Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2004
Publication Date: 6/20/2004
Citation: Stanton, T.B., Mcdowall, J.S., Rasmussen, M.A. 2004. Diverse tetracycline resistance genotypes of Megasphaera elsdenii strains selectively cultured from swine feces. Applied and Environmental Microbiology. 70(6):3754-3757. Interpretive Summary: Our problem is to find a model bacterium or "indicator organism" to be used as a standard for measuring the antibiotic resistance of bacteria in the pig's intestine. Such an indicator organism will be useful for judging the success of programs to reduce antibiotic resistance. In our last paper we identified a bacterium, Megasphaera elsdenii, in the swine intestine. M. elsdenii is highly resistant to tetracycline antibiotics. Tetracycline antibiotics have been commonly fed to swine both to prevent disease and to promote growth. In this paper, we found that swine from two organic farms that have not used antibiotics for the last four years still have high levels of resistant M. elsdenii bacteria. These M. elsdenii bacteria have six different types of genes for tetracycline resistance. These results are important for two reasons. First they show that antibiotic resistance genes not only are present but also are evolving into new genes within bacteria in the swine intestinal tract. Second, they confirm that bacteria resistant to tetracycline persist in swine even when antibiotics are not being use. Efforts to effectively reduce antimicrobial resistance will require methods or strategies other than or in addition to restricting antibiotic use. Users of this information include Federal agencies regulating antibiotic use; animal health industry; veterinary practitioners and clinicians; pharmaceutical manufacturers; and basic and applied research scientists investigating antibiotics and antibiotic alternatives.
Technical Abstract: Megasphaera elsdenii strains with high levels of tetracycline-resistance have been isolated from swine cecal samples (30). The strains carry novel, mosaic tetracycline-resistance genes, designated tet(OWO), products of recombination between tet(O) and tet(W) genes. In this study, 30 M. elsdenii strains were selectively isolated on Me109M medium from fecal samples of organically raised swine. Me109M conditions selective for M. elsdenii growth included lactate (1%, w/v) as a growth substrate, the ionophore monensin (50 ug/ml), and low pH (5.0). Under anaerobic culture conditions, total bacterial counts were 6 x 10**10 cfu/gm feces on RTC agar, a nutritionally complex, rumen-fluid based medium. On Me109M agar, M. elsdenii viable counts averaged 2.4 x 10**8 cfu/gm feces. M. elsdenii strains were analyzed for tet genes by PCR assays and by direct sequencing. Nine strains contained no detectable tet gene. Eleven strains had tet(OWO) genes. Eight strains displayed a new mosaic genotype, tet(OW), produced from a single crossover recombination between tet(O) and tet(W). Two strains contained one of two different tet(W) genes. A ruminant M. elsdenii strain contained tet(O). M. elsdenii strains with tet(OWO) genes exhibited the highest MIC's for tetracyclines (128 to > 256 ug/ml), suggesting greater resistance to tetracycline is a selective force for tet evolution by interclass recombination. Seven tet genotypes have now been described for M. elsdenii, an exemplar species and perhaps a site for tet gene evolution in the mammalian intestinal tract.