New colorimetric microdilution method for in vitro susceptibility testing of Borrelia burgdorferi against antimicrobial substances

A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isol ates, including all three genospecies pathogenic for humans. In addition, i n vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrel ia bissettii tick isolates were investigated. The applied test system is ba sed upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations ( MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, l ess than or equal to 0.125 mu g/ml), ceftriaxone (MIC range, less than or e qual to 0.015-0.06 mu g/ml), and azithromycin (MIC range, less than or equa l to 0.015-0.06 mu g/ml). Whereas tobramycin (MIC range, 8-64 mu g/ml) exhi bited little activity, spectinomycin (MIC range, 0.25-2 mu g/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of peni cillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the gen ospecies Borrelia garinii (P<0.05). Further significant differences with re spect to the MIC values of the other antimicrobial agents tested were not n oted. The colorimetric microdilution method offered the advantages of relia bility, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.