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.     

A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.     

Shiga toxin-producing Escherichia coli and enteropathogenic Escherichia coli in healthy goats in India: occurrence and virulence properties

AIMS: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India. METHODS AND RESULTS: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively. CONCLUSIONS: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.     

肠致病性大肠杆菌O45基因缺失疫苗菌株的构建及其免疫原性分析

为得到肠致病性大肠杆菌O45弱毒疫苗候选菌株,以肠致病性大肠杆菌O45为出发菌株, 利用自杀性载体pCVD442和同源重组的原理构建了O45的ler基因缺失突变菌株PEPEC O45(Δler), 并对该菌株的Vero细胞毒性、小鼠模型的安全性以及乳鼠和乳猪的被动免疫保护作用进行了研究。结果表明, O45(Δler)基因缺失突变菌株丧失了对Vero细胞的毒性作用, 并丧失了对实验小鼠的致病性, 具有良好的安全性。乳鼠和乳猪被动免疫保护性实验表明, 用该菌株分别免疫母鼠和母猪后, 乳鼠和乳猪均可通过吸吮母乳可以获得良好的被动免疫保护作用。因此本研究所构建的O45(Δler)基因缺失突变弱毒菌株可作为预防PEPEC O45感染的疫苗候选株, 为最终研制出O45的基因工程菌苗奠定基础。     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Mice vaccinated with enteropathogenic Escherichia coli ghosts show significant protection against lethal challenges

Aim: To prepare enteropathogenic Escherichia coli (EPEC) E2348/69 ghosts and investigate whether immunization with EPEC bacterial ghosts can elicit protective immune responses. Methods and Results: A recombinant plasmid with double λPL/PR‐cI857 temperature‐sensitive regulatory cassettes was constructed. The lysis gene E and/or the staphylococcal nuclease A (SNA) gene were separately inserted downstream of the two regulatory cassettes to construct the lysis plasmids pBV220::E and pBV220::E::CI‐P‐SNA. An EPEC reference strain E2348/69 (serotype O127:H6) was transformed with the lysis plasmids to produce EPEC ghosts. Mice injected with bacterial ghosts EGE (EPEC ghosts produced using lysis protein E) or EGES (EPEC ghosts produced using a combination of lysis protein E and SNA) gained weight normally and showed no clinical signs of disease. Vaccination trials showed that mice immunized with EGE or EGES were significantly protected against subsequent challenge with the wild‐type virulent parent strain, EPEC E2348/69 (42/50 and 45/50 survival, respectively); in contrast, none of the 30 control mice survived. Conclusions: Immunization with EPEC ghosts can elicit protective immune responses in BALB/c mice. Significance and Impact of the Study: EPEC ghosts may represent a promising new approach for vaccination against EPEC infection.     

Classification of perA sequences and their correlation with autoaggregation in typical enteropathogenic Escherichia coli isolates collected in Japan and Thailand

Enteropathogenic Escherichia coli (EPEC) strains produce a bundle‐forming pilus (BFP) that mediates localized adherence (LA) to intestinal epithelial cells. The major structural subunit of the BFP is bundlin, which is encoded by the bfpA gene located on a large EAF plasmid. The perA gene has been shown to activate genes within the bfp operon. We analyzed perA gene polymorphism among typical (eae‐ and bfpA‐ positive) EPEC strains isolated from healthy and diarrheal persons in Japan (n= 27) and Thailand (n= 26) during the period 1995 to 2007 and compared this with virulence and phenotypic characteristics. Eight genotypes of perA were identified by heteroduplex mobility assay (HMA). The strains isolated in Thailand showed strong autoaggregation and had an intact perA, while most of those isolated in Japan showed weak or no autoaggregation, and had a truncated perA due to frameshift mutation. The degree of autoaggregation was well correlated with adherence to HEp‐2 cells, contact hemolysis and BFP expression. Our results showed that functional deficiency due to frameshift mutation and subsequent nonsense mutation in perA reduced BFP expression in typical EPEC strains isolated in Japan.     

致病性大肠埃希菌血清型分布及对抗生素的敏感性分析

目的了解临床病例中致病性大肠埃希菌的主要血清型和对抗生素的敏感性。方法致病性大肠埃希菌的鉴定使用血清学的方法,药敏试验采用纸片扩散法,WHONET 5.0软件分析药敏结果。结果致病性大肠埃希菌的检出率为5.93%,共分离到7种血清型。在分离到的菌株中,ESBLs的检出率达45%。结论致病性大肠埃希菌是引起小儿腹泻的一种重要致病菌,应开展对致病性大肠埃希菌的检测,根据药敏结果选用合适药物。     

Oral infection with enteropathogenic Escherichia coli triggers immune response and intestinal histological alterations in mice selected for their minimal acute inflammatory responses

Enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea, is an important public health problem in Brazil and other developing countries. In vitro assays of bacterial adhesion to cultured cells are important tools for studying bacterial pathogenicity but do not reproduce all the events that occur in natural infections. In this study, the effects of oral infection with EPEC on mice selected for their minimal acute inflammatory response (AIRmin) were evaluated. Mice were orally infected with EPEC and variations in body weight, bacterial shedding and antibody production observed. The infected animals developed seric and secretory anti‐EPEC antibodies; however, neither mortality nor diarrhea was observed. Light microscopy of their intestines demonstrated histological modifications that were not present in controls. However, electron microscopy did not show bacteria attached to the intestinal epithelia to form attaching and effacing lesions, characteristic of EPEC in humans. The bacteria were detected in Peyer's patches and intestinal contents up to 5 hr post‐infection. When human anti‐EPEC secretory immunoglobulin A or avian immunoglobulin Y antibodies were administered to infected animals, they developed minor histological alterations compared with non‐treated animals. In summary, it was found that EPEC triggers immune responses and intestinal histological alterations but does not produce evidence of diarrheal disease in mice infected by the oral route. This study of EPEC experimental infection provides a better understanding of the effects of antibodies on bacterial infections and may provide a suitable model for the design and testing of immunobiological products for active or passive immunization.     

An inhibitory mechanism of action of coiled‐coil peptides against type three secretion system from enteropathogenic Escherichia coli

Human pathogenic gram‐negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled‐coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha‐helical structures that play an important role in the protein‐protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled‐coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS‐dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking‐molecular dynamic simulations, and quantum‐mechanic calculations of the various peptide‐protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled‐coil domain of the C‐terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide‐protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad‐spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.     

Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Use of repetitive DNA sequences to determine the persistence of enteropathogenic Escherichia coli in vegetables and in soil grown in fields treated with contaminated irrigation water

AIMS: Fresh fruits and vegetables are increasingly recognized as vectors for food-borne illness. On farm contamination through contaminated irrigation water is considered likely source of the pathogen for several outbreaks. The purpose of this study is to investigate the possible similarity of strains of Escherichia coli isolated from the soil and vegetables irrigated by treated wastewater. METHODS AND RESULTS: Seventy-five strains of enteropathogenic Escherichia coli isolated from vegetables, soil and irrigation water were tested for sensitivity to antibiotics and shown to be sensitive. The result of enterobacterial repetitive intergenic consensus (ERIC)-PCR shows similarities between analysed strains isolated from the three different samples. Moreover strains of E. coli isolated from vegetables over different periods of time have the same ERIC-PCR profile. CONCLUSIONS: The isolated strains of enteropathogenic E. coli can persist in soil and in vegetables growing in fields treated with contaminated irrigation water for an extended period of time. SIGNIFICANCE AND IMPACT OF THE STUDY: Contaminated irrigation water can transport pathogenic bacteria, which persists in the soil for a long period of time and contaminates the vegetables growing in the field irrigated by this contaminated water.     

Bacteriophages: A possible solution to combat enteropathogenic Escherichia coli infections in neonatal goats

Due to awareness and benefits of goat rearing in developing economies, goats' significance is increasing. Unfortunately, these ruminants are threatened via multiple bacterial pathogens such as enteropathogenic Escherichia coli (EPEC). In goat kids and lambs, EPEC causes gastrointestinal disease leading to substantial economic losses for farmers and may also pose a threat to public health via the spread of zoonotic diseases. Management of infection is primarily based on antibiotics, but the need for new therapeutic measures as an alternative to antibiotics is becoming vital because of the advent of antimicrobial resistance (AMR). The prevalence of EPEC was established using bfpA gene, uspA gene and Stx1 gene, followed by phylogenetic analysis using Stx1 gene. The lytic activity of the isolated putative coliphages was tested on multi-drug resistant strains of EPEC. It was observed that a PCR based approach is more effective and rapid as compared to phenotypic tests of Escherichia coli virulence. It was also established that the isolated bacteriophages exhibited potent antibacterial efficacy in vitro, with some of the isolates (16%) detected as T4 and T4-like phages based on gp23 gene. Hence, bacteriophages as therapeutic agents may be explored as an alternative to antibiotics in managing public, livestock and environmental health in this era of AMR.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

Characterization of typical and atypical enteroaggregative Escherichia coli in Kagoshima,Japan: biofilm formation and acid resistance

EAEC is increasingly recognized as an emerging enteric pathogen. Typical EAEC expressing the AggR regulon have been proven to be an important cause of childhood diarrhea in industrialized countries as well as in the developing world, while atypical EAEC without this regulon have not been thoroughly investigated. To investigate the bacteriological characteristics of EAEC, including both typical and atypical strains in Kagoshima, Japan, 2417 E. coli strains from Japanese children with diarrhea were screened by a quantitative biofilm assay to detect possible EAEC strains, resulting in the identification of 102 (4.2%) of these strains by the HEp‐2 cell adherence test. Virulence gene patterns, PFGE analysis and O‐serogrouping demonstrated the heterogeneity of the EAEC. The EAEC strains were classified into two groups: typical EAEC with aggR (74.5%, 76/102) and atypical EAEC without aggR (25.5%, 26/102). There was no significant difference between the typical EAEC strains (median OD₅₇₀= 0.73) and the atypical strains (median OD₅₇₀= 0.61) in biofilm formation (P= 0.17). Incidences of resistance against ampicillin, cefotaxime and tetracycline were significantly higher in the typical EAEC strains than the atypical EAEC strains (84.2% vs. 53.8%, 36.8% vs. 7.7% and 93.4% vs. 73.1%, respectively, P < 0.05). The typical EAEC strains showed significantly higher resistance ratios against HCl and lactate than the atypical strains (94.7% vs. 61.5% and 92.1% vs. 57.7%, respectively, P < 0.001). To investigate the pathogenicity of not only typical but also atypical EAEC, further bacteriological and epidemiologic studies including atypical EAEC are needed.     

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle‐forming pilus expression

Aims: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle‐forming pilus (BFP) expression. Methods and Results: Anti‐BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X‐100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram‐negative type IV‐expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl₂, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. Conclusion: The assay enables reliable identification of BFP‐expressing isolates and contributes to the differentiation of typical and atypical EPEC. Significance and Impact of the Study: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.