriptional regulation through RfaH contributes to intestinal colonization by Escherichia coli

The Escherichia coli regulatory protein RfaH contributes to efficient colonization of the mouse gut. Extraintestinal pathogenic (ExPEC) as well as non-pathogenic probiotic E. coli strains rapidly outcompeted their isogenic rfaH mutants following oral mixed infections. LPS-core and O-antigen side-chain as well as capsular polysaccharide synthesis are among the E. coli virulence factors affected by RfaH. In respect of colonization, deep-rough LPS mutants (waaG) but not capsular (kps) mutants were shown to behave similarly to rfaH mutants. Furthermore, alteration in the length of O-antigen side-chains did not modify colonization ability either indicating that it was the regulatory effect of RfaH on LPS-core synthesis, which affected intestinal colonization. Loss of RfaH did not significantly influence adhesion of bacteria to cultured colon epithelial cells. Increased susceptibility of rfaH mutants to bile salts, on the other hand, suggested that impaired in vivo survival could be responsible for the reduced colonization capacity.     

Effect of fructooligosaccharide metabolism on chicken colonization by an extra-intestinal pathogenic Escherichia coli strain

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains cause many diseases in humans and animals. While remaining asymptomatic, they can colonize the intestine for subsequent extra-intestinal infection and dissemination in the environment. We have previously identified the fos locus, a gene cluster within a pathogenicity island of the avian ExPEC strain BEN2908, involved in the metabolism of short-chain fructooligosaccharides (scFOS). It is assumed that these sugars are metabolized by the probiotic bacteria of the microbiota present in the intestine, leading to a decrease in the pathogenic bacterial population. However, we have previously shown that scFOS metabolism helps BEN2908 to colonize the intestine, its reservoir. As the fos locus is located on a pathogenicity island, one aim of this study was to investigate a possible role of this locus in the virulence of the strain for chicken. We thus analysed fos gene expression in extracts of target organs of avian colibacillosis and performed a virulence assay in chickens. Moreover, in order to understand the involvement of the fos locus in intestinal colonization, we monitored the expression of fos genes and their implication in the growth ability of the strain in intestinal extracts of chicken. We also performed intestinal colonization assays in axenic and Specific Pathogen-Free (SPF) chickens. We demonstrated that the fos locus is not involved in the virulence of BEN2908 for chickens and is strongly involved in axenic chicken cecal colonization both in vitro and in vivo. However, even if the presence of a microbiota does not inhibit the growth advantage of BEN2908 in ceca in vitro, overall, growth of the strain is not favoured in the ceca of SPF chickens. These findings indicate that scFOS metabolism by an ExPEC strain can contribute to its fitness in ceca but this benefit is fully dependent on the bacteria present in the microbiota.     

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.     

Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany

AIMS: To verify the presence of Escherichia coli Nissle 1917 as a natural isolate in swine and to characterize in vitro probiotic properties as well as in vivo persistence in a feeding experiment. METHODS AND RESULTS: During studies on the intestinal microflora of pigs, we isolated E. coli Nissle 1917 sporadically from a pig population over a period of 1 year. The identity of the isolates as E. coli Nissle 1917 was verified by serotyping, Nissle-specific PCR, macrorestriction analysis (pulsed field gel electrophoresis) and the determination of in vitro probiotic properties in invasion and adhesion assays using a porcine intestinal epithelial cell line. Both the E. coli isolates and the E. coli Nissle 1917 strain showed strong reductions in adhesion of porcine enteropathogenic E. coli and invasion of Salmonella typhimurium with epithelial cells in vitro, with a probiotic effect. Screening of five epidemiologically unlinked swine farms and two wild boar groups showed one farm positive for E. coli Nissle 1917. A feeding experiment with four piglets showed viable E. coli Nissle 1917 in the intestine of three animals. CONCLUSIONS: The results of this study suggest that the E. coli Nissle 1917 strain is already partially established in swine herds, but the colonization of individual animals is variable. SIGNIFICANCE AND IMPACT OF THE STUDY: We report natural, long-term colonization and transmission of the probiotic E. coli Nissle 1917 strain in a swine herd, characterized individual persistence and colonization properties in swine and established an in vitro porcine intestinal epithelial cell model of probiotic action. The results of this study would have implications in the use of this strain as a probiotic in swine and contribute to a better understanding of the individual nature of intestinal bacterial persistence and establishment.     

Detection of cytotoxic necrotizing factor types 1 and 2 among fecal Escherichia coli isolates from Brazilian children with and without diarrhea

The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.     

Caenorhabditis elegans as a simple model to study phenotypic and genetic virulence determinants of extraintestinal pathogenic Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause disease by invading normally sterile niches within the host body, e.g., urinary tract, blood and cerebrospinal fluid. Infections due to ExPEC strains, in particular urinary tract infections, cause considerable morbidity and significant health-care costs. The goal of our study is to evaluate whether Caenorhabditis elegans can be used as a model to study phenotypic and genetic virulence determinants of ExPEC strains. For this purpose, we used a collection of 31 E. coli strains isolated during acute extra-intestinal infections or from the feces of healthy individuals. For all strains, the phylogeny, the presence of ExPEC virulence factors, the resistance to biologically relevant stressors (bile, human serum and lysozyme), the motility, the growth rate, the virulence in C. elegans and in a murine septicaemia model has been established. The results show that there is a strong link between virulence in C. elegans and certain phenotypic and genetic virulence predictors of ExPEC strains determinable in vitro. Furthermore, there is a significant correlation between virulence of different ExPEC strains in C. elegans and in the murine model. Therefore, our results suggest that C. elegans can be used as a model to study virulence determinants of ExPEC strains.     

Virulence gene profiling of enteroaggregative Escherichia coli heat-stable enterotoxin 1-harboring E. coli (EAST1EC) derived from sporadic diarrheal patients

Between 2007 and 2009, a total of 2168 Escherichia coli strains derived from diarrheal patients, defined as putative diarrheagenic E. coli (DEC), were collected from medical institutions in Akita prefecture, Japan. Thirty five of the strains lacked typical pathogenic determinants of DEC other than astA, which encodes enteroaggregative E. coli (EAggEC) heat-stable enterotoxin 1 (EAST1). These E. coli strains are referred to as EAST1EC. Several studies have suggested a role of EAST1 in diarrhea; however, the correlation between diarrhea and the presence of astA remains inconclusive. To investigate whether EAST1EC strains derived from diarrheal patients shared pathogenic factors other than EAST1, virulence gene profiling of 12 virulence genes - iha, lpfA, ldaG, pilS, pic, pet, irp2, daa, aah, aid, cdtB and hlyA - was carried out. PCR analysis revealed that four of the 35 EAST1EC strains harbored only astA, 24 harbored genes associated with adhesins and intestinal colonization, three strains harbored the gene for α-hemolysin, and 24 strains harbored the gene for a siderophore. These results indicated that some EAST1EC strains harbor various virulence genes associated with distinct E. coli pathotypes, primarily enterohemorrhagic E. coli and EAggEC, which may represent additional pathogenic determinants of EAST1EC.     

Evolution of the iss gene in Escherichia coli

The increased serum survival gene iss has long been recognized for its role in extraintestinal pathogenic Escherichia coli (ExPEC) virulence. iss has been identified as a distinguishing trait of avian ExPEC but not of human ExPEC. This gene has been localized to large virulence plasmids and shares strong similarities with the bor gene from bacteriophage lambda. Here, we demonstrate that three alleles of iss occur among E. coli isolates that appear to have evolved from a common lambda bor precursor. In addition to the occurrence of iss on the ColV/BM virulence plasmids, at least two iss alleles occur within the E. coli chromosome. One of these alleles (designated type 3) was found to occur in the genomes of all currently sequenced ExPEC strains on a similar prophage element that also harbors the Sit iron and manganese transport system. When the prevalence of the three iss types was examined among 487 E. coli isolates, the iss type 3 gene was found to occur at a high frequency among ExPEC isolates, irrespective of the host source. The plasmid-borne iss allele (designated type 1) was highly prevalent among avian pathogenic E. coli and neonatal meningitis-associated E. coli isolates but not among uropathogenic E. coli isolates. This study demonstrates the evolution of iss in E. coli and provides an additional tool for discriminating among E. coli pathotypes through the differentiation of the three iss allele types and bor.     

Targeting virulence traits: potential strategies to combat extraintestinal pathogenic E. coli infections

Extraintestinal pathogenic Escherichia coli (ExPEC) are able to colonize, invade and induce disease in body niches outside the gastrointestinal tract. The emergence of (multi)resistant ExPEC variants calls for alternative antimicrobial strategies with a reduced pressure on selection of antibiotic resistances. We report on recent research efforts to target such virulence traits or to interfere with ExPEC colonization of extraintestinal niches in order to prevent or treat ExPEC infections and reduce the risk of further emergence of antibiotic resistances.     

Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

Use of Zebrafish to Probe the Divergent Virulence Potentials and Toxin Requirements of Extraintestinal Pathogenic Escherichia coli

Extraintestinal pathogenic E. coli (ExPEC) cause an array of diseases, including sepsis, neonatal meningitis, and urinary tract infections. Many putative virulence factors that might modulate ExPEC pathogenesis have been identified through sequencing efforts, epidemiology, and gene expression profiling, but few of these genes have been assigned clearly defined functional roles during infection. Using zebrafish embryos as surrogate hosts, we have developed a model system with the ability to resolve diverse virulence phenotypes and niche-specific restrictions among closely related ExPEC isolates during either localized or systemic infections. In side-by-side comparisons of prototypic ExPEC isolates, we observed an unexpectedly high degree of phenotypic diversity that is not readily apparent using more traditional animal hosts. In particular, the capacity of different ExPEC isolates to persist and multiply within the zebrafish host and cause disease was shown to be variably dependent upon two secreted toxins, α-hemolysin and cytotoxic necrotizing factor. Both of these toxins appear to function primarily in the neutralization of phagocytes, which are recruited in high numbers to sites of infection where they act as an essential host defense against ExPEC as well as less virulent E. coli strains. These results establish zebrafish as a valuable tool for the elucidation and functional analysis of both ExPEC virulence factors and host defense mechanisms.