Abstract
Chlorhexidine (CHX) is an essential medicine used as a topical antiseptic in skin and oral healthcare treatments. The widespread use of CHX has increased concerns regarding the development of antiseptic resistance in Enterobacteria and its potential impact on cross-resistance to other antimicrobials. Similar to other cationic antiseptics, resistance to CHX is believed to be driven by three membrane-based mechanisms: lipid synthesis/transport, altered porin expression, and increased efflux pump activity; however, specific gene and protein alterations associated with CHX resistance remain unclear. Here, we adapted Escherichia coli K-12 BW25113 to increasing concentrations of CHX to determine what phenotypic, morphological, genomic, transcriptomic, and proteomic changes occurred. We found that CHX-adapted E. coli isolates possessed no cross-resistance to any other antimicrobials we tested. Scanning electron microscopy imaging revealed that CHX adaptation significantly altered mean cell widths and lengths. Proteomic analyses identified changes in the abundance of porin OmpF, lipid synthesis/transporter MlaA, and efflux pump MdfA. Proteomic and transcriptomic analyses identified that CHX adaptation altered E. coli transcripts and proteins controlling acid resistance (gadE, cdaR) and antimicrobial stress-inducible pathways Mar-Sox-Rob, stringent response systems. Whole genome sequencing analyses revealed that all CHX-resistant isolates had single nucleotide variants in the retrograde lipid transporter gene mlaA as well as the yghQ gene associated with lipid A transport and synthesis. CHX resistant phenotypes were reversible only when complemented with a functional copy of the mlaA gene. Our results highlight the importance of retrograde phospholipid transport and stress response systems in CHX resistance and the consequences of prolonged CHX exposure.
Highlights
Chlorhexidine (CHX) is a commonly used antiseptic and disinfectant in medical, dental, and veterinary practice and it is listed as an essential medicine by the World Health Organization (World Health Organization, 2019)
CHX-adapted isolates were susceptible to all quaternary ammonium compound (QAC) cationic antiseptics we tested based on their minimum inhibitory concentration (MIC) values, as well as to all other antibiotics, including colistin (Table 2)
It is notable that CHX-adapted isolates were more susceptible to QACs cetyldimethylethylammonium bromide (CDAB) and cetytrimethylammonium bromide (CTAB) and to the aminoglycoside antibiotic tobramycin when compared to the wild-type BW25113 control (WT) (Table 2)
Summary
Chlorhexidine (CHX) is a commonly used antiseptic and disinfectant in medical, dental, and veterinary practice and it is listed as an essential medicine by the World Health Organization (World Health Organization, 2019). CHX is the active antimicrobial ingredient used in a variety of clinical antiseptics (skin, oral, and eye washes) and daily use products (cosmetics and personal hygiene products), making CHX usage widespread. Increasing resistance as well as possible antibiotic cross-resistance is very concerning given CHX’s clinical importance as a medical antiseptic and, in some cases, as a last-line debridement treatment (Brookes et al, 2020). This highlights an important knowledge gap to address regarding how intrinsic CHX mechanisms of resistance develop, especially among antimicrobial resistant species deemed to be critical priority pathogens. Despite the absence of defined CHX breakpoints, we will refer to reduced CHX susceptibility as “resistant” rather than “tolerant” based on minimum inhibitory concentration values defined for antiseptics and antibiotics (Cerf et al, 2010; Brauner et al, 2016)
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