Isolation of Tetracycline-Resistant Megasphaera elsdenii Strains with Novel Mosaic Gene Combinations of tet (O) and tet (W) from Swine

Abstract

ABSTRACT Anaerobic bacteria insensitive to chlortetracycline (64 to 256 μg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. 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 ribosomal DNA sequence similarities with M. elsdenii LC-1 T . All eight strains had tetracycline MICs of between 128 and 256 μg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet (O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 μg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 μg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii 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 present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.