Multiplex PCR for Assessment of Tetracycline Resistance (tet) Genes in Antibiotic-Resistant Soil Bacteria and the Ecological Implications

Antibiotics are becoming increasingly prevalent in bacterial communities due to clinical and agricultural misuse and overuse in their environment. As exposure increases, so does the incidence of microbial resistance. Such is the case with bacterial resistance to tetracyclines, a phenotype often acquired through the horizontal gene transfer of tet genes between bacteria. The objective of this project was to analyze the bacterial diversity of tet resistance genes in soil from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine each bacterium’s degree of resistance. The 16S rRNA gene from antibiotic-resistant isolates was amplified by PCR and sequenced to identify the isolates. All isolates’ tet genes were amplified by multiplex PCR, sequenced, and compared. Among eight isolates, three distinct species were positively identified based on their 16S rRNA sequences and four distinct tet genes were identified, though all tested susceptible to tetracycline via the Kirby-Bauer test. This project clarifies some aspects of the ecology of antibiotic resistance genes, their natural ecological function and the potential for the expansion of intrinsic multi-antibiotic resistance into new ecosystems and/or hosts.