Development of resistance to metronidazole and minocycline in vitro

By local delivery of antibiotics to periodontal pockets, very high initial concentrations are often quickly succeeded by subinhibitory concentrations, which may facilitate development of bacterial resistance. The purpose of the present study was to investigate possible development of resistance in suspected periodontal pathogens after exposure to subinhibitory concentrations of metronidazole and minocycline. The minimal inhibitory concentration (MIC) of 18 reference strains and 12 clinical isolates was determined by a broth dilution method. Subsequently, all strains with MIC < 8 micrograms/ml were exposed to serial passage on plates containing subinhibitory and gradually increasing concentrations of antibiotics, until growth was inhibited. Initially, most strains were inhibited at < or = 0.250 microgram/ml of minocycline and < or = 0.5 microgram/ml of metronidazole, though A. actinomycetemcomitans was resistant to metronidazole. After growth at subinhibitory concentrations, 8 strains survived 1-2 x and 11 stains survived 8-32 x their initial MIC of metronidazole, growing at up to 8 micrograms/ml. All A. actinomycetemcomitans survived 8-64 x their initial MIC of minocycline, growing at > or = 2 micrograms/ml, while all other strains were inhibited at < or = 0.250 microgram/ml, corresponding to a 1-8 x increase in their initial MIC. Thus, development of resistance was observed for periodontal bacteria growing at up to 64 x their initial MIC, but the final level of resistance was moderate.