Characterization of diarrheagenic Escherichia coli isolated from food in Khon Kaen, Thailand.

Four categories of 186 ready-to-eat food samples in Khon Kaen municipality, Thailand, were collected and investigated for fecal contamination by enumeration of Escherichia coli using the most probable number (MPN) method. Then, the E. coli isolates were presumptively identified as diarrheagenic E. coil by agglutinating with polyvalent O-antisera and monovalent O-antisera commonly found in diarrheagenic strains and were subsequently investigated for the presence of the recognized virulence genes for enteroaggregative (EAEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and shiga toxin-producing E. coli (STEC or EHEC) by multiplex PCR assays. All E, coli isolates were examined for antimicrobial susceptibilities by the agar disc diffusion method, and the results were compared with those obtained from clinical samples. The percentage of each type of food with E. coli, including no heat food, low heat food, high heat food, and fruit juices and beverages, was higher than accepted standards at 60.4, 46.5, 38.6 and 20%, respectively. Of 140 E. coli isolates obtained from food samples, 11 isolates (7.9%) agglutinated with 6 monovalent O-antisera, including one isolate each of O6, O8, O114 and O159, two isolates of O1, and five isolates of O157. None of the 11 isolates harbored the virulence genes for EPEC, ETEC, EAEC, EIEC and STEC. Although O157 E. coli isolates were found, the most frequent, E. coli O157:H7, was not found in this study. The astA gene, however, was found in 1 E. coli isolate that showed weakly positive agglutination against the polyvalent antisera. Approximately 50% of the 140 E. coli isolates were resistance to at least one antimicrobial agent. The resistant strains showed high resistance to tetracycline (43%), co-trimoxazole (36%), ampicillin (26%) and chloramphenicol (23%), respectively. The resistance of E. coli was high for nearly all antimicrobial agents, particularly ampicillin (76%), tetracycline (70%), co-trimoxazole (69%) and nalidixic acid (44%). The results show that nearly half of the ready-to-eat food samples evaluated in Khon Kaen Municipality had levels of E. coli higher than acceptable standards. Of the diarrheagenic E. coli classified by serogroup, almost none of the isolates had virulence genes. These results indicate the disadvantage of relying on serogrouping alone for the recognition of diarrheagenic E. coli. E. coli isolated from food may not be an enteropathogenic strain. We also found that E. coli antimicrobial resistant strains are widespread in both food and humans.

Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga / Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

La infección por Escherichia coli productor de toxina Shiga (STEC) es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH) en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.

Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The detection limit was 1.0 x 104 CFU/50 µl and the cut limit 1.0 x 105 CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures.