Combined effects of nitrate and antimicrobial compounds on in vitro subgingival biofilms

Chlorhexidine and antibiotics are commonly used as adjunct treatments for periodontitis. However, these antimicrobials can lead to microbial resistance and chlorhexidine can impair health-associated nitrate (NO₃^-) metabolism. We tested the effect of chlorhexidine (0.002%), metronidazole (16 µg/ml) and amoxicillin (0.7 µg/ml), with and without 8 mM NO₃^-, on the bacterial composition and NO₃^- metabolism of subgingival plaque samples from 12 periodontitis patients grown in vitro for 8 h. The low sublethal concentrations of amoxicillin and chlorhexidine significantly inhibited microbial growth and impaired NO₃^- reduction, whereas the physiological concentration of metronidazole did not. A lower subgingival microbial dysbiosis index (SMDI) was found in the NO₃^- condition compared with amoxicillin alone and chlorhexidine with or without NO₃^- (p < 0.05). The SMDI of the metronidazole conditions was also significantly lower than in those with chlorhexidine (p < 0.05). Moreover, NO₃^- alone or combined with metronidazole appeared to increase Neisseria spp. and Aggregatibacter spp., whilst disease-associated changes were found in the chlorhexidine and amoxicillin conditions. Adding NO₃^- to metronidazole led to health-associated changes compared with metronidazole alone. In conclusion, low levels of amoxicillin and chlorhexidine limited microbial growth, impaired NO₃^- metabolism and were linked to disease-associated microbial profiles. A dual treatment of metronidazole + NO₃^- should be further investigated in clinical studies.