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

## TL;DR

Adding nitrate with metronidazole may improve subgingival biofilm health compared to using chlorhexidine or amoxicillin alone.

## Contribution

The study introduces a novel dual treatment of metronidazole and nitrate for subgingival biofilms.

## Key findings

- Low concentrations of amoxicillin and chlorhexidine inhibited microbial growth and impaired nitrate reduction.
- Metronidazole with nitrate increased Neisseria and Aggregatibacter species.
- Nitrate combined with metronidazole showed health-associated microbial changes.

## Abstract

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.