Detection of toxB, a plasmid virulence gene of Escherichia coli O157, in enterohemorrhagic and enteropathogenic E. coli

The virulence plasmid of Escherichia coli O157 strain EDL933 carries a 10-kb putative virulence gene designated toxB. Little is known about the distribution of this gene among E. coli O157 strains or its presence in other enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) strains. We developed PCR and hybridization tools for the detection of the entire toxB sequence and investigated its presence in a collection of EHEC O157 strains and other EHEC and EPEC strains belonging to different serogroups and isolated from different sources. The EHEC O157 strains reacted with all of the PCR primers and probes used, thus indicating the presence of a complete toxB gene regardless of the human or bovine origin of the isolates. Similar positive reactions were observed for about 50% of the EHEC O26 strains tested and a few other EHEC and EPEC strains. However, the size of the DNA fragments hybridizing with the toxB probes differed from that of the positive fragments from EHEC O157, suggesting a polymorphism in the toxB genes present in the different E. coli serogroups. Moreover, several EHEC and EPEC strains belonging to different serogroups reacted with only some of the genetic tools used, suggesting either the existence of major variants of toxB or the presence of fragments of the gene. Southern blotting analysis showed that toxB sequences were located on large plasmids in EHEC and EPEC O26 as well.     

Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico.

Enteropathogenic Escherichia coli (EPEC) often exhibits localized adherence or diffuse adherence to HEp-2 cells. We recently provided evidence that HEp-2 cell-adherent or enteroadherent E. coli (EAEC) not belonging to EPEC serogroups was the cause of diarrhea among U.S. travelers to Mexico. In the present study, we looked for EAEC and EPEC in stool specimens from 154 children with acute diarrhea and 137 well children seen at several outpatient clinics in Guadalajara, Mexico. EAEC showing localized adherence (EAEC-L) was isolated from 13.0% of the patients and 0.7% of the controls (P less than 0.0001). EAEC showing diffuse adherence (EAEC-D) was recovered from 20.8% of the patients and 7.3% of the controls (P less than 0.001). EPEC was isolated from 4.5 and 6.7% of the patients and controls, respectively. Among all enteropathogens, only enterotoxigenic E. coli occurred as commonly (21.4%) as EAEC-D and EAEC-L did in children with diarrhea. Of the EAEC-L strains isolated from children with diarrhea, 20% belonged to recognized EPEC serogroups, and 3.1% of EAEC-D strains belonged to recognized EPEC serogroups. This study suggests that EAEC may be an important pediatric enteropathogen in Mexican children with diarrhea and further supports the observation that adherence to HEp-2 cells may be a marker of virulence independent of EPEC serogroup among E. coli strains.     

Clinical and genetic aspects of Shiga-like toxin production in traditional enteropathogenic Escherichia coli

Cell culture tests, DNA colony blot hybridization and polymerase chain reaction were used to examine classical enteropathogenic Escherichia coli (EPEC) for the presence of Shiga-like toxin (SLT). Fifteen of 155 strains from West Germany, originally identified as EPEC on the basis of serotyping, were shown to harbor either SLT-I or SLT-II genes. All strains that hybridized with the 20-base oligonucleotide probes which are complementary to slt-IA or slt-IIA sequences derived from the genomic DNA of enterohemorrhagic E. coli O157:H7 strain 933 produced moderate or high levels of cytotoxin in Vero and HeLa cell assays. Four additional strains of low to moderate cytotoxicity did not hybridize with either probe. Five different serogroups producing SLTs were identified: O26, O55, O111, O119 and O128. All three SLT-positive E. coli O26:H11 and four of five E. coli O111:H- isolates hybridized with a 3.4 kilobase fragment (CVD 419 probe) derived from the 60-megadalton plasmid of EHEC O157:H7. Seven of the 15 SLT-gene positive strains were associated with bloody diarrhea, six isolates were from patients with hemolytic uremic syndrome (HUS). Based on their clinical, epidemiological, pathogenic and genetic features SLT-producing E. coli among classical EPEC mimic enterohemorrhagic E. coli O157:H7 and might be considered as EHEC.     

Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia coli.

Attaching and effacing (A/E) intestinal lesions are produced by enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and RDEC-1, a pathogen of weanling rabbits. We recently identified a chromosomal locus (eae[E. coli A/E]) which is required for A/E activity in a wild-type EPEC strain. Sequences homologous to those of an eae gene probe were detected in EPEC, RDEC-1, and EHEC isolates. We report here that the eae gene is chromosomally encoded in all EPEC and EHEC strains tested and in RDEC-1. In addition, the eae probe was found to be 100% sensitive and 98% specific in detecting E. coli of EPEC serogroups that demonstrate A/E activity. Ten percent of E. coli of EPEC serogroups that hybridized with the eae probe and produced A/E activity did not hybridize with the EAF (EPEC adherence factor) probe, a plasmid-associated diagnostic probe which is currently used to identify EPEC. In addition to A/E factors, plasmid-associated adhesins also contribute to the pathogenesis of EPEC and RDEC-1. To further investigate the role of plasmid-associated adherence, a hybrid RDEC-1-EPEC strain containing the adherence plasmid of an EPEC strain in the A/E background of RDEC-1 was constructed. This hybrid strain, unlike the parent RDEC-1 strain, produced A/E lesions on human tissue culture cells, which suggests that the EPEC adherence plasmid provides tissue specificity to the hybrid strain and that the A/E factors of RDEC-1 are not host restricted.     

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.     

Nature and distribution of mucosal lesions associated with enteropathogenic and enterohemorrhagic Escherichia coli in piglets and the role of plasmid-mediated factors.

Bacterial attachment-effacement (att-eff) is emerging as an important virulence characteristic common to both enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). The contribution of the plasmid-encoded EPEC adherence factor to the production of mucosal lesions and diarrhea was investigated in gnotobiotic piglets. Bacterial att-aff in the intestinal mucosa of piglets infected with plasmid-cured EPEC strain E2348/69 (O127) was indistinguishable from that in piglets infected with the parent strain, but the distribution of lesions was different; it occurred in the small intestines of 6 of 7 piglets infected with the parent strain compared with only 2 of 11 (P = 0.006) infected with the plasmid-cured strain. Plasmid-encoded factors in EPEC and EHEC strains did not appear to contribute to bacterial competition with normal gut microflora. Of 13 strains belonging to five EPEC serogroups, O55, O142, O26, O119, and O111, 3 fulfilled the criteria for EHEC (2 O26 and 1 O111). There were three distinct patterns of bacterial association with the intestinal mucosa of infected piglets. (i) EHEC strains caused bacterial att-eff associated with extensive destruction of surface and glandular epithelia in the large intestines with little or no inflammatory response. (ii) Some EPEC strains caused severe diarrhea which correlated with the extent of bacterial att-eff in the proximal small intestine, disruption of the epithelial cell membrane, and inflammation. It is suggested that, with respect to virulent strains, this degree of involvement determines the clinical outcome. Mildly pathogenic strains (O127 and O119), in which bacterial att-eff was restricted to the distal halves of the small and large intestines, caused little or no diarrhea. In such strains, nonimmune host factors (smaller, poorly feeding, and lethargic piglets) tended to play a determining role with regard to the degree of involvement of the small intestine and hence the clinical outcome. (iii) One strain (O55) caused illness and mucosal damage which could not be accounted for by the sparse bacterial att-eff observed in the gut. Instead, bacteria penetrated into and proliferated in the lamina propria, undermining the villous tips in the small intestine. Bacterial att-eff was the most important virulence factor in most of the strains examined, but plasmid-mediated factors facilitated bacterial adhesion in the small intestine, which may explain the reduced pathogenicity of the plasmid-cured variant of strain E2348/69 for human volunteers.     

志贺毒素阴性的大肠埃希菌O157菌株的毒力因子检测

目的 研究志贺毒素阴性的大肠埃希菌0157血清群菌株可能携带的毒力因子,以确定其致病群类型。方法 对8株分离自杭州的志贺毒素阴性的大肠埃希菌0157血清群菌株及分离自美国和我国江苏的EHEC0157菌株,应用PCR技术检测stx、hly、EPEC和EHEC共有eaeA、EHEC特异的eaeA,ELISA检测ETEC的耐热和不耐热解毒素,HEP－2细胞粘附试验测定细菌与上皮细胞的粘附能力,并进行菌株质粒图谱分析。结果 8株志贺毒素阴性的大肠埃希菌0157菌株均未检出stx、hly、EHEC特异的eaeA以及ETEC的耐热和不耐热肠毒素,仅其中2株菌株携有EPEC和EHEC共有eaeA,另3株菌株对HEP－2细胞有集聚粘附能力。结论 目前我国检获的大肠埃希菌0157菌株其致病类型表现为多样性,部分菌株尚不能明确其毒力因子。EHEC0157的鉴定必须重视毒力因子的检测。     

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.     

HEp-2 cell adherence,actin aggregation,and intimin types of attaching and effacing Escherichia coli strains isolated from healthy infants in Germany and Australia

Fecal samples from healthy children under 2 years of age living in Berlin, Germany (205 infants), and Melbourne, Australia (184 infants), were investigated for the presence of attaching and effacing (AE) Escherichia coli (AEEC) strains by screening for eae (intimin) genes. Twenty-seven AEEC strains were isolated from 14 children (7.6%) from Melbourne and from 12 children (5.9%) from Berlin. The 27 AEEC strains were classified as enterohemorrhagic E. coli (one strain, producing Shiga toxin 1), typical enteropathogenic E. coli (EPEC) (one strain carrying an EPEC adherence factor [EAF] plasmid), and atypical EPEC (25 strains negative for Shiga toxins and EAF plasmids). The AEEC were divided into 18 different serotypes, O-nontypeable and O-rough strains. Typing of their intimin genes revealed the presence of intimin alpha in 6 strains, intimin beta in 11 strains, intimin gamma in 7 strains, intimin zeta in 2 strains, and intimin eta in one strain. Analysis of HEp-2 cell adherence showed diffuse adherence or localized adherence-like patterns in 26 AEEC strains; local adherence was found only with the EAF-positive strain. Ten AEEC strains showed an AE property with the fluorescent actin staining (FAS) test. The introduction of an EAF plasmid (pMAR7) converted 11 FAS-negative AEEC strains to FAS positive and increased the FAS reaction in six FAS-positive AEEC strains, indicating that the genes needed for the AE phenotype were functional in these strains. Our finding indicates that atypical EPEC strains could play a double role as strains that naturally immunize against intimin in humans and as reservoirs for new emerging human pathogenic EPEC strains.     

Cholesterol-enriched membrane microdomains are required for inducing host cell cytoskeleton rearrangements in response to attaching-effacing Escherichia coli

The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and enteropathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56- and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced alpha-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.     

Adhesive properties of enteropathogenicEscherichia coli isolated from infants with acute diarrhea in Africa

The adhesive properties of 69 enteropathogenicEscherichia coli (EPEC) strains were studied. The strains were isolated from diarrheal stools of infants in Burundi, Africa, and identified by serotyping with 12 classical EPEC O serogroup antisera. A test for adhesion to HEp-2 cells revealed that 52 % of the strains showed localized adherence and 13 % diffuse adherence. Localized adherence phenotype was found in previously described serogroups O86, O111, O125, O127, O128 and O142; strains belonging to another serogroup, O126, also exhibited localized adherence. Use of an EPEC adherence factor DNA probe in colony hybridization and in Southern blot techniques revealed that all strains exhibiting localized adherence and no strain exhibiting diffuse adherence produced a positive reaction; the genes were localized on high molecular weight plasmids (50–70 megadaltons). In vitro adhesion tests with human enterocytes performed concurrently with all 69 strains showed that only six of them adhered. These strains belonged to the O26, O117, O125, O128 and O142 serogroups. The adhesin CFA/I was detected only in the O128Escherichia coli. The strain of serogroup O142 exhibited both adhesion to human enterocytes and localized adherence to HEp-2 cells, which suggests that the adhesive systems of enterotoxigenic and enteropathogenicEscherichia coli may coexist.     

The eae gene of enteropathogenic Escherichia coli encodes a 94-kilodalton membrane protein, the expression of which is influenced by the EAF plasmid.

The production of a characteristic intestinal histopathology called attaching and effacing (A/E) lesions by enteropathogenic Escherichia coli (EPEC) is a major characteristic of EPEC pathogenesis. We previously identified a chromosomal gene (eae) of EPEC necessary for the production of A/E lesions on human tissue culture cells. Using antiserum raised to an Eae-PhoA fusion protein, we found that the eae gene encodes a 94-kDa membrane protein. This antiserum recognized a 94-kDa membrane protein in parent strain E2348/69 and a protein of similar size in E. coli HB101 carrying eae on a plasmid but did not recognize any proteins in E. coli HB101 carrying a plasmid with an internal deletion in the eae gene. Antigenically related proteins of ca. 94 kDa were detected in a collection of EPEC strains representing seven EPEC serogroups and in two EHEC strains of serotype O26:H11. Volunteer sera drawn 28 days after but not before ingestion of strain E2348/69 recognized the 94-kDa Eae protein as well as a 128-kDa Eae-PhoA fusion protein, suggesting that the Eae protein is likely to be a previously reported 94-kDa protein shown to be immunogenic in volunteers. The amount of detectable Eae protein was increased in the presence of a high-molecular-weight plasmid which is associated with the ability to produce localized adherence to tissue culture cells. These data suggest that the virulence plasmid of EPEC strain E2348/69 may have a regulatory role in the production of A/E activity.     

Examination of strains belonging to enteropathogenic Escherichia coli serogroups for genes encoding EPEC adherence factor and Vero cytotoxins

Four hundred and forty-nine strains isolated from patients with diarrhoea and belonging to 13 enteropathogenic Escherichia coli (EPEC) O serogroups were tested with a DNA probe for the EPEC adherence factor (EAF). Positive results were obtained with only 36 strains; they belonged to 10 O serogroups and flagellar typing showed they were usually of the "classical" EPEC serotypes. Thirty-four of the 36 EAF-positive strains showed localised adhesion to HEp-2 cells. The two remaining strains, of serotypes O114:H2 and O127:H4, showed low level or no adhesion to HEp-2 cells. No colonies hybridising with the EAF probe were identified in cultures from 115 faecal specimens from healthy children. Sixteen of the 449 strains hybridised with one or both probes for the Vero cytotoxin genes VT1 and VT2; 15 of the 16 strains belonged to serogroups O26 and O128. None of the strains hybridised with both the EAF and VT gene probes. These studies show that the great majority of strains belonging to EPEC O serogroups do not possess the EPEC adherence factor or carry VT genes.     

A new genetic test for the rapid identification of shiga-toxines producing (STEC), enteropathogenic (EPEC) E. coli isolates from children

Routine bacteriological techniques do not allow detection of the most frequent enteric pathogens in young children: enteropathogenic Escherichia coli (EPEC) and shigatoxinogenic E. coli (STEC/EHEC). Since there is no correlation between serotype and pathotype, a genotypic determination is therefore necessary for the identification of these pathogenic strains. We evaluated the Genotype EHEC test (Hain Life Science, Germany), a new rapid system based on DNA multiplex amplification and further hybridization for the detection of shigatoxin stx1, stx2 genes, intimin eae gene and invasin ipaH gene harbored by Shigella and enteroinvasive E. coli (EIEC). E. coli strains of various serogroups isolated from children with acute gastroenteritis, hemorrhagic colitis or hemolytic-uremic syndrome were tested. Their genotypes were first determined by standard in-house PCR. The strains collection included 11 STEC/EHEC (serogroups O157, O111, O26, O91, O-untypable) and nine EPEC (serogroups O26, O157, O55, O126, O127, O-untypable). The same strains were tested with Genotype EHEC. For all the strains, the hybridization banding pattern obtained by Genotype EHEC correlated with their expected genotypic characteristics. No specific equipment is required, except a thermocycler. Absence of electrophoresis system, of ethidium bromide staining and imaging system is a clear-cut advantage of Genotype EHEC. In addition, the short testing time (less than 2 h) optimizes treatment orientation. The Genotype EHEC test allows an easy and reliable identification of EHEC, STEC, EPEC and also EIEC. As such, it is a useful tool for the rapid diagnosis of diarrheal diseases.     

Cloning and expression of an adhesin (AIDA-I) involved in diffuse adherence of enteropathogenic Escherichia coli.

The adherence of enteropathogenic Escherichia coli (EPEC) to the small bowel mucosa is an important step in the pathogenesis of diarrheal diseases. It has been shown that many EPEC strains adhere to HEp-2 and especially HeLa cells in characteristic patterns termed localized adherence (LA) and diffuse adherence (DA). A plasmid-derived DNA fragment encoding a factor specific for LA hybridized only to EPEC strains expressing LA, which demonstrated that LA and DA are mediated by two genetically distinct adhesins. EPEC strain 2787 (O127:H27), isolated from a case of infantile diarrhea, exhibited three major properties: (i) it showed DA to HeLa cells, (ii) it carried two large (ca. 100-kilobase [kb]) plasmids and one small plasmid of about 3 kb, and (iii) no fimbriae could be detected by electron microscopy in organisms grown on agar plates or in liquid cultures. Whole isolated plasmid DNA was partially digested with EcoRI and cloned into the vector pBR322. One recombinant clone (pIB6) was found to exhibit the same DA pattern on HeLa cells as did the parent strain. This clone contained an 11-kb DNA fragment derived from the largest of the three plasmids, as shown by Southern hybridization. By deletion analysis, a 6.0-kb DNA fragment was shown to be sufficient for expression of the DA phenotype. This insert encoded the production of a 100,000-dalton protein mediating adhesion to HeLa cells.     

Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells.

Plasmid-encoded adherence factors have been shown to be important for the full expression of enteropathogenic Escherichia coli (EPEC) pathogenicity and for EPEC adhesion to cultured HEp-2 cells. EPEC strain E2348 (O127) shows localized HEp-2 cell adhesion and possesses a 60-megadalton plasmid, pMAR2. When E2348 is cured of pMAR2 it loses the ability to adhere to HEp-2 cells, while nonadherent E. coli K-12 strains P678-54 and HB101 acquire HEp-2 adhesiveness after they gain the plasmid. By electron microscopy, E2348 was seen to adhere to HEp-2 cells in a manner that closely resembled EPEC adhesion to intestinal mucosa; bacteria were intimately attached to projections of the apical HEp-2 cell membrane and caused localized destruction of microvilli. The plasmid-containing K-12 strains, on the other hand, did not show intimate attachment and there was no modification of cell surface architecture. It is concluded that plasmid pMAR2 codes for an adhesin, possibly fimbrial in nature, that promotes HEp-2 adhesion but that other chromosomally encoded factors are required for EPEC to achieve the characteristic mode of intimate cell attachment.     

2F3 monoclonal antibody recognizes the O26 O-antigen moiety of the lipopolysaccharide of enterohemorrhagic Escherichia coli strain 4276

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) organisms are groups of pathogenic strains whose infections are characterized by a typical lesion of enterocyte attachment and effacement. They are involved in enteric diseases both in humans and in animals, and EHEC strains can be responsible for hemolytic uremic syndrome in humans. Previously, it was shown that the 2F3 monoclonal antibody (MAb) is specific for the O26 EHEC and EPEC strains (P. Kerr, H. Ball, B. China, J. Mainil, D. Finlay, D. Pollock, I. Wilson, and D. Mackie, Clin. Diagn. Lab. Immunol. 6:610-614, 1999). As these groups of bacteria play an important role in pathology, the aim of this paper was to characterize the antigen recognized by the 2F3 MAb and its genetic determinant. A genomic locus containing the entire O-antigen gene cluster and half of the colanic acid gene cluster from an O26 EHEC strain was shown to be sufficient for the production of the antigen recognized by the 2F3 MAb in an E. coli DH5alpha strain. By transposon mutagenesis performed on the recombinant plasmid, all 2F3 enzyme-linked immunosorbent assay-negative mutants had their transposons inserted into the O-antigen gene cluster. The O-antigen gene cluster was also cloned from an O26 EHEC strain into the E. coli DH5alpha strain, which then produced a positive result with the 2F3 MAb. Further analysis of the type of lipopolysaccharides (smooth or rough) produced by the clones and mutants and of the O antigen of the 2F3-positive clones confirmed that the epitope recognized by the 2F3 MAb is located on the O antigen in the O26 EHEC and EPEC strains and that its genetic determinant is located inside the O-antigen gene cluster.     

Enzootic enteropathogenic Escherichia coli infection in laboratory rabbits

Enteropathogenic Escherichia coli (EPEC) is the most important cause of persistent diarrhea in children, particularly in developing countries. Animals serve as pathogenic E. coli reservoirs, and compelling evidence for cross-species EPEC transmission exists. In this report, enzootic EPEC infection associated with up to 10.5% diarrhea-associated morbidity in a large laboratory Dutch Belted rabbit colony was investigated. These rabbits were obtained from a commercial vendor and had acute diarrhea following shipment. Fecal culture of 20 rabbits yielded 48 E. coli isolates, 83% of which were eae positive. Repetitive sequence-based PCR (REP-PCR) and serologic analysis identified a single disease-associated EPEC O145:H2 strain. In sampled rabbits, EPEC-positive culture and the presence of diarrhea were significantly associated. This strain displayed a localized adherence-like HEp-2 cell adherence pattern, as seen in diarrheic human infant EPEC isolates. Treatment was instituted with the fluoroquinolone antibiotic enrofloxacin, to which all isolates were susceptible. Preshipment parenteral enrofloxacin administration reduced diarrhea-associated morbidity 22-fold and mortality 12-fold in subsequent deliveries. This report emphasizes the zoonotic potential of animal EPEC strains and the need for virulence determinant-based screening of E. coli isolates from diarrheic animals.     

Putative Adhesins of Enteropathogenic and Enterohemorrhagic Escherichia coli of Serogroup O26 Isolated from Humans and Cattle

Enterohemorrhagic Escherichia coli (EHEC) strains are responsible for food poisoning in developed countries via consumption of vegetal and animal food sources contaminated by ruminant feces, and some strains (O26, O111, and O118 serogroups) are also responsible for diarrhea in young calves. The prevalence of 27 putative adhesins of EHEC and of bovine necrotoxigenic E. coli (NTEC) was studied with a collection of 43 bovine and 29 human enteropathogenic (EPEC) and EHEC strains and 5 non-EPEC/non-EHEC (1 bovine and 4 human) O26 strains, using specific PCRs. Four “groups” of adhesins exist, including adhesins present in all O26 strains, adhesins present in most O26 strains, adhesins present in a few O26 strains, and adhesins not present in O26 strains. The common profile of EHEC/EPEC strains was characterized by the presence of loc3, loc5, loc7, loc11, loc14, paa, efa1, iha, lpfA_O26, and lpfA_O113 genes and the absence of loc1, loc2, loc6, loc12, loc13, saa, and eibG genes. Except for the lpfA_O26 gene, which was marginally associated with bovine EHEC/EPEC strains in comparison with human strains (P = 0.012), none of the results significantly differentiated bovine strains from human strains. One adhesin gene (ldaE) was statistically (P < 0.01) associated with O26 EHEC/EPEC strains isolated from diarrheic calves in comparison with strains isolated from healthy calves. ldaE-positive strains could therefore represent a subgroup possessing the specific property of producing diarrhea in young calves. This is the first time that the distribution of putative adhesins has been described for such a large collection of EHEC/EPEC O26 strains isolated from both humans and cattle.Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli strains represent two important classes of enteric pathogens that cause diarrhea in humans and animals. They have in common the ability to produce a histopathological lesion on enterocytes, called an “attaching and effacing” lesion. The intimate attachment of the bacteria to enterocytes and the localized effacement of microvilli are the main characteristics of the attaching and effacing lesion (26).EPEC strains are an important cause of infant diarrhea in developing countries and are often associated with high mortality rates (8). Human EPEC strains are subdivided into classical (type 1) and nonclassical (type 2) strains on the basis of the production of bundle-forming pili or the presence of the encoding genes. Nonclassical EPEC strains are also present in different animal species. In bovines, nonclassical EPEC strains are associated with diarrhea in young calves of up to 3 months of age (9).EHEC strains are considered to have evolved from EPEC strains through the acquisition of bacteriophages encoding Shiga toxins (Stxs) (31, 45). EHEC strains cause several clinical syndromes in humans (mainly in children and elderly people), such as diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. These have been responsible for large outbreaks in many developed countries, especially Japan, the United States, and the United Kingdom (26). Transmission can occur via consumption of vegetal and animal foodstuffs contaminated by ruminant feces (mainly cattle) (7). Some EHEC strains are also responsible for undifferentiated diarrhea in young calves of up to 3 months of age (24).EPEC and EHEC strains can belong to more than 1,000 O:H serotypes. In EHEC infections, O157:H7 is the main serotype responsible for several outbreaks and sporadic cases of hemorrhagic colitis and hemolytic-uremic syndrome, but non-O157 serogroups (such as O26, O145, O111, and O103) can also be associated frequently with severe illness in humans (5, 35). Though most, if not all, EHEC serogroups are carried by healthy animal ruminants, a few are associated with diarrhea in calves (O5, O26, O111, O118, etc.). Human and animal EPEC strains also belong to a series of O antigenic groups, including O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158 (6). Thus, several serogroups are present in both pathotypes (EHEC and EPEC) and can infect both humans and cattle. Although classical EPEC strains have always been regarded as host specific, EHEC strains have not, and the actual situation regarding nonclassical EPEC strains remains unknown.The first step in EPEC and EHEC infection is the initial adherence of bacteria to intestinal cells. This adherence step could be the basis for any host specificity via the production of colonization factors, such as the bundle-forming pilus adhesin of classical human EPEC strains.Low et al. analyzed 14 putative fimbrial gene clusters revealed by the EHEC O157:H7 Sakai sequence (21). Of these 14 putative fimbriae, several had already been described under other names, including LpfA1 (42), LpfA2 (43), F9 (20), type 1 fimbriae (32) (34), and curli fimbriae (30). Long polar fimbria (Lpf)-encoding genes had also been described previously, including lpfA_O26 and lpfA_O113, described by Toma et al. (41) and Doughty et al. (12), respectively.In addition, other putative adhesins have been described, as follows: a 67-kDa adherence-conferring protein (Iha) similar to Vibrio cholerae IrgA confers the capacity to adhere to epithelial cells in a diffuse pattern (38); Efa1 (EHEC factor for adherence), described by Nicholls et al. (27), mediates the binding of bacteria to CHO cells in vitro; ToxB, a protein encoded by a gene located on the 93-kb pO157 plasmid, is required for full adherence of the EHEC O157:H7 Sakai strain (39); Saa is an autoagglutinating adhesin identified in locus of enterocyte effacement-negative verotoxigenic E. coli strains (29); EibG is a protein responsible for the chain-like adherence phenotype of Saa-negative verotoxigenic E. coli strains (22); Paa (porcine attaching and effacing-associated) adhesin, described by An et al. (1), is involved in the early steps of the adherence mechanism of porcine EPEC strains (2); and the hemorrhagic coli pilus (HCP), whose inactivation of the main subunit (hcpA gene) reduces adherence to cultured human intestinal and bovine renal epithelial cells and to porcine and bovine gut explants, was observed in EHEC O157:H7 (46).The aim of this study was to establish the prevalence in bovine and human EPEC and EHEC strains belonging to the O26 serogroup of a total of 23 putative adhesins previously described for EHEC strains and of four fimbrial and afimbrial adhesins associated with bovine necrotoxigenic E. coli (NTEC) (36). The presence of these genes was correlated, on the one hand, with the source of isolation, and on the other hand, with EHEC/EPEC virulence factors (eae, stx₁, stx₂, and EHEC hlyA).     

Serotype, antimicrobial resistance, and adherence properties of Escherichia coli strains associated with outbreaks of diarrheal illness in children in the United States.

Since most recorded outbreaks of diarrhea in U.S. infants attributed to Escherichia coli occurred before currently available pathogenicity assays existed, we examined the characteristics of nonenterotoxigenic E. coli strains isolated from 50 outbreaks of diarrheal disease in U.S. infants between 1934 and 1987. We assayed the strains for enteropathogenic E. coli (EPEC) serotype, localized adherence (LA) and diffuse adherence to tissue cultures, the presence of EPEC adherence factor genes, Shiga-like (Vero) toxin production, and antimicrobial resistance. EPEC serotypes were identified in 28 outbreaks (56%). LA to HeLa cells was found in 23 outbreak strains and correlated 100% with the EPEC adherence factor probe. LA was observed in 21 of 28 EPEC and 2 of 22 non-EPEC strains; however, 5 of 23 strains that were LA positive for HeLa cells did not adhere to HEp-2 or HL cells. One strain was diffuse adherence positive, and none was Shiga-like toxin positive. Multiple resistance was common in EPEC (64%), LA-positive (74%), and LA-positive EPEC (76%) strains but not in others (10%). EPEC serotypes or LA was found in 60% (n = 30) of the outbreak strains. The remaining E. coli strains may represent nonpathogenic normal flora, as-yet-undefined pathogens, or pathogens that have lost virulence-associated traits during storage or subculturing.