Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2001
Publication Date: N/A
Citation: Interpretive Summary: Helical-shaped bacteria, called spirochetes, cause intestinal diseases in pigs, chickens, and possibly, in humans. The worldwide disease swine dysentery caused by an intestinal spirochete is estimated to cost the U.S. pork industry $100 million per year. One of the scientific tools that we need to study Brachyspira hyodysenteriae (B. hyo), which causes swine dysentery, is a genetic tool. If we can study and manipulate the genes of B. hyo, we can better identify and inhibit properties that enable this bacterium to cause disease. We will be better equipped to design antibiotics targeting specific weaknesses of this bacterium and to make vaccines against weakened strains. In these studies, we developed two genetic tools for working with B. hyo. First, we made special cells known as mutants. These mutants have a change in one of their genes. That change protects the cells from being killed by the antibiotic coumermycin A1. (The mutants are not dangerous because they are kept in sealed tubes and coumermycin A1 is not used to treat swine dysentery.) Second, we demonstrated that resistance to coumermycin can be passed between B. hyo cells. Using these new tools, we will be able to study antibiotic resistance transfer and ways to prevent antibiotic resistance from spreading. We will be able to genetically modify B. hyo strains so they no longer cause disease and thus can be used in formulating vaccines. This information is useful to industry and research scientists, manufacturers of vaccines and antibiotics, regulatory agency officials, and microbiologists investigating spirochete diseases.
Technical Abstract: The gyrB gene of the spirochete was isolated from a lambdaZAPII library of strain B204 genomic DNA and was sequenced. The putative protein encoded by this gene shared up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A1-resistant (Cn**r) mutant strains, both spontaneous and UV-induced, were isolated by plating B204 cells onto TSB agar plates containing 0.5 ug coumermycin A1/ml. Coumermycin A1 MIC values were 25-100 ug/ml for the resistant strains and 0.1-0.25 ug/ml for strain B204. Four Cn**r strains had single nucleotide changes in their gyrB genes, corresponding to GyrB amino acid changes of Gly78 to Ser (two strains), Gly78 to Cys, and Thr166 to Ala. When Cn**r strain 435A (Gly78 to Ser) and Cm**rKm**r strain SH (delta flaA1::cat; deltanox::kan) were cultured together in BHIS broth, cells resistant to all three antibiotics could be isolated from the co-cultures after overnight incubation. Seven Cn**rKm**rCm**r strains were tested and determined to have resistance genotypes of both strain 435A and SH. Cn**rKm**rCm**r cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to the co-cultures and their numbers increased five-fold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance is a useful selectable marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture and VSH-1 is the likely mechanism for gene transfer.