GENETIC DIVERSITY OF O157:H7 AND NON-O157 VEROCYTOTOXIGENIC ESCHERICHIA COLI INFERRED BY MULTIPLE LOCUS VARIABLE-NUMBER TANDEM-REPEAT ANALYSIS

BUSTAMANTE, ANA V.; SANSO, ANDREA M.; LUCCHESI, PAULA M. A.; PARMA, ALBERTO E.

Buenos Aires

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

VTEC Committee-Asoc. Arg. Microbiol.

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS) globally, there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). In Argentina, non-O157 strains are detected in around 40% of HUS and diarrhoea cases. Ruminants, particularly cattle, are the most common reservoirs for O157 and non-O157 VTEC strains causing infections in human as a result of foodborne and waterborne fecal contamination. A large number of VTEC serotypes have been isolated from cattle and derived food products in our country. MLVA typing, which analyses polymorphisms in variable-number of tandem repeats (VNTR) loci, is generally considered to be a robust and fast method with high discriminatory power. It has been applied in several studies for the specific typing of O157:H7 isolates, but there is a need to perform MLVA assays useful also for non-O157 VTEC. The aim of this study was to type and to analyse the genetic diversity of native strains of VTEC O157:H7 and non-O157 from Argentina, by the analysis of seven generic VNTR loci. The 64 studied strains were isolated from cattle, patients with diarrhoea and contaminated food in previous studies, between 1996 and 2002. They belong to 8 different serotypes: O20: H19 (8). O91: H21 (4), O113: H21 (4), O117: H7 (9), O145: H- (4), O171: H2 (11), O174: H21 (10), and O157: H7 (14). The reference O157: H7 strain EDL933 was used as positive control. Genetic diversity at each locus was calculated using Nei Index (D) and a dendrogram was constructed using UPGMA clustering method. All tested VTEC strains could be typed by this MLVA assay, and revealed 41 different MLVA genotypes. CVN004 (D = 0.40) and CVN015 (D = 0.38) showed the lowest number of alleles (3), whereas CNV014 (D = 0.88) was the most variable locus in the present analysis (12 alleles). One locus (CVN003) could only be amplified in O145:H- and O157:H7 isolates. The MLVA dendrogram showed two main clusters which corresponded to O157:H7 (I) and non-O157 (II), respectively. The loci that had the dominant influence on the clustering of the branch I isolates were CVN002, CVN007 and CVN015 where the common allleles #1, #6 and #5, respectively, were not seen among the O157:H7 isolates. Within the non-O157 subcluster the strains could be discriminated by alleles of loci CVN001, CVN004 and CVN014. The authors who developed this assay applied it for the typing of the ECOR collection and in a set of pathogenic E. coli and Shigella isolates. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157. The data obtained in the present study emphasize the high index of diversity in the analyzed VTEC and the need of additonal research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.