Susceptibility to pleuromutilins inBrachyspira(Serpulina)hyodysenteriae

The pleuromutilins are the only antimicrobial agents with sufficient minimum inhibitory concentration (MIC) values left to treat swine dysentery in Sweden. Other antimicrobials are either not approved for use against swine dysentery or only partly active againstBrachyspira hyodysenteriae. To date, in Sweden two pleuromutilins, tiamulin and valnemulin, are authorized for use in pigs. This study includes a comparison between MICs of tiamulin and valnemulin for Swedish field isolates ofB. hyodysenteriae, as determined by broth dilution. For different isolates the MIC of tiamulin was between 0 and 8 times higher than that of valnemulin. No resistance to pleuromutilins was recorded (tiamulin MIC range 0.031–2 μg/ml, valnemulin MIC range ≤0.016–1 μg/ml).In vitrodevelopment of tiamulin resistance was also studied. TwoB. hyodysenteriaeand twoB. pilosicolistrains became resistant to tiamulin following reiterated passages on agar containing tiamulin in increasing concentrations. The resistance emerged slowly and three of the strains that went through more than 60 passages increased their tiamulin MICs from 0.031–0.25 to more than 128 μg/ml. The tiamulin MIC for oneB. hyodysenteriaestrain that went through 29 passages increased from 0.0125 to 4 μg/ml. OneB. pilosicolistrain developed cross-resistance to valnemulin; the MIC increased from 0.25 to more than 64 μg/ml. The valnemulin MIC for oneB. hyodysenteriaestrain increased from 0.031 μg/ml to 32 μg/ml. Valnemulin MIC was not determined for theB. hyodysenteriaestrain that only went through 29 passages. The valnemulin MIC of the otherB. pilosicolistrain increased from 0.031 to 4 μg/ml.

Brachyspira (Serpulina)hyodysenteriae gyrB Mutants and Interstrain Transfer of Coumermycin A1 Resistance

ABSTRACT To further develop genetic techniques for the enteropathogen Brachyspira hyodysenteriae, thegyrB gene of this spirochete was isolated from a λZAPII library of strain B204 genomic DNA and sequenced. The putative protein encoded by this gene exhibited up to 55% amino acid sequence identity with GyrB proteins of various bacterial species, including other spirochetes. B. hyodysenteriae coumermycin A1-resistant (Cnr) mutant strains, both spontaneous and UV induced, were isolated by plating B204 cells onto Trypticase soy blood agar plates containing 0.5 μg of coumermycin A1/ml. The coumermycin A1 MICs were 25 to 100 μg/ml for the resistant strains and 0.1 to 0.25 μg/ml for strain B204. Four Cnr 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 Cnr strain 435A (Gly78 to Ser) and Cmr Kmr strain SH (ΔflaA1::catΔnox::kan) were cultured together in brain heart infusion broth containing 10% (vol/vol) heat-treated (56°C, 30 min) calf serum, cells resistant to chloramphenicol, coumermycin A1, and kanamycin could be isolated from the cocultures after overnight incubation, but such cells could not be isolated from monocultures of either strain. Seven CnrKmr Cmr strains were tested and were determined to have resistance genotypes of both strain 435A and strain SH. Cnr Kmr Cmr cells could not be isolated when antiserum to the bacteriophage-like agent VSH-1 was added to cocultures, and the numbers of resistant cells increased fivefold when mitomycin C, an inducer of VSH-1 production, was added. These results indicate that coumermycin resistance associated with agyrB mutation is a useful selection marker for monitoring gene exchange between B. hyodysenteriae cells. Gene transfer readily occurs between B. hyodysenteriae cells in broth culture, a finding with practical importance. VSH-1 is the likely mechanism for gene transfer.