Prevalence of -lactamase-producing bacteria in human periodontitis

Background and Objective Beta-lactam antibiotics prescribed in periodontal therapy are vulnerable to degradation by bacterial -lactamases. This study evaluated the occurrence of -lactamase-positive subgingival bacteria in chronic periodontitis subjects of USA origin, and assessed their in vitro resistance to metronidazole at a breakpoint concentration of 4g/mL. Material and Methods Subgingival plaque specimens from deep periodontal pockets with bleeding on probing were removed from 564 adults with severe chronic periodontitis before treatment. The samples were transported in VMGA III and then plated onto: (i) nonselective enriched Brucella blood agar (EBBA) and incubated anaerobically for 7d; and (ii) selective trypticase soy-bacitracin-vancomycin (TSBV) and incubated for 3 d in air + 5% CO2. At the end of the incubation periods, the bacterial test species were identified and quantified. Specimen dilutions were also plated onto EBBA plates supplemented with 2g/mL of amoxicillin, a combination of 2g/mL of amoxicillin plus 2g/mL of the -lactamase inhibitor clavulanic acid, or 4g/mL of metronidazole, followed by anaerobic incubation for 7d. Bacterial test species presumptively positive for -lactamase production were identified by growth on EBBA primary isolation plates supplemented with amoxicillin alone and no growth on EBBA primary isolation plates containing both amoxicillin plus clavulanic acid. A subset of such isolates was subjected to nitrocefin-based chromogenic disk testing to confirm the presence of -lactamase activity. In vitro resistance to 4g/mL of metronidazole was noted when growth of test species occurred on metronidazole-supplemented EBBA culture plates. Results Two-hundred and ninety-four (52.1%) of the study subjects yielded -lactamase-producing subgingival bacterial test species, with Prevotella intermedia/nigrescens, Fusobacterium nucleatum and other Prevotella species most frequently identified as -lactamase-producing organisms. Of the -lactamase-producing bacterial test species strains recovered, 98.9% were susceptible in vitro to metronidazole at 4g/mL. Conclusion The occurrence of -lactamase-positive subgingival bacterial species in more than half of the subjects with severe chronic periodontitis raises questions about the therapeutic potential of single-drug regimens with -lactam antibiotics in periodontal therapy. The in vitro effectiveness of metronidazole against nearly all recovered -lactamase-producing subgingival bacterial species further supports clinical periodontitis treatment strategies involving the combination of systemic amoxicillin plus metronidazole.