Characterization of Verocytotoxin-Producing Escherichia coli O178:H19, the Serotype prevalently isolated from dairy farms in Argentina

FERNÁNDEZ, DANIEL; KRÜGER, ALEJANDRA; SANZ, MARCELO E.; POLIFRONI, ROSANA; ARROYO, GUILLERMO H.; LUCCHESI, PAULA M. A.; PADOLA, NORA L.; PARMA, ALBERTO E.

Buenos Aires

Simposio; 7th INTERNATIONAL SYMPOSIUM ON SHIGA TOXIN (VEROCYTOTOXIN) - PRODUCING Escherichia coli INFECTIONS; 2009

Asociación Argentina de Microbiología; y comité internacional

Non-O157 Verocytotoxin-producing Escherichia coli (VTEC) infections can induce a range of illness, from mild gastroenteritis to critical illnesses, including haemorrhagic colitis, haemolytic uraemic syndrome (HUS), and death, either as sporadic cases or outbreaks. The serotype O178:H19 is one of the non-O157 VTEC serotypes associated with HUS in Argentina. The aim of this study was to characterize geno-phenotypically isolates belonging to serotype O178:H19. This serotype was the one isolated most frequently from dairy cattle (17/58) in four dairy farms in Argentina. For this characterization we detected the virulence factors encoding genes (vt1, vt2, ehxA, eae and saa) by a multiplex PCR and determinated vt variants by PCR and PCR-RFLP. We used monoplex PCR for csgD, csgA y crl genes (ós – regulated genes and implicated in synthesis of curli fimbriae), and Congo red plates to determine the expression of phenotype "saw" (soft and white) for absence of expression of curli in the colony, and "rdar" (red, dry and rough) for expression of this fimbriae. Also, we studied the capacity of these microorganisms to adhere and invade HEp-2 cells. We performed the inoculation of cells with 105 cfu/ml of MEM without antibiotic. To assess adherence rebounded with a topcoat trypsin and PBS and the suspension was cultured in SMAC plates. To appreciate the internalization we added MEM with gentamicin for 1 h to kill the bacteria attached to the membrane and allow only the recovery of internalized bacteria. We have found that all the isolates were vt2-positive but were negative for vt1, ehxA, eae and saa genes. Among vt2-positive isolates, vt2vha predominated (14/17) while vt2vhb and vt2EDL933 genes were detected in only two and one of 17 isolates, respectively. The variants vt2f, vt2NV206 and vt2g were not present among the analyzed VTEC isolates. The results of monoplex PCR showed that all strains had the necessary genes for the expression of the curli fimbriae, but when they were cultivated in Congo red plates, none expressed the curli fimbriae, as they showed "saw" phenotypes. Although it was not observed the production of fimbriae curli, these strains could express the fimbriae under specific stress condition. Also all the strains showed the capacity to adhere and invade HEp-2 cells. Obviously, these strains would be using other mechanisms of adhesion to epithelial cells, other than those related to intimin or Saa. The presence of vt2 variants associated with severe illness and the ability to adhere and invade, demonstrate that isolates belonging to serotype O178:H19 found in higher prevalence in dairy farms of Argentina, constitute a serious risk to public health.