Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 6, 2008
Publication Date: May 20, 2008
Citation: Stanton, T.B., Humphrey, S.B., Sharma, V.K., Zuerner, R.L. 2008. Collateral effects of antibiotics - carbadox and metronidazole induce VSH-1 and facilitate gene transfer among Brachyspira hyodysenteriae strains. Applied and Environmental Microbiology. 74(10):2950-2956. Interpretive Summary: We found that two antibiotics used to treat swine dysentery have 'collateral effects' on the anaerobic bacterium that causes swine dysentery. The name of that bacterium is Brachyspira hyodysenteriae. The antibiotics are carbadox and metronidazole. We exposed B. hyodysenteriae bacteria to subinhibitory levels of carbadox or metronidazole. Subinhibitory levels of carbadox are levels that do not inhibit growth of B. hyodysenteriae, in other words levels that do not kill the bacterial cells. When B. hyodysenteriae cells were exposed to subinhibitory levels of carbadox or metronidazole they made an unusual gene transfer agent called VSH-1. VSH-1 carries genes (DNA) from one dying B. hyodysenteriae bacterium to a living B. hyodysenteriae bacterium. We have shown that some of the genes transferred by VSH-1 are antibiotic resistance genes, genes that protect B. hyodysenteriae from the antibiotics tetracycline or tylosin. These results are important because carbadox is often used in medicated swine feeds at low or subtherapeutic levels. At levels that are subinhibitory for bacterial growth, carbadox could be stimulating gene transfer among bacteria in the intestinal tract, including the transfer of genes for antibiotic resistance. Additional experiments are needed and planned to evaluate this hypothesis. This information should be useful to the scientists and manufacturers in the antibiotic industry, to scientists investigating antibiotic resistance, to swine producers and veterinary practitioners using medicated feeds, to drug regulatory agencies.
Technical Abstract: B. hyodysenteriae is an anaerobic spirochete and the etiologic agent of swine dysentery. The genome of this spirochete contains a mitomycin C-inducible, prophage-like, gene transfer agent designated VSH-1. VSH-1 particles package random 7.5 kb fragments of the B. hyodysenteriae genome and transfer genes between B. hyodysenteriae cells. Except for mitomycin C, chemicals and environmental conditions inducing VSH-1 production are unknown. Antimicrobials used in swine management and stressors inducing traditional prophages might induce VSH-1 and thereby stimulate lateral gene transfer between B. hyodysenteriae cells. VSH-1 Inducers were presumptively identified by quantitative real time PCR (QRTPCR) to detect increased transcription of hvp38 (VSH-1 head protein gene). To confirm that increased hvp38 transcription correlated with VSH-1 virion production, VSH-1-associated 7.5 kb DNA was detected by gel electrophoresis and VSH-1 particles were detected by electron microscopy. Nine antimicrobials did not increase hvp38 transcription. By contrast, hvp38 transcription increased 16 to > 500-fold in B. hyodysenteriae cultures treated with heat shock (50 degree C, 1h), H2O2 (300 micro M), mitomycin C (10 micro g/ml), carbadox (0.5 micro g/ml), and metronidazole (1.0 micro g/ml). VSH-1 particles were detected by electron microscopy in cultures exhibiting the highest levels of hvp38 transcription. Metronidazole and carbadox were potent VSH-1 inducers. Carbadox-and metroidazole-induced VSH-1 particles transduced tylosin and chloramphenicol resistance genes. In the United States, carbadox is a common feed additive for preventing swine dysentery. The results of these studies suggest a need to evaluate VSH-1 induction and lateral gene transfer in vivo as possible collateral effects of carbadox therapy. Additionally the potentially broader impact of carbadox and metronidazole as prophage inducers on intestinal microbial ecology deserves investigation.