A-043. Novel Tetracycline Determinant Identified in Subsurface Bacteria

M. G. Brown, E. H. Mitchell, D. L. Balkwill;
Florida State Univ., Tallahassee, FL.

A novel tetracycline resistance determinant, yet to be named, has been identified in two strains of bacteria, G0887 and G0896, isolated from the deep terrestrial subsurface located at the Department of Energy Hanford Site, WA. The strains were isolated in 1992 from approximately 200 meters below land surface and then stored frozen in the Subsurface Microbial Culture Collection at Florida State University. The sediments containing the bacterial strains are thought to have been isolated from surface influence for more than 3 million years, lending to the possibility that the strains might possess novel resistance mechanisms and/or genes due to their long period of isolation from surface bacteria and lack of exposure to commercial antibiotics. The strains were initially screened for resistance to tetracycline through plating on selective media. To isolate the resistance determinant, total DNA extractions were performed and then partially digested with a restriction enzyme. The partial digest was ligated into the pEZ Seq vector from Lucigen and transformed into competent cells. The cells were plated on selective media containing a final concentration of 16 μg/ml of tetracycline, the highest clinical MIC breakpoint. The inserts from any colonies growing on the selective media were sequenced. The open reading frame with the highest similarity to known tetracycline resistance genes was determined and the nucleotide sequence was converted to an amino acid sequence. The amino acid sequences were compared to previously described tetracycline resistance determinants and showed less than 60% similarity. The new determinant sequence contains several transmembrane conserved domains suggesting that the new determinant confers resistance by active efflux.