A molecular approach for the detection and characterization of novel tetracycline resistance genes, integrons, and integron-encoded antibiotic resistance determinants in the environment

The large scale use of antimicrobial drugs in agriculture and in human medicine has been implicated as a major cause of the anti biotic resistance (ABR) problem. However, most of the research has focused on the following: (i) the clinical setting, (ii) resistance mechanisms prevailing under selective conditions and, (iii) the culturable fraction of resistant bacteria. These trends limit our understanding of the extent of the ABR problem by overlooking resistance determinants in non-culturable bacteria residing outside the clinical setting and the role they may have in future clinical threats. Additionally, knowledge on the diversity and environmental reservoirs of genetic elements involved in the capture and dissemination of ABR determinants is still limited. In order to address these problems, a PCR-based, culture-independent approach was applied to the detection, characterization, tracking, and quantification of ABR determinants and integrons (a genetic system implicated in the uptake of multiple antibiotic resistance genes) in total DNA extracted from a variety of environmental samples. Four classes of tetracycline resistance genes: tet (M), tet (O), tet (Q), and tet (36), encoding ribosomal protection proteins (RPP) were detected in DNA from soil supplemented with manure from swine fed tetracycline as growth promoter.; A novel mosaic gene and two new putative classes of RPP genes were also detected. Tetracycline resistance genes were not detected in non-agricultural reference soils. A real-time PCR assay was developed for tracking and quantifying the novel Tet 36 determinant and to determine functionality of putative RPP genes. The target sequences were traced to the animals receiving tetracycline and they also appeared to encode functional genes since their frequency increased in correlation with exposure to tetracycline. Nine variants (one new) of aminoglycoside nucleotydiltransferases and three genotypes (one new) for resistance to quaternary ammonium drugs were detected in association with class 1 integrons. Integrons were prevalent and diverse among the tested soils, but integrons encoding ABR genes were only detected in manured soils. (Abstract shortened by UMI.)...