Multiplex PCR Assay for Identification of Human Diarrheagenic
            <i>Escherichia coli</i>

Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isao; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

doi:10.1128/jcm.41.6.2669-2671.2003

Cited by 218 publications

(151 citation statements)

References 19 publications

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“…Strains biochemically identified as E. coli were differentiated as typical or atypical EPEC strains by a PCR-based method. The eae, stx and bfp-A genes were amplified using the primers eae-F (59-CTG-AACGGCGATTACGCGAA-39) and eae-R (59-CCAGACGATACG-ATC-CAG-39), stx-F (59-GAGCGAAATAATTTATATGTG-39) and stx-R (59-TGATGATGGCAATTCAGTAT-39), and bfpA-F (59-AATG-GTGCTTGCGCTTGCTGC-39) and bfpA-R (59-GCCGCTTTATC-CAACCTGGTA-39) (Toma et al, 2003;Aranda et al, 2007). The current study was approved by the Ethics Committee of the Tehran University of Medical Sciences.…”

Section: Methodsmentioning

confidence: 99%

Molecular analysis of typical and atypical enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea

Nakhjavani

Emaneini

Hosseini

et al. 2013

Journal of Medical Microbiology

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Diarrhoea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. To investigate the incidence, antimicrobial resistance and genetic relationships of enteropathogenic Escherichia coli (EPEC) in children with diarrhoea, a total of 612 stool specimens were collected in Tehran, Iran, and cultured to isolate strains of EPEC. The disc diffusion method was used to determine the susceptibility of the isolates according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of eae, stx and bfp-A genes was determined by PCR. The genetic relationships between EPEC isolates were determined by pulsed-field gel electrophoresis (PFGE). Out of the 412 strains of E. coli obtained from 612 diarrhoeal stool specimens, 23 (5.6 %) were identified as EPEC, of which seven (30.4 %) were classified as typical strains of EPEC and 16 (69.6 %) were classified as atypical. Out of the 23 EPEC isolates, 69.5 % were resistant to ampicillin, 39.1 % were resistant to tetracycline and cotrimoxazole, 30.4 % were resistant to cefpodoxime, ceftazidime, ceftriaxone and aztreonam, and 26.1 % were resistant to imipenem. The isolates were classified into 21 pulsotypes by PFGE profiles. The present study shows that typical and atypical EPEC isolates displayed considerable heterogeneity in PFGE profiles and EPEC infections were only sporadic in Tehran. Overall 69 % of isolates were resistant to at least one of the antibiotics tested. INTRODUCTIONEnteropathogenic Escherichia coli (EPEC) is one of the major causes of diarrhoea among children in developing countries (Hernandes et al., 2009;Moura et al., 2009). The pathogenesis of EPEC depends on the locus of enterocyte effacement (LEE), a chromosomal pathogenicity island. The LEE contains a number of different genes, including eae, which has an essential role in inducing a characteristic lesion formation in the intestinal epithelium, termed an attaching and effacing (A/E) lesion (Elliott et al., 1998;Vallance & Finlay 2000;Kaper et al., 2004). The eae gene encodes intimin, a 94 kDa outer-membrane protein that is responsible for the intimate adherence between bacterial and enterocyte membranes (Vallance & Finlay, 2000;Trabulsi et al., 2002). EPEC strains are classified as typical or atypical, according to the presence or absence of the E. coli adherence factor plasmid (EAF) that carries the bfpA gene, which encodes the bundle-forming pili (Hernandes et al., 2009). The most typical EPEC strains belong to the classic O : H serotypes and are eae-and bfpA-positive (Ochoa et al., 2008). The atypical EPEC isolates are eaepositive and bfpA-and stx (the gene encoding shiga-like toxin)-negative. EPEC are among the most important pathogens infecting children under 2 years of age in the developing world (Ochoa et al., 2008). Recent studies indicate that atypical EPEC is more prevalent than typical Abbreviations: EPEC, enteropathogenic Escherichia coli; LEE, locus of enterocyte effacement; PFGE, pulsed-field gel electrophore...

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Section: Methodsmentioning

confidence: 99%

Molecular analysis of typical and atypical enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea

Nakhjavani

Emaneini

Hosseini

et al. 2013

Journal of Medical Microbiology

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“…DNA extracted with a DNeasy TM Tissue Kit (QIAGEN, Hilden, Germany), or boiled enrichment culture was used as templates. Oligonucleotide primers (Table 2) for PCR were as follows: SK1, SK2 for eae (Oswald et al, 2000), Vtcom-u, Vtcomd for stx (Yamasaki et al, 1996), LT L , LT R for elt (Toma et al, 2003), and AL65, AL125c for est (Toma et al, 2003). PCR was performed under the following conditions: initial denaturation for 5 min at 94 C, 30 cycles of 1 min at 94 C, 1 min at 55 C, 1 min at 72 C, and a final extension step of 7 min at 72 C. PCR amplicons were electrophoresed on 2% SeaKem GTG agarose gel (Takara, Otsu, Shiga, Japan) in 13 TAE buffer.…”

Section: Isolation and Identification Of E Colimentioning

confidence: 99%

Genetic Characteristics and Antimicrobial Resistance of Escherichia Coli From Japanese Macaques (Macaca Fuscata) in Rural Japan

Ogawa

Yamaguchi

Suzuki

et al. 2011

Journal of Wildlife Diseases

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ABSTRACT:Escherichia coli was isolated from wild and captive Japanese macaques (Macaca fuscata) to investigate the risk of zoonotic infections and the prevalence of antimicrobial-resistant Escherichia coli in the wild macaque population in Shimokita Peninsula, a rural area of Japan. We collected 265 fresh fecal samples from wild macaques and 20 samples from captive macaques in 2005 and 2006 for E. coli isolation. The predominant isolates were characterized by serotyping, virulence gene profiling, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and microbial sensitivity tests. In total, 248 E. coli strains were isolated from 159 fecal samples from wild macaques, and 42 E. coli were isolated from 17 samples from captive macaques. None of the virulence genes eae, stx, elt, and est were detected in any of the isolates. The relatedness between wild-and captive-derived isolates was low by serotyping, PFGE, and plasmid profiling. Serotypes O8:H6, O8:H34, O8:H42, O8:HUT, O103:H27, O103:HNM, and OUT:H27 were found in wild macaque feces; serotypes O157:H42 and O119:H21 were recovered from captive macaques. Oand H-serotypes of the 26 isolates were not typed by commercial typing antisera and were named OUT and HUT, respectively. Twenty-eight isolates had no flagellar antigen, and their H-serotypes were named HNM. Similarity of PFGE patterns between wild-derived isolates and captivederived isolates was ,70%. No plasmid profile was shared between wild-derived and captivederived isolates. The prevalence of antimicrobial-resistant E. coli was 6.5% (n562) in wild macaques, and these isolates were resistant to cephalothin. We conclude that wild Japanese macaques in Shimokita Peninsula were unlikely to act as a reservoir of pathogenic E. coli for humans and that antimicrobial-resistant E. coli in wild macaques may be derived from humans.

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“…El diagnostico de ECD es difícil, para identificarlas correctamente deben diferenciarse de miembros no patógenos de la biota normal, generalmente por determinación de los serotipos, lo cual no es determinante, pues muchas de estas bacterias pueden intercambiar material genético y con ello genes de virulencia, por lo que en los últimos años se ha elegido los métodos moleculares como la reacción en cadena de la polimerasa (PCR) para la detección con resultados rápidos, fiables, con alta sensibilidad y especificidad (7,8) basada en la identificación de la presencia de genes de virulencia específicos, que están ausentes en cepas no patógenas (9,10).…”

Section: Introductionunclassified

Molecular detection of diarrheogenic Escherichia coli in pediatric patients with acute diarrheal syndrome in Paraguay

Weiler

Orrego

Álvarez

et al. 2017

Mem. Inst. Investig. Cienc. Salud

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R E S U M E NLa Escherichia coli diarreogénica (ECD) se ha clasificado con base en criterios clínicos, epidemiológicos y moleculares en cinco grupos, cada uno con factores de virulencia específicos. El objetivo fue determinar la prevalencia de ECD en pacientes pediátricos con enfermedad diarreica aguda del Laboratorio Central de Salud Publica en el periodo 2012-2015. Se procesaron muestras de heces con síndrome diarreico agudo, provenientes de pacientes pediátricos, en los cuales se buscó algún gen de virulencia ECD utilizando métodos convencionales de siembra y screening molecular, mediante PCR múltiple con cebadores diseñados específicamente para amplificar los genes de virulencia elt, est, eae, stx, ipaH y aggR. Del total de muestras analizadas, 13% (180/1379) de las muestras presentó algún factor de virulencia compatible con algún patotipo ECD con mayor predominio en niños de 1 a 3 años. La frecuencia de los distintos patotipos fue la siguiente: 61 (34%) ETEC, 40 (22%) EAEC, 41 (23%) EPEC, 27 (15%) EIEC, 7 (4%) STEC y 3 (2%) ETEC/EAEC, 1 (0.5%) ETEC/EAEC/EIEC. El porcentaje de E. coli diarreogénicas detectado tiene similitud con lo reportado en otros países de la región, lo que nos indica que estos patógenos son parte importante de la etiología de la enfermedad diarreica aguda infecciosa en la población infantil en nuestro país. Se debe destacar que para el diagnóstico de las diferentes categorías ECD, es necesario disponer de un procedimiento diagnóstico específico dirigido a la detección de los factores de virulencia utilizando métodos moleculares o métodos inmunológicos.Palabras clave: Escherichia coli diarreogénicas, Enfermedad Diarreica Aguda, PCR.

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Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

Cited by 218 publications

References 19 publications

Molecular analysis of typical and atypical enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea

Molecular analysis of typical and atypical enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea

Genetic Characteristics and Antimicrobial Resistance of Escherichia Coli From Japanese Macaques (Macaca Fuscata) in Rural Japan

Molecular detection of diarrheogenic Escherichia coli in pediatric patients with acute diarrheal syndrome in Paraguay

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