Detection of subtilase cytotoxin gene in strains of Verocytotoxigenic Escherichia coli from Argentina

LUCCHESI, PAULA M. A.; GRANOBLES, CLAUDIA V.; SUAREZ, LORENA; SANSO, A. MARIEL; KRÜGER, ALEJANDRA; PARMA, ALBERTO E.

Buenos Aires

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

Asociación Argentina de Microbiología y Comité internacional

Verocytotoxins are regarded as the main virulence factors of verocytotoxigenic Escherichia coli (VTEC), however subtilase cytotoxin (SubAB) also contributes to endothelial damage. SubAB is the prototype of a new family of AB5 cytotoxins and, as well as verocytotoxins, has Vero cell cytotoxic activity. The global distribution of this toxin is unknown. The aim of this study was to detect the gene of subtilase cytotoxin in VTEC obtained in Argentina from different sources (cattle and  bovine meat) and belonging to different serotypes.  These strains have been selected from a well characterized, previously described strain collection of our Laboratory. Data for vt1, vt2, eae, saa, and ehxA were used to select representative strains, allowing assessment of potential associations among subAB, other virulence factors and serotypes. In order to test 95 VTEC strains for the presence of the gene of subtilase cytotoxin, a previously described PCR multiplex which detects subA, vt2 and vt1 was used. PCR products have a size of 556, 255 and 180 bp, respectively. The reference E. coli strain EDL933, producer of VT1 and VT2, but lacking subtilase, was used as negative control for subA. DNA was obtained by boiling bacterial cultures. Amplification products were visualized in agarose gels, stained with ethidium bromide. We found that 21 strains were positive for subA, and belonged to serotypes O2:H5, O20:H19, O39:H49, O79:H19, O88:H21, O113:H21, O141:H8, O178:H19, ONT:H7, ONT:H8 and ONT:H19. As expected, subA was not found in eae-positive strains. In relation with the presence/absence of other genes that can also be also megaplasmid-encoded, we found two combinations: subA+/ ehxA+/saa+ (20 strains) and subA+/ ehxA-/saa+ (one strain). Strains positive for saa were not necesarily subA-positive. These results reveal the presence of cytotoxin subtilase gene in Argentinian VTEC isolates and confirm once again the great genetic variability of megaplasmids.