Genetic diversity of verocytotoxigenic Escherichia coli 0178:H19 isolated from dairy farms in Argentina.

BUSTAMANTE A. V., SANSO A. M., FERNÁNDEZ D., PADOLA N. L, LUCCHESI P. M. A. & PARMA A. E.

Buenos Aires

Simposio; 7th International Symposium on Shiga Toxin (Verocytotoxin) – producing Escherichia coli infections (VTEC2009); 2009

Asociación Argentina de Microbiología

Cattle are an important reservoir of verocytotoxigenic E. coli (VTEC) and represent a significant source from where this organism can enter the environment and also the food chain. E. coli O178:H19 was one of the VTEC serotypes detected with higher prevalence in cattle from four dairy farms sampled during different seasons along a 12-month period. The aim was to study the genetic diversity of these VTEC O178:H19 isolates obtained from cattle on dairy farms A, B, C and D. Eighteen isolates belonging to this serotype were analyzed. Ten of them corresponded to the winter sampling, four to autumn and also four to spring samplings. An initial screening by randomly amplified polymorphic DNA analysis (RAPD) was done, using the 15-mer oligonucleotide M13. DNA fingerprints were compared visually and each RAPD profile was defined by the presence or absence of bands at particular positions on the gel. Patterns were considered different when at least one polymorphic band could be detected. Furthermore, the isolates were typed by a multiple-locus variable-number tandem-repeat analysis (MLVA). Seven loci VNTR were analysed by PCR and amplification products were run in denaturing polyacrylamide gels and then stained with silver nitrate. Genetic diversity at each locus was calculated using Nei Index and a dendrogram was constructed using UPGMA.Four profiles were determined by RAPD, one of them was shared by isolates from the four dairy farms. Only one profile was present among isolates from farms A and B, two in those from farm C and three in farm D.In the MLVA assay, three out seven loci were polymorphic, presenting from two to seven alleles and diversity index values from 0.24 to 0.75. Nine different profiles could be identified. Two main clusters (I and II), with subclusters (I1 –I7 and II1-II2), were observed. Within clusters, MLVA profiles differed in one locus, indicating that they consist of closely related strains. Between clusters, however, MLVA profiles are due to polymorphisms in three loci. Almost all isolates (15/16) grouped into MLVA cluster I belonged to a same RAPD profile. Profiles belonging to cluster I were present among the four dairy farms. Furthermore, profiles belonging to cluster II were also present in farms C and D. In winter, when more isolates were obtained, more genetic profiles were observed both by RAPD and MLVA. One of these RAPD profiles was also present in isolates from autumn and spring.Strain diversity was observed within and among farms. Closely related E. coli O178:H19 isolates were present among the four dairy farms, but also, unrelated isolates were found in two farms (C and D). This study highlights the clonal diversity of the O178:H19 serotype present in Argentine dairy farms. Cattle are an important reservoir of verocytotoxigenic E. coli (VTEC) and represent a significant source from where this organism can enter the environment and also the food chain. E. coli O178:H19 was one of the VTEC serotypes detected with higher prevalence in cattle from four dairy farms sampled during different seasons along a 12-month period. The aim was to study the genetic diversity of these VTEC O178:H19 isolates obtained from cattle on dairy farms A, B, C and D. Eighteen isolates belonging to this serotype were analyzed. Ten of them corresponded to the winter sampling, four to autumn and also four to spring samplings. An initial screening by randomly amplified polymorphic DNA analysis (RAPD) was done, using the 15-mer oligonucleotide M13. DNA fingerprints were compared visually and each RAPD profile was defined by the presence or absence of bands at particular positions on the gel. Patterns were considered different when at least one polymorphic band could be detected. Furthermore, the isolates were typed by a multiple-locus variable-number tandem-repeat analysis (MLVA). Seven loci VNTR were analysed by PCR and amplification products were run in denaturing polyacrylamide gels and then stained with silver nitrate. Genetic diversity at each locus was calculated using Nei Index and a dendrogram was constructed using UPGMA.Four profiles were determined by RAPD, one of them was shared by isolates from the four dairy farms. Only one profile was present among isolates from farms A and B, two in those from farm C and three in farm D.In the MLVA assay, three out seven loci were polymorphic, presenting from two to seven alleles and diversity index values from 0.24 to 0.75. Nine different profiles could be identified. Two main clusters (I and II), with subclusters (I1 –I7 and II1-II2), were observed. Within clusters, MLVA profiles differed in one locus, indicating that they consist of closely related strains. Between clusters, however, MLVA profiles are due to polymorphisms in three loci. Almost all isolates (15/16) grouped into MLVA cluster I belonged to a same RAPD profile. Profiles belonging to cluster I were present among the four dairy farms. Furthermore, profiles belonging to cluster II were also present in farms C and D. In winter, when more isolates were obtained, more genetic profiles were observed both by RAPD and MLVA. One of these RAPD profiles was also present in isolates from autumn and spring.Strain diversity was observed within and among farms. Closely related E. coli O178:H19 isolates were present among the four dairy farms, but also, unrelated isolates were found in two farms (C and D). This study highlights the clonal diversity of the O178:H19 serotype present in Argentine dairy farms.