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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.

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