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.     

Target genes for virulence assessment of Escherichia coli isolates from water, food and the environment

The widespread species Escherichia coli includes a broad variety of different types, ranging from highly pathogenic strains causing worldwide outbreaks of severe disease to avirulent isolates which are part of the normal intestinal flora or which are well characterized and safe laboratory strains. The pathogenicity of a given E. coli strain is mainly determined by specific virulence factors which include adhesins, invasins, toxins and capsule. They are often organized in large genetic blocks either on the chromosome ('pathogenicity islands'), on large plasmids or on phages and can be transmitted horizontally between strains. In this review we summarize the current knowledge of the virulence attributes which determine the pathogenic potential of E. coli strains and the methodology available to assess the virulence of E. coli isolates. We also focus on a recently developed procedure based on a broad-range detection system for E. coli-specific virulence genes that makes it possible to determine the potential pathogenicity and its nature in E. coli strains from various sources. This makes it possible to determine the pathotype of E. coli strains in medical diagnostics, to assess the virulence and health risks of E. coli contaminating water, food and the environment and to study potential reservoirs of virulence genes which might contribute to the emergence of new forms of pathogenic E. coli.     

Virulence genotypes of Escherichia coli strains from bacteremia in relation to phylogeny

Bacteremia is the principal way of dissemination of local infections to distant organs. Escherichia coli bacteremia is almost always clinically significant, suggesting an increased risk of developing sepsis syndrome. Fifty-one E. coli bloodstream human isolates were analyzed using PCR technique for several molecular markers associated with extraintestinal virulence, and their phylogenetic group assignment, taking into account the link between the phylogenetic background and the intrinsic virulence of this species. Sixteen virulence genotypes have been identified, the majority of the blood isolates carrying the association of two genes. The genes encoding type 1 fimbria and aerobactin had the highest prevalence. As a confirmation of other studies, the strains assigned to E. coli phylogenetic group B2 exhibited the highest concentration of virulence genes, and represented almost half of the clinical blood isolates. The multifactorial virulence of E. coli strains isolated from invasive infections reflects a phylogenetic inheritance, and supports the concept of ExPEC pathotype as a subset of E. coli population involved in human infectious diseases. The surveillance of geographical variation of E. coli pathogenic clones is useful for epidemiological analysis.     

Genetic Characterization of Escherichia coli O157:H7 Strains Isolated from the One-Humped Camel (Camelus dromedarius) by Using Microarray DNA Technology

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.     

Detection system for Escherichia coli-specific virulence genes: absence of virulence determinants in B and C strains.

We describe a rational approach to simultaneously test Escherichia coli strains for the presence of known virulence genes in a reverse dot blot procedure. Specific segments of virulence genes of E. coli designed to have similar hybridization parameters were subcloned on plasmids and subsequently amplified by PCR as unlabeled probes in amounts sufficient to be bound to nylon membranes. Various pathogenic isolates and laboratory strains of E. coli were probed for the presence of virulence genes by labeling the genomic DNA of these strains with digoxigenin and then hybridizing them to the prepared nylon membranes. These hybridization results demonstrated that besides the E. coli K-12 safety strain derivatives, E. coli B and C strains are also devoid of genes encoding any of the investigated virulence factors. In contrast, pathogenic E. coli control strains, used to evaluate the method, showed typical hybridization patterns. The described probes and their easy application on a single filter were shown to provide a useful tool for the safety assessment of E. coli strains to be used as hosts in biotechnological processes. This approach might also be used for the identification and characterization of clinically significant E. coli isolates from human and animal species.     

Genetic characterization of Escherichia coli O104 isolates from different sources in the United States

Escherichia coli O104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagic E. coli and those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producing E. coli serotypes.     

Use of representational difference analysis to identify Escherichia coli O157-specific DNA sequences

Whereas several important virulence factors in Escherichia coli O157 have been identified, studies suggest they are not always essential and are probably insufficient to account for the severe clinical manifestation of E. coli O157 infection. Identification of putative virulence determinants is crucial to the understanding of bacterial pathogenesis and genomic comparison analysis may aid the characterisation of unidentified virulence attributes. In this study, representational difference analysis (RDA) was used for genomic comparison of E. coli O157 with the proposed ancestral strain, E. coli O55. Unique E. coli O157 gene sequences were isolated and one, termed RDA-1, taken forward for further analysis. Southern blotting with labelled RDA-1 as a probe showed it to be present in 77% of E. coli O157 isolates and absent in all non-E. coli O157 screened. Sequence flanking RDA-1 was obtained from a genomic clone identified by hybridisation, and contained an open reading frame predicted to encode a novel iron-regulated outer membrane protein.     

The role of capsular antigens in serum resistance and in vivo virulence of Escherichia coli

Abstract To identify critical microbial determinant(s) in serum resistance and in vivo virulence, 5 strains of Escherichia coli with different combinations of K and O types were examined along with their unencapsulated mutants. All 3 E. coli strains possessing the K1 capsule were considerably more resistant to normal human serum in vitro and more virulent in newborn rats than their isogenic mutants lacking the K1 capsule. In contrast, two K5 encapsulated strains and their unencapsulated mutants were essentially the same for their degree of serum resistance and in vivo virulence. These findings suggest that for the demonstration of serum resistance and in vivo virulence, a capsule is required for E. coli strains encapsulated with K1, whereas certain E. coli strains encapsulated with K5 may not need a capsule.     

Role of the 25 kDa major outer membrane protein of Legionella pneumophila in attachment to U-937 cells and its potential as a virulence factor for chick embryos

The gene encoding the 25 kDa major outer membrane protein (MOMP) of Legionella pneumophila was transformed into Escherichia coli JM 83 and the resultant E. coli LP 116 clone expressed the Legionella-MOMP. Compared with the parent E. coli strain, the clone showed a fivefold increase in opsonin-independent binding to U-937 cells. Furthermore, this gene was incorporated by electroporation into a low virulence derivative of Leg. pneumophila which showed reduced expression of the MOMP but enhanced expression of a 31 kDa protein in the OMP profile. After electroporation, the attenuated strain showed an increased expression of the MOMP while the 31 kDa protein was eliminated and virulence for the chick embryo was re-established. The use of a monoclonal antibody specific for the MOMP abolished virulence and adherence. These studies suggest that the 25 kDa MOMP of Leg. pneumophila serves as an adhesive molecule for host cells and that this protein plays a major role in the virulence of the organism for the chick embryo.     

Genetics of hemolysin of Escherichia coli.

The expression of alpha-hemolysin is a property frequently associated with Escherichia coli extraintestinal infections. We have examined the genetic basis for hemolysin expression by an E. coli strain isolated from a human urinary tract infection. The genes necessary for hemolysin synthesis were found to be chromosomal and to map near the ilv gene cluster. Isogenic hly+ and hly derivatives were also prepared and tested for virulence in the chicken embryo model system. Hemolysin was found to be necessary but not in itself sufficient for E. coli virulence in this in vitro model.     

Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P

External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.     

Differences in virulence among Escherichia coli O157:H7 strains isolated from humans during disease outbreaks and from healthy cattle

Escherichia coli O157:H7 causes life-threatening outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans and significant economic loss in agriculture and could be a potential agent of bioterrorism. Although the prevalence of E. coli O157:H7 in cattle and other species with which humans have frequent contact is high, human infections are relatively uncommon, despite a low infectious dose. A plausible explanation for the low disease incidence is the possibility that not all strains are virulent in humans. If there are substantial differences in virulence among strains in nature, then human disease may select for high virulence. We used a gnotobiotic piglet model to investigate the virulence of isolates from healthy cattle and from humans in disease outbreaks and to determine the correlation between production of Shiga toxin 1 (Stx1) and Stx2 and virulence. Overall, E. coli O157:H7 strains isolated from healthy cattle were less virulent in gnotobiotic piglets than strains isolated from humans during disease outbreaks. The amount of Stx2 produced by E. coli O157:H7 strains correlated with strain virulence as measured by a reduction in piglet survival and signs of central nervous system disease due to brain infarction. The amount of Stx1 produced in culture was not correlated with the length of time of piglet survival or with signs of central nervous system disease. We suggest that disease outbreaks select for producers of high levels of Stx2 among E. coli O157:H7 strains shed by animals and further suggest that Stx1 expression is unlikely to be significant in human outbreaks.     

Loss of virulence genes in Escherichia coli populations during manure storage on a commercial swine farm

Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in Escherichia coli during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (n = 392) carried estB, fedA, stx(2e), astA, paa, aida-I, and sepA at lower frequencies than isolates obtained from fresh feces (n = 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal E. coli population was initially dominated by a single genotype, all isolates of which carried fedA and aida-I. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of E. coli that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by E. coli. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.     

Characterization of antibiotic-resistant and potentially pathogenic Escherichia coli from soil fertilized with litter of broiler chickens fed antimicrobial-supplemented diets

The objective of this study was to characterize antimicrobial resistance and virulence determinants of Escherichia coli from soil amended with litter from 36-day-old broiler chickens ( Gallus gallus domesticus ) fed with diets supplemented with a variety of antimicrobial agents. Soil samples were collected from plots before and periodically after litter application in August to measure E. coli numbers. A total of 295 E.?coli were isolated from fertilized soil samples between August and March. Antibiotic susceptibility was determined by Sensititre, and polymerase chain reaction was performed to detect the presence of resistance and virulence genes. The results confirmed that E.?coli survived and could be quantified by direct plate count for at least 7 months in soil following litter application in August. The effects of feed supplementation were observed on E.?coli numbers in November and January. Among the 295 E.?coli, the highest antibiotic resistance level was observed against tetracycline and β-lactams associated mainly with the resistance genes tetB and bla(CMY-2), respectively. Significant treatment effects were observed for phylogenetic groups, antibiotic resistance profiles, and virulence gene frequencies. Serotyping, phylogenetic grouping, and pulsed-field gel electrophoresis confirmed that multiple-antibiotic-resistant and potentially pathogenic E.?coli can survive in soil fertilized with litter for several months regardless of antimicrobials used in the feed.     

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.     

Lack of the RNA chaperone Hfq attenuates pathogenicity of several Escherichia coli pathotypes towards Caenorhabditis elegans

Escherichia coli is an important agent of Gram-negative bacterial infections worldwide, being one of the leading causes of diarrhoea and urinary tract infections. Strategies to understand pathogenesis and develop therapeutic compounds include the use of the nematode Caenorhabditis elegans as a model for virulence characterization and screening for novel antimicrobial entities. Several E. coli human pathotypes are also pathogenic towards C. elegans, and we show here that lack of the RNA chaperone Hfq significantly reduces pathogenicity of VTEC, EAEC, and UPEC in the nematode model. Thus, Hfq is intrinsically essential to pathogenic E. coli for survival and virulence exerted in the C. elegans host.     

DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains

ColV plasmids have long been associated with the virulence of Escherichia coli, despite the fact that their namesake trait, ColV production, does not appear to contribute to virulence. Such plasmids or their associated sequences appear to be quite common among avian pathogenic E. coli (APEC) and are strongly linked to the virulence of these organisms. In the present study, a 180-kb ColV plasmid was sequenced and analyzed. This plasmid, pAPEC-O2-ColV, possesses a 93-kb region containing several putative virulence traits, including iss, tsh, and four putative iron acquisition and transport systems. The iron acquisition and transport systems include those encoding aerobactin and salmochelin, the sit ABC iron transport system, and a putative iron transport system novel to APEC, eit. In order to determine the prevalence of the virulence-associated genes within this region among avian E. coli strains, 595 APEC and 199 avian commensal E. coli isolates were examined for genes of this region using PCR. Results indicate that genes contained within a portion of this putative virulence region are highly conserved among APEC and that the genes of this region occur significantly more often in APEC than in avian commensal E. coli. The region of pAPEC-O2-ColV containing genes that are highly prevalent among APEC appears to be a distinguishing trait of APEC strains.     

Virulence of Escherichia coli Serotypes for Mice

A study was undertaken to determine whether virulence in mice could be used to assess the pathogenicity of a variety of Escherichia coli serotypes. Sixty-one E. coli strains isolated from animals, poultry, or humans were serotyped to determine their O, K, and H antigens, and were administered to mice via the intraperitoneal route with and without a mucin adjuvant. The ld(50) dose was then determined for each serotype. The results indicated that the source of the serotype may be associated with virulence for mice. Serotypes isolated from nonenteric, systemic sources showed a greater virulence for mice inoculated intraperitoneally than did the enteric and the nonenteric, nonsystemic (localized) isolates. It was observed that not all serotypes belonging to a specific serogroup were virulent for mice and that the presence or absence of a K antigen had no effect on the virulence of strains of one serotype.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Virulence characteristics of Escherichia coli isolates from children with urinary tract infections

The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common infecting agent in children and adults of both sexes. In an attempt to evaluate the intrinsic virulence of E. coli uroisolates from children, 54 strains were assessed by using PCR for the presence of five representative genetic determinants coding for adherence systems (pap, sfa/foc, afa), and toxins (hly and cnf). The prevalence of pap, sfa/foc and afa genes was 55%, 54%, and 44%, respectively. Hemolysin-encoding gene hly was detected in 55% strains, while cnf was exhibited by 35% of the screened E. coli isolates. Among the 39 PCR positive strains isolated from children's urine cultures the co-occurrence of the various targeted virulence genes was detected in 30 strains, the virulence profiles identified suggesting the presence of their localization on chromosomal regions known as pathogencity-associated islands. The rapid and reliable detection of the intrinsic virulence potential by this molecular approach could be very useful when evaluating the importance of microorganism pathogenicity versus host's susceptibility for developing an overt symptomatology of infection.