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PubMed2002

Virulence properties of Escherichia coli faecal strains isolated in Poland from healthy children and strains belonging to serogroups O18, O26, O44, O86, O126 and O127 isolated from children with diarrhoea.

Four hundred and twenty-seven Escherichia coli isolates from 427 cases of infantile diarrhoea in Poland, belonging to serogroups O18, O26, O44, O86, O126 and O127 and 150 E. coli isolates from 52 healthy children were examined for selected virulence properties. The presence of the plasmid pAA, a plasmid encoding enterohaemolysin, the genes encoding intimin (eae), bundle-forming pili (bfp), Shiga toxins I and II (stxI, stxII) and cytotoxic necrotising factor types 1 and 2 (cnfl, cnf2) was investigated by PCR. Adhesion to HEp-2 cell monolayers was also tested and selected strains were investigated for the presence of P-fimbriae and haemolytic activity. Typical enteropathogenic E. coli isolates (typical EPEC, strains possessing eae and bfp, but not stx) were not found. The particular classes of E. coli among 427 isolates from ill children were: atypical EPEC (eae+ bfp, stx-), 21.3%; Shiga toxin-producing E. coli (STEC), 0.7%; diffusely adherent E. coli (DAEC), 4%; enteroaggregative E. coli (EAEC), 16.9%; necrotoxic E. coli type 1 (NTEC1), 0.2%; and cell-detaching E. coli (CDEC), 29%. With the exception of STEC, all the above classes of E. coli were found among the isolates from healthy children which comprised: atypical EPEC 8.0%, DAEC 6.7%, EAEC 17.3%, NTEC1 14.0% and CDEC 40.0%. Cell detachment (CD) was significantly associated with 3-h haemolytic activity. There was also strong correlation between haemolytic activity (Hly) and the presence of P-fimbriae. No correlation was found between the presence of the cnf1 gene and CD, Hly or P-fimbriae.

Adhesins, Bacterial↗

PubMedDate not supplied

[Association of patterns of Escherichia coli adherence to HEp-2 cells with acute and persistent diarrhea].

In Brazil diarrhea is the cause of approximately 15% of death among infants. Enteropathogenic E coli is the most important bacterial agent causing acute diarrhea, which is defined as less than 14 days of duration. About 30% of these cases may evolve to persistent diarrhea, defined as lasting more than 14 days. In this work it was carried out a case-control study including 34 children under 2 years of age, and admitted to hospital facilities in São Paulo for rehydration therapy. Thirty-four age matched children hospitalized in the same facilities, and presenting no gastrointestinal symptoms were included as controls. Stool samples were analyzed for the presence of bacterial pathogens (diarrheagenic E coli, Shigella, Salmonella, Yersinia, and Campylobacter), protoparasytes, rotavirus, and enteric adenovirus. The E coli strains isolated were analyzed for their ability to adhere to HEp-2 cultured cells, in a 3 h adhesion assay. Search for homology with DNA probes for localized adherence (EAF, eaeA probes), AA (enteroagregative adherence) (AA probe), and diffuse adherence (F1845, AIDA-I probes) was carried out by the colony hybridization method. Twenty-four of the cases were acute diarrhea and 10 persistent diarrhea. Strains with localized adherence were associated with acute and persistent diarrhea. About 23.5% of E coli were associated with typical Enteropathogenic E coli strains (EAF+, eaeA+). Enteroaggregative E coli (EAggEC) (AA+) was isolated only from cases and in similar frequency for acute and persistent diarrhea. Diffusely adherent E coli (DAEC) which did not hybridize with the diffuse adherence probes were isolated among cases and controls. E coli eaeA+ with localized-like adherence was isolated from cases in a frequency three times higher than in controls, suggesting that it may really have a pathogenic potential.

Acute Disease↗

PubMed2003

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

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

Actins↗

PubMed2005

Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types.

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.

Adhesins, Bacterial↗

PubMed2003

Enteropathogenic Escherichia coli O157 strains from Brazil.

We describe two serogroup O157 Escherichia coli strains from Brazilian infants with diarrhea. A variety of assays indicate that these strains belong to the enteropathogenic, not the enterohemorrhagic, pathotype. These strains possess a novel bfpA allele encoding the type IV pilin characteristic of typical enteropathogenic E. coli strains. Our results emphasize the pitfalls of classifying pathogenic E. coli by serogroup.

Adhesins, Bacterial↗

PubMed2003

Genotypic and phenotypic characterization of attaching and effacing Escherichia coli (AEEC) isolated from children with and without diarrhoea in Londrina, Brazil.

Attaching and effacing Escherichia coli (AEEC) have been implicated in diarrhoea in humans in several countries. A total of 919 E. coli strains, isolated from 125 children with diarrhoea and 98 without diarrhoea, was investigated by PCR for the presence of the EAF, bfp, eae and stx genes. Thirty-four of these isolates were found to carry the eae gene; they were isolated from 27 (79.4 %) children with diarrhoea and seven (20.6 %) controls, in the city of Londrina, Brazil. These strains were investigated for their genotypic and phenotypic characteristics. Different genetic profiles were observed; strains containing the eae gene alone were most common (47.1 %). The characteristic genetic profile of typical enteropathogenic E. coli (EPEC), eae, bfp and EAF, was only found in isolates from children with diarrhoea. The stx gene was not detected in any of the 34 strains studied. Ten (29.4 %) strains were negative in the fluorescent actin-staining test. Localized adhesion (LA) was the most common pattern of adhesion (44.1 %), followed by the aggregative adhesion (AA) (23.5 %) and localized adhesion-like (LAL) (14.7 %) patterns. The results showed a strong association between strains presenting the LA pattern and diarrhoea. Forty-seven per cent of the strains studied belonged to classical O-serogroups of EPEC. The most common serotype found was O119 : H6; these isolates all showed the LA pattern, were positive for fluorescent actin-staining and were associated with diarrhoea. Intimin beta was detected in seven strains, four of which belonged to serotype O119 : H6 and three to serotype ONT : H7; all were associated with diarrhoea. On the other hand, intimin epsilon was detected in two strains of serotype O111 : H38 and one of serotype ONT : H19, isolated from children without diarrhoea. To our knowledge, this is the first report of the occurrence of intimin epsilon in strains of E. coli isolated from humans in Brazil.

Adhesins, Bacterial↗

PubMed1983

The clonal nature of enteropathogenic Escherichia coli strains.

Enteropathogenic Escherichia coli belonging to O groups O111 and O55 and isolated from cases of infantile diarrhea in 26 countries all over the world during 1950-1960 were examined for their outer-membrane protein (OMP) patterns; nine O:H serotypes of O111 and 11 serotypes of O55 were represented. Characteristic biotypes have earlier been described as closely associated with the most common O:H serotypes: O111:H2, O111:H12, O55:H6, and O55:H7. Different OMP patterns characterized each of the common O:H serobiotypes. The OMP patterns can be looked upon as another set of stable phenotypic characters in addition to the serotype antigens and the fermentative characters. The description of geographically widely spread, stable sero-/bio-/OMP types supports the concept that the typical enteropathogenic E coli strains also have a clonal connection.

Bacterial Outer Membrane Proteins↗

PubMed1976

The prevalence of enterotoxigenic Escherichia coli in the feces of calves with diarrhea.

A study was undertaken to determine the prevalence of enterotoxigenicity among Escherichia coli isolated from calves with diarrhea and from a control group of normal calves. The test organisms consisted of 200 E. coli recovered from scouring calves less than two weeks of age and 100 E. coli from normal calves. The enterotoxigenicity of the cultures was evaluated by three methods, namely, injection of ligated segments of piglet intestine, injection of ligated segments of calf intestine and oral inoculation of suckling mice. Live cutures of all the test organisms were used for the ligated intestine studies whereas sterile broth culture supernatants were used in the suckling mouse tests. Of the isolates from scouring calves, 36% were enterotoxigenic in the piglet intestine and 28% in the calf intestine. Amongst the isolates from normal calves, none was enterotoxigenic in the piglet intestine and one was enterotoxigenic in the calf test system. The ligated piglet intestine was considered unsuitable for determining the enterotoxigenicity of bovine E. coli, whereas the ligated calf intestine test was satisfactory and correlated completely with the suckling mouse test. The enterotoxigenic E. coli of bovine origin produced an enterotoxin that resembled the heat stable enterotoxin of typical porcine enteropathogenic E. coli.

Animals↗

PubMed1997

HEp-2 cell adherence patterns, serotyping, and DNA analysis of Escherichia coli isolates from eight patients with AIDS and chronic diarrhea.

Three morphologic patterns of interaction between bacteria and enterocytes have been observed in colonic biopsy specimens from AIDS patients with chronic diarrhea in the United States. The DNA encoding virulence factors and the HEp-2 cell adherence patterns of Escherichia coli strains isolated from the stools of eight symptomatic AIDS patients were compared with those of five control strains with known adherence patterns. One clinical isolate from a patient with attaching-and-effacing enteropathy displayed the localized adherence attaching-and-effacing pattern typical of enteropathogenic E. coli on HEp-2 cells, five isolates displayed the "stacked-brick" aggregative adherence pattern typical of enteroaggregative E. coli strains, and one isolate showed the pattern characteristic of diffusely adherent E. coli. One patient's isolate displayed features of all three patterns. No clinical isolate hybridized with standard probes for enteropathogenic, enteroaggregative, diffusely adherent, enterotoxigenic, and enteroinvasive E. coli strains. Thus, isolates from symptomatic AIDS patients in the United States can display the same interactive patterns with HEp-2 cells as the agents of pediatric or traveler's diarrhea, but lack their typical virulence factors.

Acquired Immunodeficiency Syndrome↗

PubMed2026

Parallel evolutionary trajectories rewire enteropathogenic Escherichia coli adhesion to restore host attachment.

Enteropathogenic Escherichia coli (EPEC) causes disease in children, presenting as chronic diarrhea that can impair physical and cognitive development. The attachment of typical EPEC (tEPEC) to the gut epithelium via bundle-forming pili (BFP) is a key factor in its virulence. Yet, infections by atypical EPEC (aEPEC), which lack BFP, have become increasingly common. To investigate how aEPEC recover host-attachment in the absence of BFP, we performed experimental evolution using a non-adherent E. coli, constructed to mimic the ancestor of aEPEC, and selected adherent progeny. Highly adherent variants evolved through phase-variable activation of type I fimbriae (T1F), followed by two alternative trajectories: bacterial filamentation, which increases T1F avidity, or point mutations in the T1F adhesin FimH that enhance ligand affinity. Extending our analysis to the genomes of 327 aEPEC strains isolated from infected patients revealed that similar FimH mutations are common. We further demonstrated experimentally that these naturally occurring variants often increase epithelial-attachment. Our findings implicate T1F in aEPEC pathogenesis and suggest it may be clinically relevant for anti-adhesion therapy. More broadly, these results indicate that impaired host-attachment can be rapidly compensated by upregulating and optimizing an alternative adhesin, and that combining experimental evolution with comparative genomics can reveal evolutionary trajectories occurring in nature.

Bacterial Adhesion↗

PubMed1994

Escherichia coli serogroup O111 includes several clones of diarrheagenic strains with different virulence properties.

Genetic variation among isolates of Escherichia coli O111 obtained mostly from patients with diarrhea in Brazil was assessed by multilocus enzyme electrophoresis to characterize chromosomal genotypes and by gene probes and adherence assays to characterize virulence properties. Among the 152 isolates, we resolved 16 distinct electrophoretic types (ETs), which differed on average at 40% of the enzyme loci. We identified four major bacterial O111 clones of different disease classes: ET 12, which includes the bulk of the enteropathogenic E. coli strains, typically showing localized adherence and intimate attachment in tissue culture assays; ET 1, which includes strains with a different set of virulence markers; ET 9, which includes strains that show intimate attachment but lack localized adherence and Shiga-like toxin genes; and ET 8, which includes strains that are Shiga-like toxin producers and have the corresponding traits of enterohemorrhagic E. coli. Enteroaggregative strains constituted ET 10 and also occurred in ET 1. Isolates of the major clones were found in South and North America and matched in ET and virulence factors to previously described diarrheagenic clones that are widely disseminated in the human population. Because the major clones are genetically distantly related and exhibit different combinations of virulence factors, we hypothesize that they have distinct mechanisms of pathogenesis. The results indicate that genetic divergence of bacteria with the O111 antigen, as measured by allelic variation in enzyme loci, is accompanied by divergence in virulence properties of clones so that identification and classification of pathogenic E. coli strains cannot be based solely on serotyping or a single virulence factor.

Bacterial Adhesion↗

PubMed2002

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil.

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the São Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.

Animals↗

PubMed2004

Diarrheagenic Escherichia coli categories among the traditional enteropathogenic E. coli O serogroups--a review.

The so called enteropathogenic Escherichia coli (EPEC) O serogroups include typical and atypical EPEC, enterohaemorrragic E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli. The aim of this article is to review the composition of each O serogroup and the major serotypes, clones, and additional virulence characteristics of each of these diarrheagenic categories. Their adherence patterns and genetic relationships are also presented. The review is based on the study of 805 strains of serogroups O26, O55, O86, O111, O114, O119, O125, O126, O1127, O128, and O142 most of which isolated in Sao Paulo from children with diarrhea between 1970 and 1990. Since some O serogroups include more than one diarrheagenic category O serogrouping only should be abandoned as a diagnostic method. However serotyping is a reliable method for those serotypes that correspond to clones.

Adhesins, Escherichia coli↗

PubMed2004

Activation of NF-kappaB in intestinal epithelial cells by E. coli strains isolated from the colonic mucosa of IBD patients.

BACKGROUND AND AIMS: The involvement of bacteria in the pathogenesis of inflammatory bowel disease has been discussed for several years. In this study we evaluated the ability of E. coli isolates from inflamed and noninflamed colonic mucosa to activate NF-kappaB. MATERIALS AND METHODS: Fifteen bacterial strains from inflamed and six from noninflamed colonic tissues from IBD patients. Their ability to induce NF-kappaB activation was examined in vitro by gel-shift assays. The activation of the TNF-alpha promoter was determined by reporter gene assays. Bacterial isolates were characterized by invasion assays, electron microscopy, and PCR. RESULTS: Four of 15 E. coli bacterial isolates from inflamed IBD tissues induced NF-kappaB activity in intestinal epithelial cells as determined by gel-shift assays. NF-kappaB activation was only seen with living bacteria but not with heat-inactivated cells. Isolates from noninflamed tissues and a wild-type E. coli control strain induced a weaker or no activation. Reporter gene assays with a construct comprising a luciferase gene driven by the TNF-alpha promoter revealed that isolates from Crohn's disease patients induced a stronger activation of the TNF-alpha gene than isolates from ulcerative colitis patients. The isolated bacteria invaded HT-29 cells, although typical virulence genes for enteropathogenic, enterhemorrhagic, or enteroinvasive E. coli, i.e., eae, tir, EspA, Per (A-C), ipaC, were not detected in these cells. Bacterial invasion was additionally confirmed by electron microscopy examination. CONCLUSION: Our results indicate that E. coli strains can be found in the mucosa of some IBD patients which are able to activate NF-kappaB similar to known pathogenic strains. The absence of several virulence genes in these cells suggests that they are members of the luminal flora which acquire as yet unidentified virulence determinants and are therefore involved in the pathophysiology of IBD.

Colon↗

PubMed2003

Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of attaching and effacing Escherichia coli.

Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.

Adhesins, Escherichia coli↗

PubMed2004

Alpha 1-antitrypsin binds to and interferes with functionality of EspB from atypical and typical enteropathogenic Escherichia coli strains.

Enteropathogenic Escherichia coli (EPEC), including diffusely adhering atypical E. coli, strains use a type III secretion system to deliver effector proteins into the membrane and cytoplasm of infected cells. The E. coli secreted proteins A, B, and D (EspA, EspB, and EspD) are required for the formation of the characteristic attaching and effacing (A/E) lesions. EspB and EspD are thought to form a translocation pore in the host cell membrane through which effector proteins are injected into the host cytosol. Besides its function in pore formation, EspB has been found in the cytosol and implicated to function as an effector protein. We screened for putative host cell proteins interacting with EspB of atypical EPEC strains and identified alpha(1)-antitrypsin (AAT) as a binding partner for EspB. AAT binds to EspB in pull-down and overlay experiments and also to EspD in overlay experiments. In agreement with the role of EspB and EspD in pore formation, EPEC-mediated hemolysis of red blood cells is strongly reduced by AAT in a concentration-dependent manner, indicating that AAT interferes with type III secretion by inhibiting the formation of the translocation pore. This is further supported by a decreased actin polymerization after infection of HeLa or CaCo-2 cells with EPEC in the presence of physiologically relevant concentrations of AAT. In this study, we identify AAT as a new binding partner for EspB and EspD, suggesting a previously unappreciated role for AAT in host cell defense against EPEC infections and potentially also against other bacterial pathogens.

Actins↗

PubMed2006

Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil).

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).

Acute Disease↗

PubMed2004

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.

Child, Preschool↗