Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.     

Rapid categorization of pathogenic Escherichia coli by multiplex PCR

A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli(EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli(EPEC); invE for enteroinvasive E. coli(EIEC); elt, estp, and esth for enterotoxigenic E. coli(ETEC); CVD432 and aggR for enteroaggregative E. coli(EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR system, all 12 targeted genes (stx1, stx2, eaeA, invE, elt, estp, astA, esth, bfpA, aggR, EAF, and CVD432) were amplified in a single PCR reaction in one tube and detected by electrophoresis. Using our multiplex PCR, the 208 clinically isolated strains of diarrheagenic E. coli in our laboratory were successfully categorized and easily analyzed for the presence of virulence plasmids.     

Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli

To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.     

Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1

Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C(8) reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH(2)-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1-C2 and C3-C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.     

Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan

In the present study, we examined the prevalence and characteristics of CTEC among diarrheal children in Japan during a year-long surveillance study. A PCR-RFLP assay for the detection and differentiation of five types of E. coli cdtB gene (types I through V) was developed, and 362 stool specimens collected from patients reporting to pediatric departments in two hospitals were analyzed. Of the 35 samples (9.7%) that were positive for the cdtB gene, 21 were positive for cdt-I , three for cdt-II , four for cdt-III , three for cdt-IV and four samples were positive for cdt-V , as determined by different molecular techniques. The recovery of CTEC having cdt alleles was a little less, which included 19 with cdt-I , one cdt-II , three cdt-III, three cdt-IV and four with cdt-V . Among 30 CTEC strains isolated, the majority of them (43%) belonged to serogroup O2. The other virulence genes such as astA , cnf1 , eaeA , cnf2 and bfpA genes were detected in 14 (47%), 11 (37%), four (13%), three (10%) and one (3.3%) strains of CTEC, respectively. However, the other common virulence-associated genes specific for DEC were not detected in these strains. Interestingly, an untypable cdt gene was detected by PCR-RFLP in Providencia alcalifaciens . Our data indicate that CTEC may be associated with diarrheal children in Japan and most of them do not belong to a conventional enteropathogenic pathovar and thus differ from strains isolated in developing countries.     

Escherichia coli Expressing EAST1 Toxin Did Not Cause an Increase of cAMP or cGMP Levels in Cells, and No Diarrhea in 5-Day Old Gnotobiotic Pigs

Background

Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.

Methodology/Principal Findings

In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.

Conclusion/Significance

Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.     

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.     

Pathogenesis of enteroaggregative Escherichia coli infection

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in 'typical EAEC strains'. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis.     

Atypical enteropathogenic Escherichia coli genomic background allows the acquisition of non-EPEC virulence factors

Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I (group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli (EHEC)]; cluster II (group A) also contains enteroaggregative and diffusely adherent E. coli ; cluster III (group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV (group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background – especially the strains of phylogenetic group B1 – that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.     

Pathotyping Escherichia coli by using miniaturized DNA microarrays

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.     

禽致病性大肠杆菌IMT5155 疑似毒力基因在人源及动物源大肠杆菌中的分布

[目的]检测禽致病性大肠杆菌IMT5155自分泌黏附素基因等具有代表性的疑似毒力基因在不同来源大肠杆菌中的分布,为进一步研究其致病机理提供依据.[方法]采用PCR和Dot blot,检测疑似毒力基因在不同地区(101株大肠杆菌中国分离株和121株大肠杆菌德国分离株)、不同来源(人源、禽源及猪源)大肠杆菌中的分布,并分析其和大肠杆菌系统进化分群的关系.[结果]自分泌黏附素基因B11等11个疑似毒力基因在禽致病性大肠杆菌中分布率较高,阳性率分别为:A1 36.4%(32/88)、A8 53.4%(47/88)、A1063.6%(56/88)、B1137.5%(33/88)、F3 59.1%(52/88)等,且疑似毒力基因主要存在于大肠杆菌B2进化群中.值得注意的是,D1、E9和F11基因片段在新生儿脑膜炎大肠杆菌中有较高的分布率,分别为60%(6/10)、80%(8/10)和90%(9/10),而在新生儿脑膜炎大肠杆菌中未检测到B11基因.[结论]自分泌黏附素B11等疑似毒力基因与禽致病性大肠杆菌关系密切,但疑似毒力基因D1、E9和F11与新生儿脑膜炎大肠杆菌密切相关,提示禽致病性大肠杆菌可能是新生儿脑膜炎大肠杆菌的毒力基因储库.     

A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.     

Identification of unconventional intestinal pathogenic Escherichia coli isolates expressing intermediate virulence factor profiles by using a novel single-step multiplex PCR

Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for.     

Insights into a multidrug resistant Escherichia coli pathogen of the globally disseminated ST131 lineage: genome analysis and virulence mechanisms

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.     

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.     

Thermoregulation-dependent expression of Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12.

Resistance to the bactericidal action of normal human serum is one of the characteristics of virulent Yersinia enterocolitica. This property is attributable to the virulence plasmid harbored by pathogenic strains of the species. Serum resistance in Y. enterocolitica is thermoregulated, and its expression correlates well with the presence of virulence plasmid-encoded outer membrane proteins. To further examine the biochemical basis underlying resistance, we cloned a large segment (ca. 30 kilobases) of virulence plasmid DNA and studied the expression of plasmid-encoded outer membrane proteins in a serum-sensitive strain of Escherichia coli. The presence of the 160-kilodalton Y. enterocolitica-derived outer membrane protein 1 on E. coli transformants conferred a high degree of hydrophobicity, autoagglutinability, and resistance to serum killing. All of these properties were thermoregulated in E. coli with fidelity, suggesting that a functional thermoregulatory element was present in the cloned DNA. Elimination of protein 1 from the outer membrane of E. coli transformants by insertional inactivation of the structural gene with a Kanr gene cassette abrogated all of these properties and returned the serum-sensitive phenotype.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

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.     

Identification of entero-aggregative Escherichia coli based on surface properties

Twenty-nine strains of Escherichia coli that adhere to HEp-2 cells with a'stacked brick' pattern (EAggEC), and four nonadherent control strains, wereexamined for the ability to hybridize with gene probes for aggregative (AA) and diffuse (DA)HEp-2 cell adhesion phenotypes. These strains were also tested for the ability to express an 18 kDa membrane-associated outer- membrane protein (MAP), to agglutinate erythrocytes, and toproduce a pellicle during broth culture. Thirteen of the 29 HEp-2 adherent strains of E. coli hybridized with the gene probes for both AA and DA, and expressed an 18 kDa outermembrane protein (OMP) which was antigenically related to the MAP expressed by strains of E. coli O126:H27. The strains that did not carry the additional DA genes did notexpress an 18 kDa OMP. Although strains of EAggEC share the ability to adhere to HEp-2 cellswith a stacked brick pattern, these strains exhibit a diverse range of physical and biochemicalproperties. From the results of this study, it was concluded that currently, the possession ofEAggEC genes or the ability to adhere to HEp-2 cells in a stacked brick formation, remain theonly reliable means of identifying EAggEC.