Evaluation and use of a micro-broth dilution procedure for testing sensitivity of fermentative avian mycoplasmas to antibiotics

A micro-broth dilution procedure is described for testing the sensitivity of glucose-fermenting avian mycoplasmas to antibiotics. A systematic evaluation was made of potential sources of variation that could affect the reliability of this procedure. Strain WVU 1853 of Mycoplasma synoviae (MS) was used as a standard culture and was tested against the antibiotics erythromycin, spiramycin, streptomycin, tetracycline, tiamulin, and tylosin. Inoculum density, duration of incubation before the test was read, and initial pH of the medium significantly affected test results. Type of serum and concentration of pig serum in the medium had little effect, provided the test was read when the phenol red indicator in the medium of a culture control (without antibiotic) had changed from deep red (pH 7.8) to orange-yellow (pH 7). The presence of bacterial inhibitors in the medium, the solvents used to dissolve some antibiotics, the use of thawed or actively growing cultures, and the number of passages of the culture before testing did not appear to be significant causes of test variation. By controlling important sources of variation, a standardized test was developed that gave reproducible results. The standardized test was used to determine the sensitivity to antibiotics of 49 strains of M. gallisepticum (MG), eight strains of MS, five strains of M. pullorum, seven strains of M. gallinaceum, and one strain of M. columborale. Minimum inhibitory concentrations of the macrolide antibiotics were lower for the majority of MG strains than for the other Mycoplasma species tested. However, some strains of MG showed acquired resistance to the macrolide antibiotics. These strains also showed increased resistance to lincomycin and tiamulin.