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

Abstract

<b>Objectives:</b> Chlorhexidine digluconate (chlorhexidine) and Listerine^® mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in <i>Neisseria gonorrhoeae</i>. We performed <i>in vitro</i> challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials. <b>Methods:</b> A customized morbidostat was used to subject <i>N. gonorrhoeae</i> 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. <b>Results:</b> 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 (<i>mtrR</i>) and the <i>mlaA</i> gene, part of the maintenance of lipid asymmetry (Mla) system. <b>Conclusion:</b> Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in <i>N. gonorrhoeae</i>. This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry.