Typical 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.

Molecular and phenotypic typing of enteropathogenic Escherichia coli isolated in childhood acute diarrhea in Abuja, Nigeria.

Enteropathogenic Escherichia coli (EPEC) causes infectious diarrhea among children in developing countries. However, in Nigeria, due to limited laboratory resources, the genetic diversity of its virulence factors, which includes its intimin subtypes remain undefined. This study aims to evaluate the serotypes, antibiotic resistance and the intimin subtypes genes associated with EPEC strains isolated from children with acute diarrhea in Abuja, Nigeria. EPEC isolates from diarrheic children aged 0-60 months in Abuja, Nigeria were analyzed. PCR for EPEC virulence gene, Hep-2 cell Adherence and serotyping were performed. EPEC strains were further subtyped by PCR reactions for the identification of intimin subtype genes: α (alpha), β (beta), γ1 (gamma-1), and έ (epsilon). Antibiotic resistance and ESBL production was determined by CLSI guidelines. Overall, 18 (4.5%) out of 400 children with acute diarrhea had EPEC infection. Typical EPEC strains were detected in 14 (3.5%) whereas 4 (1.1%) were atypical EPEC. Fifteen (83.3%) of the EPEC isolated belonged to β intimin subtype gene, while the remaining 3 EPEC isolates possessed the intimin έ subtype. No α and γ intimin subtypes were detected. Traditional EPEC serotypes O114:H14 were detected only in tEPEC strains. Marked resistant to β-lactam agents were observed but no ESBL-producing tEPEC or aEPEC was detected. This is the first report of intimin subtype genes in Abuja, Nigeria. These data will be useful in facilitating the characterization of intimin variants of EPEC and some Shiga toxin-producing E. coli (STEC) in humans and other animal species.

Detection of eae, bfpA, espA Genes on Diarrhoeagenic Strains of Escherichia coli Isolates

The Enteropathogenic Escherichia coli (EPEC) is one of pathogenic strain of diarrheagenic E. coli group in children andinfant that occurs in developing countries. The significant virulence factors in pathogenic EPEC are eaeA (E. coli attachingeffacing), bfpA (bundle-forming pilus A) and espA (encoding secreted protein A) genes. The use of DNA probes to detect thevirulence genes in E. coli in Indonesia is not common yet. In this experiment the gene fragments of eae, bfpA, and espA were usedas probes to detect the EPEC among E. coli isolates from stool specimensin of diarrheic children attending Public Health Centersin Yogyakarta. The DNA samples were isolated from 49 diarrheagenic E. coli isolates. The DNA probes of eae, bfpA and espAwere obtained by amplification of DNA fragment of EPEC O126 using PCR technique. Furthermore, those probes were used toidentify the presence of those genes among E. coli isolates using hybridization technique. The results showed that 42 (85.7%)isolates were espA+, 25 isolates (51%) were eaeA+ (EPEC strains). Therefore among 25 isolates of EPEC, 20 isolates (80 %)among EPEC were bfpA+ (typical EPEC strains).Keywords : DNA probe, eae, bfpA, espA, EPEC.

Genetic analysis of enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid reveals a new deletion within the EAF probe sequence among O119 typical EPEC strains.

BackgroundEnteropathogenic Escherichia coli (EPEC) are classified into typical and atypical strains based on the presence of the E. coli adherence factor (EAF) plasmid. The EAF plasmid contains the bfp (bundle-forming pilus) operon and the perABC (plasmid encoded regulator) gene cluster. A 1-kb cryptic region of EAF plasmid has been widely used as a genetic probe for EPEC detection. However, some EPEC strains may harbor an EAF plasmid lacking the EAF probe sequence, which makes the differentiation between typical and atypical a complex task. In this study, we report the genetic analysis of the EAF plasmid-encoded genes in a collection of EPEC clinical isolates.MethodsA total of 222 EPEC clinical isolates, which were previously classified as typical (n = 70) or atypical (n = 152) by EAF probe reactivity, were screened for the presence of different EAF sequences by PCR and DNA hybridization.ResultsAll typical strains possessed intact bfpA and perA genes, and most of them were positive in the PCR for EAF probe sequence. However, a subset of 30 typical strains, 22 of which belonged to O119 serogroup, presented a 1652 pb deletion in the region between 1093-bp downstream perC and 616-bp of the EAF fragment. The bfpA, bfpG, and per genes were found in all typical strains. In addition, 32 (21 %) atypical strains presented the perA gene, and 20 (13.2 %) also presented the bfpA gene. Among the 32 strains, 16 belonged to the O119:H2, O119:HND, and ONT:HND serotypes. All 32 atypical strains contained perA mutation frameshifts and possessed an IS1294 element upstream of the per operon as detected by PCR followed by restriction fragment length polymorphism (RFLP) typing and multiplex PCR. Among the 20 bfpA probe-positive strains, eight O119 strains possessed deletion in the bfp operon at the 3′end of bfpA due to an IS66 element.ConclusionOur data show that typical O119 strains may contain a deletion within the EAF probe sequence not previously reported. This new finding suggests that care should be taken when using the previously described EAF PCR assay in epidemiological studies for the detection of typical O119 strains. In addition, we were able to confirm that some atypical strains carry vestiges of the EAF plasmid.

BfpL is essential for type IV bundle-forming pilus biogenesis and interacts with the periplasmic face of BfpC

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea among infants in developing countries. The bundle-forming pilus (BFP), a type IV pilus found on the surface of EPEC, is essential for full virulence of typical EPEC strains. The machinery for BFP assembly and function is encoded by an operon of 14 genes. Here we investigate the role in pilus biogenesis of BfpL, a small protein with a single N-terminal predicted transmembrane domain reminiscent of pilin-like proteins. We confirmed that a bfpL mutant lacks BFP, and associated auto-aggregation and localized adherence phenotypes. Furthermore, we found that a double mutant unable to express both the putative retraction ATPase BfpF and BfpL also lacks BFP and associated phenotypes, distinguishing BfpL from pilin-like proteins. Western blots of sheared pilus preparations did not suggest that BfpL is a component of BFP. Topology studies using C-terminal truncations and a dual reporter revealed that most of the BfpL protein resides in the periplasm. Further, we demonstrated through yeast two-hybrid assays and confirmed by fluorescence anisotropy that BfpL interacts with the periplasmic face of BfpC. Thus, BfpL has a function distinct from those of pilin-like proteins and is instead part of an inner-membrane subassembly complex that is believed to extract bundlin, the main pilus subunit, from the inner membrane to be incorporated into BFP.

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.

Bundle-forming pilus retraction enhances enteropathogenicEscherichia coliinfectivity

Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.

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.

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.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.

Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli

Atypical enteropathogenic Escherichia coli (EPEC) comprise an important group of paediatric pathogens. Atypical EPEC have reservoirs in farm and domestic animals where they can be either commensal or pathogenic; serogroup O26 is dominant in humans and animals. Central to intestinal colonization by EPEC is the translocation of the type III secretion system effector Tir into enterocytes, which following phosphorylation (Tir-Yp) recruits Nck to activate the N-WASP actin signalling cascade. The authors have recently shown that typical EPEC strains, belonging to the EPEC-2 lineage, carry a tir gene encoding Tir-Yp and can also use the alternative TccP2 actin-signalling cascade. The aim of this study was to determine if tccP2 is found in atypical EPEC isolated from human and farm animals. tccP2 was found at a frequency of 41% in non-O26 EPEC isolates and in 82.3% of the O26 strains. TccP2 of human and animal strains show high level of sequence identity. It is shown that most strains carry a tir gene encoding Tir-Yp. In addition the authors identified two new variants of tir genes in EPEC O104:H12 and NT:H19 strains.

Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: identification of two novel intimin variants (μB and ξR/β2B)

A total of 71 enteropathogenic Escherichia coli (EPEC) strains isolated from children with diarrhoea in Montevideo, Uruguay, were characterized in this study. PCR showed that 57 isolates carried eae and bfp genes (typical EPEC strains), and 14 possessed only the eae gene (atypical EPEC strains). These EPEC strains belonged to 21 O : H serotypes, including eight novel serotypes not previously reported among human EPEC in other studies. However, 72% belonged to only four serotypes: O55:H- (six strains), O111:H2 (13 strains), O111:H- (14 strains) and O119:H6 (18 strains). Nine intimin types, namely, alpha1 (two O142 strains), beta1 (29 strains, including 13 O111:H2 and 14 O111:H-), gamma1 (three O55:H- strains), theta (five strains, including three strains with H40 antigen), kappa (two strains), epsilon1 (one strain), lambda (one strain), muB (six strains of serotypes O55:H51 and O55:H-) and xiR/beta2B (22 strains, including 18 O119:H6) were detected among the 71 EPEC strains. The authors have identified two novel intimin genes (muB and xiR/beta2B) in typical EPEC strains of serotypes O55:H51/H- and O119:H6/H-. The complete nucleotide sequences of the novel muB and xiR/beta2 variant genes were determined. PFGE typing after XbaI DNA digestion was performed on 44 representative EPEC strains. Genomic DNA fingerprinting revealed 44 distinct restriction patterns and the strains were clustered in 12 groups. Only 15 strains clustered in six groups of closely related (similarity>85%) PFGE patterns, suggesting the prevailing clonal diversity among EPEC strains isolated from children with diarrhoea in Montevideo.

Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.

Evaluation of Multiplex PCRs for Diagnosis of Infection with Diarrheagenic Escherichia coli and Shigella spp

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.

Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea.

The virulence profiles of most atypical enteropathogenic Escherichia coli (EPEC) strains are unknown. A total of 118 typical and atypical strains of EPEC serotypes and non-EPEC serogroups isolated from children with or without acute diarrhea who were from different cities in Brazil were examined for virulence-associated markers and adherence to HEp-2 cells, and also had random amplified polymorphic DNA (RAPD) analysis performed. Atypical strains were identical to typical strains with regard to the virulence factors encoded on the locus of enterocyte effacement (LEE). In contrast with typical EPEC strains, none of the atypical strains reacted with the bfpA probe, and half of the strains hybridized with the perA probe. Most atypical strains presented Tir sequences that correlated with enteropathogenic or enterohemorrhagic E. coli (98%), had LEE inserted in either selC or pheU (88%), and presented a typeable intimin (52%). Eighteen new serotypes were found in the EPEC strains. Atypical and typical EPEC strains belonged to different RAPD clusters. Most atypical strains showed a localized-like adherence pattern (61.5%). Of the non-LEE-encoded virulence factors, enteroaggregative E. coli heat-stable enterotoxin was noted most frequently (45%) and was significantly associated with diarrhea (P=.01). Thus, this virulence marker may be used as an additional tool for the diagnosis of truly atypical pathogenic strains.

Adhesion of enteropathogenic Escherichia coli to host cells.

Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.

Metabolism of phenylpropionic acid in enteropathogenic Escherichia coli belonging to serogroup O111 and its application for diagnosis

We evaluated a biochemical assay based on the ability to metabolise β-phenylpropionic acid (PPA) as a diagnostic aid in the identification of typical enteropathogenic Escherichia coli (EPEC) strains. A total of 1061 E. coli strains of serogroups O55, O111, and O119 were initially characterised regarding their H types (serotypes) and the presence of EPEC DNA sequences, eae, EAF, and bfpA. In case of the serogroup O111 strains, 84.6% carried the typical EPEC markers, and the great majority of those (98.1%) were PPA-positive. In contrast, only 0.9% of the serogroups O55 and O119 strains carrying the typical EPEC markers (53.6% and 75.4%, respectively) were PPA-positive. We conclude that the PPA test is a useful method to detect typical EPEC strains only among strains of the O111 serogroup.

Molecular identification of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea in Londrina, Parana, Brazil

In this work, the prevalence of enteropathogenic Escherichia coli (EPEC) in children in Londrina-PR, Brazil, was evaluated by means of digoxigenin-labelled DNA probes which identify the plasmid responsible for EPEC adherence factor (EAF), and virulence genes for EPEC as bundle-forming pilus (bfp) and E. coli attaching-effacing factor (eae). In addition, the isolated strains were serotyped and tested for adherence to HEp-2 cells. From 102 children with diarrhoea, 19 strains hybridized with at least one probe, and eleven of them were identified as typical EPEC because they hybridized with the three probes used, showed a localized adherence (LA) pattern, and presented no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype O119:H6 (43%); in four strains O antigens could not be determined using antisera against O1 to O173, they were all ONT:H7 (29%); one strain belonged to O111:H6. Three strains were classified as atypical EPEC: O26H-, O111:H9 and O119:HNT. Strains O26H- and O111:H9 hybridized with the eae probe only and showed localized adherence like (LAL) pattern; strain O119:HNT hybridized with the bfp and eae probes, and showed a localized adherence/diffuse adherence (LA/DA) pattern after 6 h. A DA pattern was observed in two strains isolated from children with diarrhoea (ONT:H11 and O142:H34), which hybridized with the eae probe. From 46 controls, five strains hybridized with one or two probes, but none hybridized with all probes or presented the LA pattern. Three strains with the DA pattern hybridized with the eae probe. No EPEC strain belonging to classical EPEC serotypes was isolated from faeces of control children.

Unidentified serogroups of enteropathogenic Escherichia coli (EPEC) associated with diarrhoea in infants in Londrina, Parana, Brazil

Digoxigenin-labelled DNA probes were used to characterise enteropathogenic Escherichia coli (EPEC) isolated in Londrina (Brazil) from faeces samples of 102 children with diarrhoea, and the results were compared with those obtained by serogrouping and adherence to HEp-2 cells. The probes employed detect the gene coding EPEC adherence factor (EAF) and the virulence genes for bundle-forming pilus (bfp) and entero-attaching-effacing (eae) factor. Twenty-one isolates hybridised with at least one probe, and 11 of them were classified as typical EPEC because they hybridised with all three probes, showed a pattern of localised adherence (LA) and carried no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype 0119:H6 and one to O111:H6; O antigens could not be determined in four strains with antisera against 01-0173. All typical EPEC strains carried a 70-MDa plasmid plus two other large plasmids. These data showed that typical EPEC virulence traits may be found in strains not belonging to classical serogroups/serotypes and that molecular identification is required for studying the epidemiology of diarrhoea in children.