Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87 percent of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Pathotypic Characterization of Enterocyte Effacement-related LEE Genes in EHEC and EPEC Isolated from Diarrheal Patients

Attaching and effacing Escherichia coli (AEEC) cause enteric infections in humans and animals. Attaching indicates the intimate attachment of bacteria to the enterocyte, and effacing relates to the localized effacement of brush border microvilli. Enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) infections are characterized by the formation of attaching and effacing (AE) lesion on the intestinal epithelial cells. Therefore, they are often grouped together as AEEC. Development of multiplex PCR allowed us to type five of the most important genes implicated in the formation of the AE lesion. A total of 60 AEEC strains isolated from diarrheal patients were investigated by multiplex PCR for the presence of the insertion site of locus of enterocyte effacement (LEE) and LEE-related (eae, tir, espA, espB, and espD) genes. Associating the results of LEE genes typing in the AEEC strains, three different pathotypes are determined: eae(gamma)-tir(gamma)-espA(gamma)-espB(gamma)-espD(gamma) (O157:H7), eae(beta)-tir(beta)-espA(beta)-espB(beta)-espD(beta) (O26:H11), and eae(alpha)-tir(alpha)-espA(alpha)-espB(alpha)-espD(alpha) (O55:H6). These results indicate that AEEC are a heterogenous groups of organisms.

Genotypic and Phenotypic Characterization of Enteropathogenic Escherichia coli Isolated from Diarrheal Patients in Gwangju

Enteropathogenic Escherichia coli (EPEC) have been implicated in human diarrhea in several countries. Central to EPEC-mediated disease is its ability to cause intestinal lesions, known as attaching and effacing (A/E) lesion. We investigated 92 EPEC strains isolated from patients with diarrhea in Gwangju for their genotypic and phenotypic characteristics. Sixteen (17.4%) of all strains were found to be typical EPEC because they were bfpA gene positive by PCR. The most of typical EPEC isolates (87.5%) showed a localized adhesion (LA) pattern in Hep-2 cell adherence assay, whereas, only 11 atypical EPEC isolates (14.5%) were adhered to Hep-2 cells in a localized manner. Thirteen of the EPEC strains studied belonged to classical O-serogroups of EPEC and 7 isolates were classified as nonclassical EPEC serogroup and the other isolates could not be serotyped with our antisera. The subtypes of eae, tir, espA and espB genes which are major virulence genes concerned of A/E lesion on chromosome were analyzed by multiplex PCR for finding the original resource. The results showed that the composition of these genes subtypes was homogenous and heterogenous in 12 and 26 isolates, respectively. The others were non-determined type in terms of the gene subtype because of genetic diversity of intimin-coding eae genes. Our findings indicated that EPEC isolates from patients with diarrhea were diverse genetically and phenotypically, which require further study in regard to their virulence and epidemiological significance.

Caracterización geno-fenotípica de aislados de Escherichia coli AEEC de pacientes pediátricos con procesos diarreicos infecciosos en la ciudad de La Paz: implicancias para el diagnóstico y epidemiología de las enfermedades diarreicas agudas / Geno-Phenotypic characterization of aeec Escherichia coli isolated from children with infectious diarrheal diseases in La Paz: relevancy for the diagnosis and epidemiology of acute diarrheal diseases

En el presente estudio se realizó la caracterización de Escherichia coli Enteropatogénica (EPEC) y Escherichia coli Enterohemorrágica (EHEC), dos categorías patogénicas de E. Coli, causantes de la lesión de adherencia y esfacelación (EAEE), en muestras de heces diarreicas de niños menores a 5 años. El perfil patogénico de EAEE se realizo mediante el análisis por PCR, de los genes intimina (eae), bundlina (bfpA) y toxinas siga (stx1 y stx2). Estas pruebas, se complementaron con ensayos fenotípicos de la resistencia a antibióticos, fermentación de sorbitol y producción de b-D-glucoronidasa. La prevalencia de EAEE fue del 7% con preponderancia de las cepas EPEC (95%) sobre EHEC. Se encontró una mayor proporción (83%) de cepas EPEC atípicas que típicas. Un alto porcentaje de los aislados de EPEC es resistente a más de 5 antibióticos analizados. La frecuencia de multiresistencia a bloques de 5 y 2 antibióticos sugiere que la resistencia es transmisible por vía horizontal. La correlación entre la pertenencia a un serogrupo particular de EPEC y las características genotípicas, mostró heterogeneidad en el perfil de patogenicidad tanto entre un mismo como entre diferentes serogrupos, demostrando que el diagnostico de DEC mediante serotipificación no es útil en nuestro medio. Los aislados de EHEC, se caracterizan por presentar una marcada susceptibilidad a los antibióticos. Se reporta la presencia de los serogrupos O157 y O6. Este estudio, constituye el primer reporte en nuestro medio sobre la determinación y caracterización geno-fenotípica de EPEC y EHEC por métodos moleculares. En conjunto, los datos obtenidos tienen relevancia para el diagnostico, tratamiento y estudio de la epidemiología de AEEC en las EDA en Bolivia.

In this study, enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. Coli, two E. Coli categories causing attaching and effacing lesions, were isolated and characterized from children with diarrhea less than 5 years of age. The AEEC pathogenic profile was analyzed by PCR for the presence of the intimin (eae), bundle-forming pilus (bfpA) and Shiga toxin (stx, stx2) genes. Phenotypic analysis for the presence of antibiotic multi-resistance, sorbitol fermentation and B-D glucoronidase were also performed. AEEC prevalence was 7%. EPEC accounted for 95% of the isolates of which 83% were atypical. A high percentage of EPEC isolates is resistant to more than 5 antibiotics. The multi-resistance frequency to 5 and 2 antibiotics suggest antibiotic resistance transmission by lateral transfer. The lack of correlations between EPEC serogroups and genotypic strain profile demonstrates that serological DEC diagnosis is not useful for local isolates. EHEC isolates were remarkably susceptible to most of the antibiotics tested. The isolation of 0157 and 06 serogroups is reported. This is the first report of EPEC and EHEC molecular strain characterization. The results described are relevant for EAEE diagnosis, treatment and epidemiology of diarrheal diseases in Bolivia.