AUTHOR=Laumen Jolein G. E. , Van Dijck Christophe , Manoharan-Basil Sheeba S. , Abdellati Saïd , De Baetselier Irith , Cuylaerts Vicky , De Block Tessa , Van den Bossche Dorien , Xavier Basil B. , Malhotra-Kumar Surbhi , Kenyon Chris 

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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.776909

DOI=10.3389/fmicb.2021.776909

ISSN=1664-302X

ABSTRACT=<p><bold>Objectives:</bold> Chlorhexidine digluconate (chlorhexidine) and Listerine<sup>®</sup> mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in <italic>Neisseria gonorrhoeae</italic>. We performed <italic>in vitro</italic> challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials.</p><p><bold>Methods:</bold> A customized morbidostat was used to subject <italic>N. gonorrhoeae</italic> reference strain WHO-F to dynamically sustained Listerine<sup>®</sup> or chlorhexidine pressure for 18 days and 40 days, respectively. Cultures were sampled twice a week and minimal inhibitory concentrations (MICs) of Listerine<sup>®</sup>, chlorhexidine, ceftriaxone, ciprofloxacin, cefixime and azithromycin were determined using the agar dilution method. Isolates with an increased MIC for Listerine<sup>®</sup> or chlorhexidine were subjected to whole genome sequencing to track the evolution of resistance.</p><p><bold>Results:</bold> We were unable to increase MICs for Listerine<sup>®</sup>. 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 (<italic>mtrR</italic>) and the <italic>mlaA</italic> gene, part of the maintenance of lipid asymmetry (Mla) system.</p><p><bold>Conclusion:</bold> Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in <italic>N. gonorrhoeae</italic>. This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry.</p>