Atypical Enteropathogenic Escherichia Coli Strains Research Articles

Molecular genotyping of Escherichia coli o111 isolated from children with diarrhea in rural and urban areas

Introduction: Enteropathogenic Escherichia coli (EPEC) is a group of bacteria associated with infantile diarrhea. EPEC is defined as being typical (tEPEC) or atypical (aEPEC) depending on whether it presents the EPEC adherence factor (EAF) plasmid or not; tEPEC presents EAF. Objective. Carry out a retrospective study of E. coli strains isolated from children with diarrhea in both rural and urban areas to determine if they belong to tEPEC or aEPEC groups. Material & Methods: Strains that had been reported to be E. coli were analyzed by PCR using specific primers and 188 anti-O sera and 53 anti-H sera. The presence of eae, bfp, eafA, perA, espC and espP genes associated with virulence was analyzed, as was the presence of arpA, chuA, yjaA and TspE4 to define phylogroups. Results: Fifty-three cultures of E. coli isolated between 1985 and 2003 from 35 children under five years of age with severe diarrhea were analyzed. Twelve (34.3%) children came from a rural area and 23 (65.7%) from an urban area. Serologic typing identified the following serotypes: O111ab:NM (67.9%), O111ab:H2 (20.8%), O111ab:H12 (9.4%) and O111ac:H8 (1.9%). The presence of eae was positive in all 53 strains, while bfp and perA was detected in 38 (71.7%) strains and eaf in 35 (66%) strains. The combination of eae/bfp used to determine typical strains was positive in 38 (71.7%) strains and negative in 15 (28.3%) strains. Analysis by year and region showed that between 1985 and 1987, 35 (66%) strains were typical and 4 (7%) atypical in the rural area and urban area1. The same analysis between 1994 and 2003 in the urban areas showed that 11 (20.8%) strains were atypical, which were eae positive but eaf/bfp/perA negative, and 3 (5.7%) strains were typical. Phylogenetic grouping identified B1 and A in 38 (71.7%) and 9 (17%) strains respectively, and 2 (3.8%) each of B2, D and F. Conclusion. Typical EPEC strains were more prevalent in both the rural and urban areas between 1985 and 1987. In contrast, the presence of atypical EPEC strains was more prevalent in urban areas in more recent years. Analysis showed that the strains fell predominantly into commensal phylogroups.

Characterization of Antimicrobial Susceptibility, Extended-Spectrum β-Lactamase Genes and Phylogenetic Groups of Enteropathogenic Escherichia coli Isolated from Patients with Diarrhea.

ObjectivesInfectious diarrhea is one of the most common causes of pediatric death worldwide and enteropathogenic Escherichia coli (EPEC) is one of the main causes. There are 2 subgroups of EPEC, typical and atypical, based on the presence or absence of bundle forming pili (bfp), of which atypical EPEC is considered less virulent, but not less pathogenic. Antimicrobial resistance towards atypical EPEC among children is growing and is considered a major problem. In this study the pattern of antibiotic resistance in clinical isolates was determined.MethodsUsing 130 isolates, antibiotic resistance patterns and phenotypes were assessed, and genotypic profiles of extended spectrum β-lactamase (ESBL) production using disc diffusion and PCR was carried out. Phylogenetic groups were analyzed using quadruplex PCR.ResultsThere were 65 E. coli isolates identified as atypical EPEC by PCR, among which the highest antibiotic resistance was towards ampicillin, followed by trimethoprim-sulfamethoxazole, and tetracycline. Multidrug resistance was detected in 44.6% of atypical EPEC isolates. Around 33% of isolates were determined to be extended spectrum β-lactamase producers, and in 90% of isolates, genes responsible for ESBL production could be detected. Moreover, the majority of atypical EPEC strains belonged to Group E, followed by Groups B1, B2 and C.ConclusionHigh rates of multidrug resistance and ESBL production among atypical EPEC isolates warrant periodical surveillance studies to select effective antibiotic treatment for patients. It is considered a critical step to manage antibiotic resistance by avoiding unnecessary prescriptions for antibiotics.

Genetic and Virulence Characteristics of a Hybrid Atypical Enteropathogenic and Uropathogenic Escherichia coli (aEPEC/UPEC) Strain.

Hybrid strains of Escherichia coli combine virulence traits of diarrheagenic (DEC) and extraintestinal pathogenic E. coli (ExPEC), but it is poorly understood whether these combined features improve the virulence potential of such strains. We have previously identified a uropathogenic E. coli (UPEC) strain (UPEC 252) harboring the eae gene that encodes the adhesin intimin and is located in the locus of enterocyte effacement (LEE) pathogenicity island. The LEE-encoded proteins allow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) to form attaching and effacing (A/E) lesions in enterocytes. We sought to characterize UPEC 252 through whole-genome sequencing and phenotypic virulence assays. Genome analysis unveiled that this strain harbors a complete LEE region, with more than 97% of identity comparing to E2348/69 (EPEC) and O157:H7 Sakai (EHEC) prototype strains, which was functional, since UPEC 252 expressed the LEE-encoded proteins EspB and intimin and induced actin accumulation foci in HeLa cells. Phylogenetic analysis performed comparing 1,000 single-copy shared genes clustered UPEC 252 with atypical EPEC strains that belong to the sequence type 10, phylogroup A. Additionally, UPEC 252 was resistant to the bactericidal power of human serum and colonized cells of the urinary (T24 and HEK293-T) and intestinal (Caco-2 and LS174T) tracts. Our findings suggest that UPEC 252 is an atypical EPEC strain that emerges as a hybrid strain (aEPEC/UPEC), which could colonize new niches and potentially cause intestinal and extraintestinal infections.

Captive wildlife from India as carriers of Shiga toxin-producing, Enteropathogenic and Enterotoxigenic <i>Escherichia coli</i>

Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) make up an important group of pathogens causing major animal and public health concerns worldwide. The aim of this study was to determine the prevalence of different pathotypes of E. coli in captive wildlife. We analyzed 314 fresh fecal samples from captive wildlife, 30 stool swabs from animal caretakers, and 26 feed and water samples collected from various zoological gardens and enclosures in India for the isolation of E. coli, followed by pathotyping by multiplex PCR. The overall occurrence rate of E. coli was 74.05% (274/370). The 274 E. coli isolates were pathotyped by multiplex PCR targeting 6 genes. Of them, 5.83% were pathotyped as EPEC, 4.74% as STEC, and 1.09% as ETEC. The 16S rRNA genes from the selected isolates were amplified, sequenced, and a phylogenetic tree was constructed. The phylogenetic tree exhibited indiscriminate genetic profiling and some isolates from captive wild animals had 100% genetic identity with isolates from caretakers, suggesting that captive wildlife may serve as a reservoir for infection in humans and vice-versa. The present study demonstrates for the first time the prevalence of these E. coli pathotypes in captive wildlife in India. Our study suggests that atypical EPEC strains are more frequent than typical EPEC strains in captive wildlife. Discovering the implications of the prevalence of these pathotypes in wildlife conservation is a challenging topic to be addressed by further investigations.

Typical & atypical enteropathogenic Escherichia coli in diarrhoea & their role as carrier in children under five.

Background & objectives:Multidrug-resistant enteropathogenic Escherichia coli (EPEC) is responsible for a large number of cases of infantile diarrhoea in developing countries, causing failure in treatment with consequent health burden and resulting in a large number of deaths every year. This study was undertaken to determine the proportion of typical and atypical EPEC in under five children with diarrhoea and controls, their function as a carriage and to identify virulent genes associated with them.Methods:During the study period, 120 stool samples including 80 from controls children were collected and analyzed for the presence of EPEC using standard bacteriological methods. Isolates were subjected to antimicrobial testing by disc diffusion method. Isolates confirmed as E. coli by phenotypic method were further tested for the presence of attaching and effacing (eae) and bundle-forming pilus (bfpA) genes by real-time SYBR Green-based polymerase chain reaction.Results:All isolates were tested for the presence of EPEC. The frequency of typical EPEC was 20 and 16.25 per cent whereas the frequency of atypical EPEC strains was 5 and 23.75 per cent in patients and controls, respectively (P< 0.05) and bfpA was seen in 45 and 18.75 per cent isolates of diarrhoeal patients and controls, respectively.Interpretation & conclusions:Our results showed that typical EPEC was a common cause of diarrhoea, but at the same time, atypical EPEC was emerging as colonizers in the intestine of children with and without diarrhoea in and around Delhi. Children can be considered asymptomatic carriers of these pathogens and can transmit them to other susceptible children. Adequate steps need to be taken to stop these strains from developing and spreading further.

Virulence factors and antimicrobial resistance in Escherichia coli strains isolated from hen egg shells

Eggs may contain extraintestinal pathogenic (ExPEC) and diarrheogenic (DEC) Escherichia coli which in addition may carry antibiotic resistance. The wide use of biocides and disinfectants in the food industry may induce biocide tolerance in bacteria. The aim of the present study was to evaluate biocide tolerance and antibiotic resistance in E. coli from hen egg shells. A total of 27 isolates obtained from a screening of 180 eggs were studied. Seven isolates carried both eae and bfpA genes of typical enteropathogenic E. coli (EPEC) strains, while 14 isolates only carried eae associated with atypical EPEC strains. Shiga toxin genes stx and stx2 were detected in four isolates. Heat-stable and heat-labile enterotoxin genes as well as aggR were also detected. Several isolates had minimum inhibitory concentrations (MICs) that were higher than the wild-type for the biocide hexadecylpyridinium chloride (HDP, 18.52%) or the commercial disinfectant P3 oxonia (OX, 14.81%). Antibiotic resistance was detected for ampicillin (37.03%), streptomycin (37.03%), tetracycline (37.03%), chloramphenicol (11.11%), nalidixic acid (18.51%) and trimethoprim-sulfamethoxazole (14.81%). Eight isolates (29.63%) were biocide tolerant and antibiotic resistant. Efflux pump genes detected included acrB (96.29%), mdfA (85.18%) and oxqA (37.03%), in addition to quaternary ammonium compound (QAC) resistance genes qacA/B (11.11%) and qacE (7.40%). Antibiotic resistance genes detected included blaCTX-M-2 (22.22%), blaTEM (3.70%), blaPSE (3.70%), tet(A) (29.63%), tet(B) (29.63%), tet(C) (7.40%), tet(E) (11.11%), aac(6′)-Ib (3.70%), sul1 (14.81%), dfrA12 (3.70%) and dfrA15 (3.70%). Most isolates (96.30%) carried more than one genetic determinant of resistance. The most frequent combinations were efflux pump components acrB and mdfA with tetracycline resistance genes (33.33% of isolates). Isolates carrying QAC resistance genes also carried between 4 and 8 of the additional antimicrobial resistance genes investigated. Regardless of biocide tolerance and antibiotic resistance, all isolates were sensitive to carvacrol (0.25%), thymol (0.125%) and trisodium phosphate (1 to 1.5%), but they exhibited a heterogeneous response to sodium lactate and lysozyme-EDTA combinations that apparently were not related with antibiotic resistance. Results from the study reveal not only a low incidence of biocide tolerance but also the presence of multiple resistance strains carrying multiple genetic determinants of resistance.

Isolation of atypical enteropathogenic Escherichia coli from chicken and chicken-derived products

Atypical enteropathogenic Escherichia coli (EPEC) strains from chicken and chicken-derived products were isolated and characterised.The strains presented a wide variety of serotypes, some have been reported in other animal species (O2:H40, O5:H40) and in children with diarrhoea (O8:H-). Most of the strains carried intimin β.The results indicate that chicken and chicken products are important sources of atypical EPEC strains that could be associated with human disease, and highlight the need to improve hygiene practices in chicken slaughtering and meat handling.

Isolation of atypical enteropathogenic and shiga toxin encoding Escherichia coli strains from poultry in Tehran, Iran.

The purpose of this study was to investigate the prevalence of enteropathogenic Escherichia coli (EPEC) and shiga toxin producing E. coli (STEC) strains in healthy broilers in Iran. STEC and EPEC strains as diarrheagenic E. coli are among the most prevalent causative agents in acute diarrhea. Domestic animals, mainly cattle and sheep, have been implicated as the principal reservoirs of these pathotypes; however their prevalence among the broilers is varied among different countries. A total of 500 cloacal swab samples from broilers of five different poultry houses (A-E) were collected to investigate the presence of stx1, stx2, hly, eae, and bfp virulence genes among the E. coli isolates by polymerase chain reaction. The shiga toxin encoding strains were evaluated serologically to detect their interaction with a commercial antiserum against O157 antigen. Out of the 500 collected samples, 444 E. coli strains were isolated. Three strains (0.67%) presented at least one of the studied virulence genes (stx2, hly and eae), two strains were identified as STEC (stx2 (+), O157:nonH7) and one as an atypical EPEC strains (eae (+) bfp (-)). The study established the presence of STEC and atypical EPEC in healthy broilers in Iran. Poultry might serve as vectors for transmission of pathogenic E. coli to human populations.

Investigation of enteropathogenic Escherichia coli and Shiga toxin-producingEscherichia coli associated with hemolytic uremic syndrome in İzmir Province, Turkey.

The purpose of this study was to investigate Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains originating from diarrheagenic patients. A total of 102 patients with diarrhea between October 2012 and January 2013 were enrolled in this study. Multiplex and standard polymerase chain reactions were performed to detect and distinguish STEC and EPEC strains. O serotyping of EPEC was carried out by monovalent antisera. The O and H serotyping of STEC strains was performed at the Refik Saydam Institute, Ankara. A total of 5 (3.42%) strains were identified as STEC, and 3 strains (2.05%) were atypical EPEC. One of the STEC serotypes was O157:H7 carrying VT1, Stx1A, and escv genes. The other STEC strain was identified as O174:H21, which is associated with hemolytic uremic syndrome and consists of VT2 and Stx2A genes. One of the EPEC and three of the STEC serotypes were nontypeable. The serotypes of the atypical EPEC strains were identified as O114 and O26. To the best of our knowledge, this is the first report of O174:H21 from the İzmir region that was shown to be a Shiga toxin-producing non-O157 serotype of STEC.

Phenotypic and genotypic characteristics associated with biofilm formation in clinical isolates of atypical enteropathogenic Escherichia coli (aEPEC) strains.

BackgroundBiofilm formation by enteropathogenic Escherichia coli (EPEC) have been recently described in the prototype typical EPEC E2348/69 strain and in an atypical EPEC O55:H7 strain. In this study, we sought to evaluate biofilm formation in a collection of 126 atypical EPEC strains isolated from 92 diarrheic and 34 nondiarrheic children, belonging to different serotypes. The association of biofilm formation and adhesin-related genes were also investigated.ResultsBiofilm formation occurred in 37 (29%) strains of different serotypes, when the assays were performed at 26°C and 37°C for 24 h. Among these, four strains (A79, A87, A88, and A111) formed a stronger biofilm than did the others. The frequency of biofilm producers was higher among isolates from patients compared with isolates from controls (34.8% vs 14.7%; P = 0.029). An association was found between biofilm formation and expression of type 1 fimbriae and curli (P < 0.05). Unlike the previously described aEPEC O55:H7, one aEPEC O119:HND strain (A111) formed a strong biofilm and pellicle at the air-liquid interface, but did not express curli. Transposon mutagenesis was used to identify biofilm-deficient mutants. Transposon insertion sequences of six mutants revealed similarity with type 1 fimbriae (fimC, fimD, and fimH), diguanylate cyclase, ATP synthase F1, beta subunit (atpD), and the uncharacterized YjiC protein. All these mutants were deficient in biofilm formation ability.ConclusionThis study showed that the ability to adhere to abiotic surfaces and form biofilm is present in an array of aEPEC strains. Moreover, it seems that the ability to form biofilms is associated with the presence of type 1 fimbriae and diguanylate cyclase. Characterization of additional biofilm formation mutants may reveal other mechanisms involved in biofilm formation and bring new insights into aEPEC adhesion and pathogenesis.

Characterization of virulence genes in typical and atypical enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea in infants and children in developing countries. The aim of this study was to investigate the frequency of EPEC in children with diarrhea by polymerase chain reaction (PCR) method targeting the eae gene. The presence of virulence gene including bfpA was also investigated by PCR, for the differentiation of typical and atypical EPEC strains. We also sought to determine the presence of virulence gene including efa-1, fimH and lpfDo113 of the isolated strains. Stool samples were collected from 313 less than 5 years old children with diarrhea at Shahid Beheshti Hospital, Kashan, Iran, from November 2009 to May 2010. Specimens were examined by PCR test to determine the eae gene to detect of EPEC pathotype and also the frequency of genes coding virulence factors of EPEC such as bfpA, efa-1, fimH and lpfDo113 were analyzed. Results showed that 51 cases (28.6%) out of 178E. coli isolated were EPEC. The percentage of isolated EPECs carrying virulence genes were bfpA (tEPEC) (11.8%), efa1 (2%), fimH (98%) and lpfDo113 (13.7%). Variable frequency of EPEC virulence factors isolated from children with diarrhea needs further studies for creating epidemiological map of diarrheal infections caused by EPEC.   Key words: Enteropathogenic Escherichia coli, virulence genes, diarrhea, children, polymerase chain reaction.

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.

Virulence gene profiling of enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains: a basis for molecular risk assessment of typical and atypical EPEC strains

BackgroundEnterohaemorrhagic E. coli (EHEC) can cause severe disease such as bloody diarrhoea and haemolytic uraemic syndrome in humans. Besides production of Shiga toxins, the presence of LEE (eae-gene) and non-LEE (nle) encoded effector genes harboured on O-islands OI-122, OI-71 and OI-57 is associated with EHEC virulence and their frequency in outbreaks. Genes encoded by the EHEC-plasmid are putative virulence markers of EHEC. EHEC-plasmids, LEE and non-LEE effector genes have also been detected in some strains of enteropathogenic E. coli (EPEC). The objective of this study was to analyze the relationship between EHEC and EPEC for virulence genes encoded by genomic O-islands and by the EHEC-plasmids.ResultsNle genes ent/espL2, nleB and nleE (OI-122), nleA, nleF and nleH1-2 (OI-71), nleG5-2 and nleG6-2 (OI-57), espK (CP-933N) and the EHEC-plasmid encoded genes ehxA, espP, etpD and katP were searched in 73 typical and in 235 atypical enteropathogenic E. coli (EPEC) strains. Typical and atypical EPEC each fall into two clusters. Cluster 1 typical (n = 46) and atypical (n = 129) EPEC strains were characterized by the presence of OI-122 encoded genes and grouped together with 64 investigated EHEC strains. Cluster 2 typical (n = 27) and atypical (n = 106) strains grouped together with 52 LEE-negative, Shiga toxin-producing E. coli (STEC) and with 21 apathogenic E. coli strains. Typical EPEC Cluster 1 strains belonged to serotypes frequently involved in severe illness and outbreaks in children (O111:H2, O114:H2, O55:H6, O127:H6 and O142:H6). Atypical EPEC Cluster 1 strains were characterized by serotypes related to EHEC (O26:H11, O55:H7, O145:H28, O103:H2 and O103:H25).ConclusionThe OI-122 encoded nleB gene was found to be most closely associated with Cluster 1 strains and may serve as a diagnostic tool for the identification of virulent EHEC and EPEC seropathotypes. OI-71 encoded genes nleA, nleF and nleH1-2 are less associated with Cluster 1 strains. EHEC-plasmid, OI-57 and CP-933 associated genes showed only weak similarities with virulent Cluster 1 EHEC and EPEC strains.

Molecular and phenotypic characterization of atypical enteropathogenic Escherichia coli serotypes isolated from children with and without diarrhea

We characterized 36 atypical enteropathogenic Escherichia coli (EPEC) serotypes isolated from children with and without diarrhea in Iran. Because the identification of atypical EPEC based on biochemical features is rather difficult and time consuming, we used a combination of three approaches, including a polymerase chain reaction-based method, culture adherence assay, and the restriction analysis of fliC gene (fliC-restriction fragment length polymorphism), to identify E coli serotypes. To distinguish typical and atypical EPEC strains, the presence of EPEC attaching effacing A gene (eaeA) gene and EPEC-attaching factor (EAF) plasmid were analyzed. All E coli strains were identified based on the detection of the eaeA(+), bundle-forming pili A gene (bfpA(-)), EAF(-) or eaeA(-), bfpA(+), EAF(-) profiles and the absence of stx (encoded for shiga toxin) gene as atypical EPEC. All strains studied belonged to 5 atypical EPEC serogroups and 15 serotypes based on the virulence profiles. Of 36 atypical EPEC serotypes, 22 (61.2%) and 14 (38.8%) strains isolated from diarrheal and healthy cases, respectively. O142:H48 (19.5%) and O111:H21 (11.1%) serotypes were the most prevalent isolates, followed by serotypes O111: H(-) and O86:H48 (5.6% each). The characteristics of the atypical EPEC serotypes from children with diarrhea were significantly different from those without diarrhea. The compilation of data on atypical EPEC strains presented here indicates the importance of a combined approach of conventional and molecular tests to study the virulence and epidemiology of EPEC serotypes in human subjects.

Isolation of atypical enteropathogenic Escherichia coli from children with and without diarrhoea in Delhi and the National Capital Region, India

A total of 17 typical and atypical enteropathogenic Escherichia coli (EPEC) were isolated from 396 children with and without diarrhoea. Out of 12 EPEC isolates from patients with diarrhoea, 3 (25 %) were atypical EPEC while 9 (75 %) were typical EPEC. It was observed that atypical EPEC strains had colonized the intestines of healthy children and its isolation rates were higher in healthy children than in children with diarrhoea. Interestingly all of the atypical EPEC isolates carried a megaplasmid, mostly comparable with the size of EPEC adherence factor (EAF) encoding gene but no virulence gene was detected in this megaplasmid. Studies also indicated that multidrug resistance EPEC are emerging and all the atypical EPEC strains showed significantly less resistance to all antimicrobial agents used in this study than typical EPEC. This study also supports the opinion that Shiga toxin-producing E. coli does not pose a major threat to human health in India. Subtyping analysis reveals that eae-α1, eae-β2 and eae-λ could be common EPEC subtypes prevalent in children with diarrhoea in Delhi. The present study is believed to be the first report of the detection of atypical EPEC from children without diarrhoea and records of isolation of eae-γ1, eae-γ2 and the rare eae-λ subtype in India. The data also indicated that typical EPEC are a common cause of diarrhoea and atypical EPEC are emerging as colonizers of the intestine of children with and without diarrhoea in Delhi and the National Capital Region, India.

Allelic variability of critical virulence genes (eae, bfpA and perA) in typical and atypical enteropathogenic Escherichia coli in Peruvian children

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infantile diarrhoea in developing countries. The aim of this study was to describe the allelic diversity of critical EPEC virulence genes and their association with clinical characteristics. One hundred and twenty EPEC strains isolated from a cohort diarrhoea study in Peruvian children were characterized for the allele type of eae (intimin), bfpA (bundlin pilin protein of bundle-forming pilus) and perA (plasmid encoded regulator) genes by PCR-RFLP. Atypical EPEC strains (eae+, bfp-) were the most common pathotype in diarrhoea (54/74, 73 %) and control samples from children without diarrhoea (40/46, 87 %). Overall, there were 13 eae alleles; the most common were beta (34/120, 28 %), theta (24/120, 20 %), kappa (14/120, 12 %) and mu (8/120, 7 %). There were five bfpA alleles; the most common were beta1/7 (10/26), alpha3 (7/26) and beta5 (3/26). There were three perA alleles: beta (8/16), alpha (7/16) and gamma (1/16). The strains belonged to 36 distinct serogroups; O55 was the most frequent. The gamma-intimin allele was more frequently found in diarrhoea episodes of longer duration (>7 days) than those of shorter duration (3/26, 12 % vs 0/48, 0 %, P<0.05). The kappa-intimin allele had the highest clinical severity score in comparison with other alleles (P<0.05). In Peruvian children, the virulence genes of EPEC strains are highly variable. Further studies are needed to evaluate additional virulence markers to determine whether relationships exist between specific variants and clinical features of disease.

Clonal Relationship among Atypical Enteropathogenic Escherichia coli Strains Isolated from Different Animal Species and Humans

Forty-nine typical and atypical enteropathogenic Escherichia coli (EPEC) strains belonging to different serotypes and isolated from humans, pets (cats and dogs), farm animals (bovines, sheep, and rabbits), and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. A close clonal relationship between human and animal isolates was found by MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out since the transmission dynamics between the reservoirs are not yet clearly understood.

The Flagella of an Atypical Enteropathogenic Escherichia coli Strain Are Required for Efficient Interaction with and Stimulation of Interleukin-8 Production by Enterocytes In Vitro

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.

Detection of Cytolethal Distending Toxin and Other Virulence Characteristics of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients in Republic of Korea

Cytolethal distending toxins (CDTs) represent an emerging family of newly described bacterial products that are produced by a number of pathogens. The genes encoding these toxins have been identified as a cluster of three adjacent genes, cdtA, cdtB, and cdtC, plus 5 cdt genetic variants, designated as cdt-I, cdt-II, cdt-III, cdt-IV, and cdt- V, have been identified to date. In this study, a general multiplex PCR system designed to detect Escherichia coli cdts was applied to investigate the presence of cdt genes among isolates. As a result, among 366 E. coli strains, 2.7% were found to carry the cdtB gene. In addition, the use of type-specific primers revealed the presence of cdt-I, cdtIV, and cdt-V types of the cdt gene, yet no cdt-II or cdt- III strains. The presence of other virulence genes (stx1, stx2, eae, bfp, espA, espB, and espD) was also investigated using a PCR assay. Among the 10 cdtB gene-positive strains, 8 were identified as CDT-producing typical enteropathogenic E. coli (EPEC) strains (eae(+), bfp(+)), whereas 2 were identified as CDT-producing atypical EPEC strains (eae(+), bfp(-)). When comparing the cytotoxic activity of the CDT-producing typical and atypical EPEC strains, the CDT-producing atypical EPEC strains appeared to be less toxic than the CDT-producing typical EPEC strains.

Contribution of the pst-phoU Operon to Cell Adherence by Atypical Enteropathogenic Escherichia coli and Virulence of Citrobacter rodentium

Strains of enteropathogenic Escherichia coli (EPEC) generally employ the adhesins bundle-forming pili (Bfp) and intimin to colonize the intestine. Atypical EPEC strains possess intimin but are negative for Bfp and, yet, are able to cause disease. To identify alternative adhesins to Bfp in atypical EPEC, we constructed a transposon mutant library of atypical EPEC strain E128012 (serotype O114:H2) using TnphoA. Six mutants that had lost the ability to adhere to HEp-2 cells were identified, and in all six mutants TnphoA had inserted into the pstSCAB-phoU (Pst) operon. To determine if the Pst operon is required for adherence, we used site-directed mutagenesis to construct a pstCA mutant of E128012. The resultant mutant showed a reduced ability to adhere to HEp-2 cells and T84 intestinal epithelial cells, which was restored by trans-complementation with intact pstCA. To determine if pst contributes to bacterial colonization in vivo, a pstCA mutation was made in the EPEC-like murine pathogen, Citrobacter rodentium. C57BL/6 mice infected perorally with the pstCA mutant of C. rodentium excreted significantly lower numbers of C. rodentium than those given the wild-type strain. Moreover, colonic hyperplasia and diarrhea, which are features of infections with C. rodentium, were not observed in mice infected with the pstCA mutant but did occur in mice given the trans-complemented mutant. As mutations in pst genes generally lead to constitutive expression of the Pho regulon, our findings suggested that the Pho regulon may contribute to the reduced virulence of the pstCA mutants. To investigate this, we inactivated phoB in the pstCA mutants of EPEC E128012 and C. rodentium and found that the phoB mutation restored the adherent phenotype of both mutant strains. These results demonstrate that Pst contributes to the virulence of atypical EPEC and C. rodentium, probably by causing increased expression of an unidentified, Pho-regulated adhesin.