Sub-Inhibitory Concentrations of Chlorhexidine Induce Resistance to Chlorhexidine and Decrease Antibiotic Susceptibility in Neisseria gonorrhoeae .

Objectives: Chlorhexidine digluconate (chlorhexidine) and Listerine ^® mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in Neisseria gonorrhoeae . We performed in vitro challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials. Methods: A customized morbidostat was used to subject N. gonorrhoeae reference strain WHO-F to dynamically sustained Listerine ^® or chlorhexidine pressure for 18 days and 40 days, respectively. Cultures were sampled twice a week and minimal inhibitory concentrations (MICs) of Listerine ^® , chlorhexidine, ceftriaxone, ciprofloxacin, cefixime and azithromycin were determined using the agar dilution method. Isolates with an increased MIC for Listerine ^® or chlorhexidine were subjected to whole genome sequencing to track the evolution of resistance. Results: We were unable to increase MICs for Listerine ^® . Three out of five cultures developed a 10-fold increase in chlorhexidine MIC within 40 days compared to baseline (from 2 to 20 mg/L). Increases in chlorhexidine MIC were positively associated with increases in the MICs of azithromycin and ciprofloxacin. Low-to-higher-level chlorhexidine resistance (2-20 mg/L) was associated with mutations in NorM. Higher-level resistance (20 mg/L) was temporally associated with mutations upstream of the MtrCDE efflux pump repressor ( mtrR ) and the mlaA gene, part of the maintenance of lipid asymmetry (Mla) system. Conclusion: Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in N. gonorrhoeae . This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry.
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
(Copyright © 2021 Laumen, Van Dijck, Manoharan-Basil, Abdellati, De Baetselier, Cuylaerts, De Block, Van den Bossche, Xavier, Malhotra-Kumar and Kenyon.)