In vitro antimicrobial susceptibility testing of Borrelia burgdorferi: a microdilution MIC method and time-kill studies

Abstract

The susceptibility of Borrelia burgdorferi, the causative agent of Lyme borreliosis, to various antimicrobial agents varies widely among published studies. These differences are probably due in part to variations in susceptibility testing techniques and growth endpoint determinations. We developed a microdilution method for determining the MICs of antibiotics against B. burgdorferi. The method incorporated BSK II medium, a final inoculum of 10(6) cells per ml, and a 72-h incubation period and was found to be simple and highly reproducible. A variety of antibiotics and strains of B. burgdorferi and one strain of Borrelia hermsii were examined by this method. MICs of penicillin, ceftriaxone, and erythromycin for the B31 strain of B. burgdorferi were 0.06, 0.03, and 0.03 microgram/ml, respectively. We compared the MICs obtained by the microdilution method with those obtained by a macrodilution method using similar criteria for endpoint determinations and found the values obtained by both methods to be in close agreement. To further investigate the bactericidal activities of penicillin, ceftriaxone, and erythromycin against strain B31, we used subsurface plating to determine MBCs and we also performed time-kill studies. The MBCs of penicillin, ceftriaxone, and erythromycin were 0.125, 0.03, and 0.06 micrograms/ml, respectively. Time-kill curves demonstrated a greater than or equal to 3-log10-unit killing after 72 h with penicillin, ceftriaxone, and erythromycin; ceftriaxone provided the greatest reduction in CFU. The described methods offer a more standardized and objective approach to susceptibility testing of B. burgdorferi.