Virulence profiles of Shiga toxin-producing Escherichia coli and other potentially diarrheagenic E.coli of bovine origin, in Mendoza, Argentina

This study described a group of strains obtained from a slaughter house in Mendoza, in terms of their pathogenic factors, serotype, antibiotype and molecular profile. Ninety one rectal swabs and one hundred eight plating samples taken from carcasses of healthy cattle intended for meat consumption were analyzed. Both the swab and the plate samples were processed to analyze the samples for the presence of virulence genes by PCR: stx1, stx2, eae and astA. The Stx positive strains were confirmed by citotoxicity assay in Vero cells. The isolates were subsequently investigated for their O:H serotype, antimicrobial susceptibility and molecular profile by Random Amplification of Polymorphic DNA (RAPD). Twelve E.coli strains were identified by their pathogenicity. Nine were from fecal origin and three from carcasses. Three strains carried the stx1 gene, three the stx2 gene, two carried eae and four the astA gene. The detected serotypes were: O172:H-; O150:H8; O91:H21; O178:H19 and O2:H5. The strains showed a similarity around 70% by RAPD. Some of the E.coli strains belonged to serogroups known for certain life-threatening diseases in humans. Their presence in carcasses indicates the high probability of bacterial spread during slaughter and processing.

Evaluation of a multiplex PCR method to detect enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) has been implicated in sporadic diarrhea in children and adults and has been identified as the cause of several outbreaks worldwide. The HEp-2 test remains the gold standard for identification of this pathotype. A 60-65 MDa plasmid encodes the aggregative adherence fimbriae (AAF/I and AAF/II), a transcriptional activator (aggR gene), the enteroaggregative heat-stable enterotoxin EAST1 (astA gene) and a cytotoxin (Pet). The standard assay for EAEC is performed only in research laboratories, because it is expensive, labor intensive and time-consuming. The Polymerase Chain Reaction (PCR) offers the possibility of rapid diagnosis. In the current study, a multiplex PCR assay which checks aggR and astA genes was designed. Eigthy-eight E. Coli strains, isolated from children with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by aggR-astA PCR. A strong correlation between the presence of the specific marker aggR and the reference test was found. The astA gene had a similar distribution between aggregative and localized strains, indicating that this gene could not be considered as a marker of EAEC. We conclude that aggR may be used to identify EAEC, using the PCR method as a screening test.

Characterization of enteroaggregative Escherichia coli strains isolated from children with acute diarrhea, in Mendoza, Argentina

Enteroaggregative Escherichia coli (EAEC) is an increasingly recognized cause of diarrhea in children in developing and developed countries. EAEC is recognized by a characteristic aggregative pattern of adherence to human epithelial (HEp-2) cells cultured in vitro. This is the gold standard assay. The aggregative phenotype is associated with the presence of a 65 MDa plasmid (pAA) that also encodes several other putative virulence factors, such as the aggregative adherence fimbria I (AAF/I) and the enteroaggregative heat-stable enterotoxin (EAST1). The objective of this work was to evaluate the application of PCR (polymerase chain reaction) to identify EAEC strains in cases of acute diarrhea. A total of 87 E. coli strains, isolated from patients under 2 years of age with acute diarrhea in Mendoza, Argentina, were characterized by the reference method (HEp-2 assay), and by AAF/I- and EAST1-PCR. PCR sensitivity and specificity in comparison with the cell culture assay showed 94.4 sensitivity and 78.26 specificity. EAST1- and AAF/I-PCR could be recommended as a screening test, applicable to epidemiologic studies.

Acumulación de actina y adherencia a células HEp-2 de cepas de Escherichia coli aisladas de niños con diarrea en Mendoza, Argentina / Actin accumulation and HEp-2 cell adherence assay for Escherichia coli isolated from children in Mendoza, Argentina

Entre los niños menores de 2 años atendidos en el Hospital Infantil H. Notti de Mendoza, Argentina, es frecuente la diarrea causada por E. coli. La identificación se realiza por medio de marcadores bioquímicos y seroagrupamiento con antisueros que incluyen la mayoría de los serogrupos tradicionales de E. coli enteropatógeno (EPEC). Aunque se ha comprobado que existen serogrupos O y serotipos H de E. coli comúnmente asociados a diarrea infantil, en algunos estudios existe discrepancia tanto en relación a su presencia en controles sanos, en cuanto a su clasificación como EPEC en estudios genotípicos con sondas de ADN. En este trabajo se comparó la información proporcionada por la metodología estándar con los patrones de adherencia a células HEp-2, de cepas obtenidas de coprocultivos de 140 niños menores de 2 años con diarrea aguda, prolongada y crónica. Se hizo un grupo control de 40 casos sin diarrea. Las cepas se clasificaron según serología positiva (+) y negativa (-) frente a antisueros polivalentes. Se identificaron 3 tipos de adherencia: localizada (LA), difusa (DA) y EAagg (agregativa). Además, se realizó la prueba denominada "Fluorescence Actin Staining" (FAS) que pone en evidencia la lesión característica de EPEC, denominada "attaching-effacing" (A/E) producida por la formación de un pedestal de filamentos de actina en el sitio de adhesión bacteriana, en tejido intestinal y células en cultivo. En diarreas agudas, la adherencia fue relacionada con la serología + (P=0,001). La presencia de LA en diarreas fue del 11 por ciento con predominio significativo en niños menores de 12 meses (P=0,0001) y serología + (P=0,0001). El 80 por ciento de estas cepas dieron positiva la prueba FAS. El patrón DA apareció en diarreas (29 por ciento P=0,0001) y controles (2,5 por ciento); EAgg sólo en diarreas (20 por ciento P=0,0001). Este trabajo postula que la patogenicidad no estaría restringida a serogrupos. Debido a la frecuencia del aislamiento de cepas enteroadherentes de las que no se conoce bien la patogenia y que no son detectadas por las pruebas fenotípicas estándares, sería importante complementar al laboratorio clínico con técnicas que evidencien factores de virulencia