Detección del gen "stx2" en muestras ambientales y evaluación de su variabilidad

Abstract

Shiga toxin producing "Escherichia coli" have emerged recently as human pathogens. The main virulence factors are the Shiga toxins (which are bacteriophage encoded), the external membrane protein called intimin, which is responsible for the adhesion of the bacterium to the intestinal epithelium and the hemorrhagic hemolysin. The aim of the study was to analyse the prevalence of the "stx2 " gene, the detection, isolation and characterization of the "stx2 "gene-carrying bacteria from aquatic environments and the characterization of the "stx2 " bacteriophages which are integrated in the genome of these bacteria. A combination of the NMP and the nested PCR method was used to evaluate the prevalence of the "stx2 "gene in raw municipal sewage and animal wastewater. The "stx2 " gene was detected in all the samples with a constant ratio between "stx2 " and the bacterial indicators analysed. 144 strains were isolated by colony blot hybridisation. 72 "E. Coli" O157 strains were isolated using IMS, CT-SMAC and the immunological detection of the O157 antigen. These strains were biochemically characterized and a total of 65 "stx2 " and 28 O157 strains were chosen as representatives for further studies. All the strains were identified as "E. Coli"belonging to 36 diferent serotypes. These strains antibiotics were mostly resitant to sulfonamides, tetracycline, cloramfenicol, thrimethroprim. All the strains carried one or two "stx2 "gene variants. The "stx1 "gene was detetcted in 28% and 20% of the isolated strains of urban sewage and bovine slaughterhouse. The eae gene was detected in "E. Coli"O157:H7, and the ehxA gene in 7%, 46% and 11% of municipal sewage, bovine slaughterhouse, and mixed slaughterhouse wastewater origin, respectively. The "saa" gene was detected in 5 strains. Production of Stx2 protein was detected mainly in strains from bovine origin. A greater percentage of strains expressed the "stx1 "gene. Most of the isolated strains harbored "stx2 " bacteriophages in their genome (mainly strains from bovine slaughterhouse origin), which carried different "stx2 "variants, presented variability in the RFLP analysis as well as in the infection to different hosts. The diversity of "stx2 " bacteriophages observed could contribute to the dispersion of the gene stx2 between the different bacterial populations.