Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/2003
Publication Date: 7/20/2003
Citation: STANTON, T.B., HUMPHREY, S.B. ISOLATION OF TETRACYCLINE-RESISTANT MEGASPHAERA ELSDENII STRAINS WITH NOVEL MOSAIC GENE COMBINATIONS OF TET(O) AND TET(W) FROM SWINE. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 2003. V. 69. P. 3874-3882. Interpretive Summary: We grew bacteria resistant to the antibiotic tetracycline from the intestines of swine fed and not fed tetracycline. One of the tetracycline-resistant bacteria was identified as Megasphaera elsdenii, an important and common bacterium in the intestines of humans and animals. The M. elsdenii bacteria contain a new type of tetracycline resistance gene. The gene was a mosaic gene, made from pieces of different tetracycline genes. These findings are significant for two reasons. They indicate that some molecular tests for tetracycline genes can incorrectly identify the mosaic genes. The show that the swine intestinal tract is not only a place where antibiotic-resistant bacteria live but also a place where antibiotic resistance genes evolve.
Technical Abstract: Strains of anaerobic bacteria insensitive to high concentrations of chlortetracylcine (64 and 256 mg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline (400 g/T). A nutritionally complex, rumen-fluid based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large coccus morphology, rapid growth on lactate, and 16S rDNA sequence similarities with M. elsdenii LC-1T. All eight strains had tetracycline MIC values between 128-256 mg/ml and gave a positive reaction for the tet(O) gene in PCR assays for 14 different tet classes. By contrast, three ruminant M. elsdenii strains recovered from 25-year-old culture stocks had tetracycline MICs of 4 mg/ml and did not contain tet genes. The tet genes of tetracycline-resistant M. elsdenii strains 7-11 and 14-14 were amplified and cloned into E. coli. Both genes conferred tetracycline resistance (MIC = 32-64 mg/ml) on recombinant E. coli strains. The sequences of the cloned resistance genes revealed the 7-11 and 14-14 genes represent two different mosaic genes formed by interclass (double crossover) recombination events involving tet(O) and tet(W). One or the other genotype was detected in the eight M. elsdenii strains by PCR assays. These findings suggest a role for the commensalistic microbiota not only in the preservation and dissemination of resistance in the intestinal tract but also in the evolution of antibiotic resistance.