Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.     

Comparison of ruminant and human attaching and effacing Escherichia coli (AEEC) strains

The presence of 12 genes associated with virulence in human attaching and effacing Escherichia coli (AEEC) was studied within a collection of 20 enterohemorrhagic E. coli (EHEC) and 206 atypical enteropathogenic E. coli (EPEC) isolated from ruminants. In addition, virulence genes and the clonal relationship of 49 atypical EPEC O26 strains isolated from humans and ruminants were compared to clarify whether ruminants serve as a reservoir of atypical EPEC for humans. A great diversity in the content of virulence gene was found. Thus, the espH, espG and map genes were detected in more than 85% of ruminant AEEC strains; the tccP2, espI, efa1/lifA, ehxA and paa genes were present in 50-70% of strains; and other genes such as tccP, espP, katP and toxB were detected in <25% of strains. EHEC strains contained more virulence genes than atypical EPEC strains. Our results suggest for the first time that the efa1/lifA gene is associated with diarrhea in newborn ruminants and that the AEEC strains with the H11 flagellar antigen are potentially more virulent than the non-H11 AEEC strains. Importantly, we identified a new intimin variant gene, eaeρ, in three ruminant atypical EPEC strains. The comparison of ruminant and human EPEC O26 strains showed that some ruminant strains possess virulence gene profiles and pulse-field gel electrophoresis pulsotypes similar to those of human strains. In conclusion, our data suggest that atypical EPEC is a heterogeneous group with different pathogenic potential and that ruminants could serve as a reservoir of atypical EPEC for humans.     

Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome

Enteropathogenic Escherichia coli (EPEC) previously were identified in poult enteritis-mortality syndrome (PEMS)-affected turkeys and associated as a cause of this disease. In the present study, the prevalence of EPEC in PEMS-affected turkeys was examined retrospectively with archived tissues and intestinal contents collected from 12 PEMS-affected turkey flocks in 1998. Formalin-fixed intestinal tissues were examined by light and electron microscopy for attaching and effacing (AE) lesions characteristic of EPEC, and frozen (-75 C) intestinal contents were examined for presence of EPEC. Escherichia coli isolates were characterized on the basis of epithelial cell attachment, fluorescent actin staining (FAS) test, and presence of E. coli attaching/effacing (EAE), shigalike toxin (SLT) type I, SLT II, and bundle-forming pilus (BFP) genes by polymerase chain reaction procedures. EPEC isolates were examined for pathogenicity and ability to induce AE lesions in experimentally inoculated young turkeys. AE lesions were identified by light microscopy in Giemsa-stained intestines from 7 of 12 PEMS-affected turkey flocks. Lesions consisted of bacterial microcolonies attached to epithelial surfaces with epithelial degeneration at sites of attachment and inflammatory infiltration of the lamina propria. Electron microscopy confirmed the identity of AE lesions in six of seven flocks determined to have AE lesions by light microscopy. EPEC were identified in 4 of 12 flocks on the basis of the presence of EAE genes a nd absence of SLT I and SLT II genes; all isolates lacked BFP genes. EPEC isolates produced AE lesions and variable mortality in turkeys coinfected with turkey coronavirus. In total, EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases on the basis of identification of AE lesions and/or EPEC isolates. These findings provide additional evidence suggesting a possible role for EPEC in the pathogenesis of PEMS.     

Infections with enteropathogenic Escherichia coli (EPEC) in cattle. 1. Comparison of different animal models and a cell culture system for the establishment of a detection system for "attaching and effacing" (AE) lesions]

In several animals species, enteropathogenic Escherichia coli (EPEC) were described as agents causing diarrhea. The histopathogenic pattern of EPEC is due to a typical adherence to enterocytes, called "attaching and effacing" (AE). This lesions are characterized by the formation of pedestals, cups and a marked loss of microvilli on enterocytes. In view of using an "in vitro system" (HeLa-cell culture) to test the adherence of EPEC, we first tested bovine EPEC in several laboratory animals. Various strains of mice, one day chicks (peroral) and a three day old calf (ligated intestinal loops) were inoculated with a bovine pathogenic EPEC (S 102-9). The adherence of EPEC "in vivo" was histologically, electron microscopically and bacteriologically investigated and compared to adherence to HeLa cell cultures. AE-lesions were found on calf enterocytes as well as on HeLa-cells, no lesions were seen in mice and chicks. The ligated intestinal loop test seems to be a useful model to compare "in vivo" to "in vitro" adherence.     

Incidence of common DNA sequences in bovine and porcine Escherichia coli strains causing diarrhoea

Seventeen bovine and 56 porcine Escherichia coli isolates from cases of diarrhoea and from healthy animals were examined for DNA sequences homologous to the genes for verocytotoxins (VT), enterotoxins and human enterohaemorrhagic E coli/enteropathogenic E coli (EHEC EPEC) sequences. VT-1 was the most common toxin among the bovine isolates and VT-2 the most common in the porcine isolates. No isolates had homologous sequences to enteropathogenic adherence factor, but 71.2 per cent hybridised to the DNA probe encoding specific EHEC sequences, and 95.9 per cent showed homology with a 23 kb DNA fragment common to EHEC and EPEC plasmids.     

Colonisation of conventional weaned pigs by enteropathogenic Escherichia coli (EPEC) and its hazard potential for human health

Enteropathogenic Escherichia coli (EPEC) bacteria frequently cause severe enteric diseases primarily in children and in young rabbits. Their pathogenicity for pigs has been tested by oral infection of colostrum-deprived newborn, and of severely immunosuppressed weaned pigs, but colonisation of conventional weaned pigs by porcine EPEC has not been experimentally studied. EPEC show similarities to enterohaemorrhagic E. coli (EHEC) additionally carrying shiga toxin genes integrated into the chromosome by lambdoid phages. We have demonstrated earlier that the porcine EPEC prototype strain P86-1390 (O45) could be transduced in vivo (in ligated loops of weaned pigs), by Stx2 phage derived from a human EHEC. Thus, the ability of this porcine EPEC strain to colonise conventional weaned pigs under farming conditions became a question of relevance to human health. To clarify this question, four intragastric infection experiments were performed on a total of 95 conventional weaned pigs. The EPEC P86-1390 and other well-characterised porcine EPEC strains were applied to 54 pigs, leaving 41 weaned pigs as negative controls. In three experiments moderate predispositions were applied: coinfections with enterotoxigenic E. coli (ETEC) or with low-virulence TGE coronavirus, application of fumonisin B1 with a normal therapeutic dose of dexamethasone, and the increase of soybean protein concentration in the feed. A total of 41 weaned pigs served as negative controls inoculated with a commensal porcine E. coli. Housing conditions simulated the farm environment. As an overall result, ileal segments of 18.5% of infected pigs were shown to be colonised by EPEC, while no EPEC were detected in the ilea of controls. Among predisposing factors occurring on farms, feed protein content increased by 20% (26.3% crude protein, provided by 48% soybean meal) seemed to enhance EPEC colonisation and resulted in the mobilisation of spontaneous latent EPEC/ETEC infection. The results indicate that under normal farm conditions porcine EPEC may colonise conventional weaned pigs by inducing ileal attaching effacing (AE) lesions with reasonable frequency, without clinical signs. The results also suggest that conventional weaned pigs may represent undetected reservoirs of porcine EPEC, potentially giving rise to the emergence of new types of EHEC due to natural transduction by Stx phages.     

Allelic types of long polar fimbriae in bovine and human Escherichia coli O157 strains

Long polar fimbriae (Lpf) are recently discovered adhesins and increasingly important genetic markers of pathogenic Escherichia coli strains. The presence and genotype diversity of Lpf operons was screened in a collection of 97 Escherichia coli O157 strains representing different pathotypes, isolated from healthy cattle (n = 43) and human patients (n = 54) in several countries. Individual structural genes of Lpf were scanned by PCR, and allelic variants were detected with a recently developed typing scheme. Ninety-five strains carried at least one whole Lpf operon (genes lpfABCD and/or lpfABCDE). The 64 enterohaemorrhagic (EHEC) and 24 enteropathogenic (EPEC) strains all carried two Lpf operons, allele 3 of lpfA1 and allele 2 of lpfA2, a combination characteristic of the O157:H7/NM serotype. Out of the 9 bovine atypical (AT; stx-, eae-) strains, 7 carried one complete Lpf operon, allele 1 of lpfA2. The atypical strains belonged to main phylogenetic groups A and B1, while the EHEC and EPEC strains were from group D. Lpf variants carried by the 72 strains of the Escherichia coli Reference Collection (ECOR) were determined with the same typing scheme. Alleles were detected in 25 strains, of which 6 were found negative for the respective Lpf operons in earlier studies. The marker value of the Lpf allelic combination for the O157:H7/NM serotype was confirmed, and further evidence was given for the presence of at least two different genetic lineages of atypical bovine E. coli O157 strains.     

Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea

Nineteen Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups were isolated from calves with diarrhea in Paraná State, Brazil, and studied for virulence markers associated with EPEC or enterohemorrhagic E. coli (EHEC). The 19 isolates belonged to 12 serotypes with isolates of O26:H11, O119:H25 and O114:H⁻ being the most prevalent. Localized adherence (LA) was demonstrated for 37% of the isolates, consisting of all four O26:H11, both O114:H⁻ and one O114:H40 isolates. All the LA strains were positive in the fluorescent-actin staining (FAS) test and possessed attaching-effacing E. coli (eae) sequences, but only O114 strains hybridized with the EPEC adherence factor (EAF) probe. None of the strains produced Shiga-like toxins (Verotoxin). Only the O26:H11 strains hybridized with the EHEC plasmid specific (CVD419) probe and were enterohemolytic, properties associated with EHEC strains. This investigation demonstrates that among the bovine strains isolated only those of serogroup O114 behaved as typical EPEC.     

Initial adherence of EPEC,EHEC and VTEC to host cells

Initial adherence to host cells is the first step of the infection of enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC) and verotoxigenic Escherichia coli (VTEC) strains. The importance of this step in the infection resides in the fact that (1) adherence is the first contact between bacteria and intestinal cells without which the other steps cannot occur and (2) adherence is the basis of host specificity for a lot of pathogens. This review describes the initial adhesins of the EPEC, EHEC and VTEC strains. During the last few years, several new adhesins and putative colonisation factors have been described, especially in EHEC strains. Only a few adhesins (BfpA, AF/R1, AF/R2, Ral, F18 adhesins) appear to be host and pathotype specific. The others are found in more than one species and/or pathotype (EPEC, EHEC, VTEC). Initial adherence of EPEC, EHEC and VTEC strains to host cells is probably mediated by multiple mechanisms.     

Polymerase chain reaction typing of genes of the locus of enterocyte effacement of ruminant attaching and effacing Escherichia coli

The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain’s belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.     

Domestic Cats Constitute a Natural Reservoir of Human Enteropathogenic Escherichia coli Types

Feces of 70 diarrhoeic and 230 non‐diarrhoeic domestic cats from São Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non‐diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae‐θ (three strains), eae‐κ (n = 3), eae‐α1 (n = 2), eae‐ι (n = 2), one eae‐α2, eae‐β1 and eae‐η each, and two were not typeable. The majority of the EPEC isolates adhered to HEp‐2 cells in a localized adherence‐like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.     

Phylogenetic background of attaching and effacing <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from animals

Detection and distribution of eae gene in forty-four attaching and effacing Escherichia coli (AEEC) strains of animal origin were investigated. Association of distinct intimin alleles with phylogenetic background were assessed among strains in comparison with different serogroups. Phylogenetic analysis showed that 31 EHEC/eae+ STEC strains belong to groups A, B1 and E, 13 EPEC strains segregated in B1 and B2. Moreover, group A possessed the eae gamma2/theta type, group B1 the eae beta1, eae kappa, eae zeta, and eae epsilon types, group B2 the eae alpha1, eae alpha2 and eae iota types, while the group E possessed the eae gamma1 type. The presence of numerous eae-types show that EPEC and EHEC/eae+ STEC tested have a high genetic homology within each phylogenetic group.     

Antibiotic resistance,ESBL genes,integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum β-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1–15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for bla_TEM and bla_CTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.     

Enhancement of enteropathogenic Escherichia coli pathogenicity in young turkeys by concurrent turkey coronavirus infection

In a previous study, turkey coronavirus (TCV) and enteropathogenic Escherichia coli (EPEC) were shown to synergistically interact in young turkeys coinfected with these agents. In that study, inapparent or mild disease was observed in turkeys inoculated with only TCV or EPEC, whereas severe growth depression and high mortality were observed in dually inoculated turkeys. The purpose of the present study was to further evaluate the pathogenesis of combined TCV/EPEC infection in young turkeys and determine the role of these agents in the observed synergistic interaction. Experiments were conducted to determine 1) effect of EPEC dose, with and without concurrent TCV infection, and 2) effect of TCV exposure, before and after EPEC exposure, on development of clinical disease. Additionally, the effect of combined infection on TCV and EPEC shedding was determined. No clinical sign of disease and no attaching and effacing (AE) lesions characteristic of EPEC were observed in turkeys inoculated with only EPEC isolate R98/5, even when turkeys were inoculated with 10(10) colony forming units (CFU) EPEC (high dose exposure). Only mild growth depression was observed in turkeys inoculated with only TCV; however, turkeys inoculated with both TCV and 10(4) CFU EPEC (low dose exposure) developed severe disease characterized by high mortality, marked growth depression, and AE lesions. Inoculation of turkeys with TCV 7 days prior to EPEC inoculation produced more severe disease (numerically greater mortality, significantly lower survival probability [P < 0.05], increased frequency of AE lesions) than that observed in turkeys inoculated with EPEC prior to TCV or simultaneously inoculated with these agents. Coinfection of turkeys with TCV and EPEC resulted in significantly increased (P < 0.05) shedding of EPEC, but not TCV, in intestinal contents of turkeys. These findings indicate that TCV infection predisposes young turkeys to secondary EPEC infection and potentiates the expression of EPEC pathogenicity in young turkeys.     

Ingestion and killing of Streptococcus agalactiae by bovine granulocytes in the presence of natural opsonins

An enzyme-linked immunosorbent assay (ELISA) demonstrated the presence of naturally acquired antibodies against Streptococcus agalactiae in normal bovine serum (NBS). In milk wheys, ELISA values were much lower than in sera. Pre-colostral calf serum (PCS) was shown to lack antibodies to type II and III S. agalactiae. The opsonic requirements of 10 human and 10 bovine strains were investigated by evaluating the phagocytosis-induced reduction of the incorporation of radiolabeled thymidine by streptococci. Antibodies present in NBS were required for the efficient ingestion of both human and bovine isolates type II by bovine granulocytes. Three out of five type III bovine isolates were opsonized in the absence of specific antibodies (opsonization by PCS) and type II and III bovine isolates did not require complement opsonization. By contrast, inactivation of complement reduced phagocytosis of human isolates and only one type III strain of human origin was opsonized by PCS. These findings suggest that human isolates had higher opsonic requirements. The phagocytic killing of 6 type III strains (5 mastitis isolates and the reference typing strain) was investigated. Opsonization by normal serum enabled bovine blood granulocytes to ingest and kill S. agalactiae. Nevertheless, greater than or equal to 35% of bacteria remained viable at the end of the phagocytosis incubation in 10% NBS. Heat treatment of serum decreased the efficacy of killing for only 3 of the 6 tested strains. An IgG2 fraction of normal adult bovine serum promoted active ingestion, which was still increased in the presence of PCS. Normal wheys displayed large variations in their ability to promote ingestion of S. agalactiae by blood granulocytes. The promoting effect was systematically less than that of serum from the same cow, and this can be related to the lower ELISA values found in wheys.     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.     

High prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 from cattle in selected regions of Japan

The prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 was examined in bovine faeces. EHEC O157 was isolated from the faeces of 42 (13.0%) of 324 cattle. Of the 4 farms and the facilities tested, the 3 farms and the facilities were found positive for EHEC O157. The highest isolation rate among the farms was 33.7%. The prevalence of EHEC O157 in heifers was higher than that in calves and other cattle. No cattle positive for EHEC O157 showed any clinical signs except 2 calves with diarrhea in a veterinary hospital. Almost all isolates possessed the stx gene, and Stx-positive strains carrying both stx(1) and stx(2) genes were predominant. These results indicate that EHEC O157 are distributed in bovine faeces, and that dairy and beef farms in selected regions of Japan are heavily contaminated with the organisms.     

The possible influence of LuxS in the in vivo virulence of rabbit enteropathogenic Escherichia coli

Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.     

Secretion of virulence factors by Escherichia coli.

In order to interact with their host, pathogenic strains of E. coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease. Since E. coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes. E. coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems. In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes. It is a one-step mechanism. The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage. In E. coli, the best studied type I secreted protein is haemolysin. In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence. The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV). The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second. The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins. The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells. Such a system is found in attaching and effacing E. coli and in diffusely adhering E. coli.