Characterization of verocytotoxin- producing Escherichia coli O178:H19, the serotype prevalently isolated from dairy farms in Argentina

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

Centro Cultural Borges - C.A.B.A

Congreso; VTEC2009 -7th. International Symposium on Shiga Toxin (verocytotoxin)- Producing Escherichia coli Infections – Buenos Aires; 2009

Asociación Argentina de Microbiología

Keywords: VTEC, O178:H19. dairy farms, variants, adherence,  curli fimbriae.  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 in outbreaks. Strains of serotype O178:H19 have been associated with cases of HUS. The aim of this study was to characterize geno-fenotypically isolates belonging to the serotype O178:H19. This serotype was isolated most frequently from dairy cattle (17/58) in four dairy farms from Argentina. For this characterization we determine the virulence factors encoding genes (vt1, vt2, ehxA, eae and saa) by multiplex PCR  and vt variants by PCR and PCR-RFLP. We used monoplex PCR for csgD, csgA y crl genes and Congo red plates to determine the expression of curli fimbriae. Also, we studied the capacity of adherence and invasion of this serotype in HEp-2 cells. We worked on these cell lines grown in MEM at 37 º C with 5% CO2. We performed the inoculation of cells with 105 cfu / ml of MEM without antibiotic. The plates were incubated in the same conditions for 3 h and were washed with PBS. To assess adherence rebounded with a topcoat trypsin and PBS. The suspension obtained was planted in SMAC plates. To appreciate the internalization, after 3 h of incubation was washed with PBS and 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 of the isolates were vt2-positive but none of them contained 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 vt2fg 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 these strains were cultivated in Congo red plates, none expressed the curli fimbriae, showed saw phenotypes. Although it was not observed the production of fimbriae curli, these strains could be expressed the fimbriae under specific stress condition. Also all the strains showed to capacity of adherence and invasion in HEp-2 cells. Obviously these strains have other mechanisms of union with epithelial cells, other than intimin or Saa. The results of this study demonstrate that the serotype O178: H19 found in higher prevalence in dairy farms of Argentina, is a serious risk to public health.