Phenotypic and Multi-Omics Characterization of Escherichia coli K-12 Adapted to Chlorhexidine Identifies the Role of MlaA and Other Cell Envelope Alterations Regulated by Stress Inducible Pathways in CHX Resistance.

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.

Antiseptic quaternary ammonium compound tolerance by gram-negative bacteria can be rapidly detected using an impermeant fluorescent dye-based assay.

Biocides such as quaternary ammonium compounds (QACs) are potentially important contributors towards bacterial antimicrobial resistance development, however, their contributions are unclear due to a lack of internationally recognized biocide testing standards. Methods to detect QAC tolerance are limited to laborious traditional antimicrobial susceptibility testing (AST) methods. Here, we developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to discriminate QAC susceptibility among Gram-negative Enterobacterales and Pseudomonadales species. RFDMIA uses a membrane impermeant fluorescent dye, propidium iodide, in a 30-min 96-well fluorescent microplate-based assay where cell suspensions are exposed to increasing QAC concentrations. Our results demonstrate that RFDMIA can discriminate between QAC-susceptible and QAC-adapted Escherichia coli tolerant phenotypes and predict benzalkonium and cetrimide tolerance in all species tested except for intrinsically fluorescent Pseudomonas aeruginosa. RFDMIA identified a close association to minimum inhibitory concentration values determined by broth microdilution AST and increasing fluorescent dye emission values. RFDMIA emission values and scanning electron microscopy results also suggest that CET-adapted E. coli isolates have a CET dependence, where cells require sub-inhibitory CET concentrations to maintain bacilliform cell integrity. Overall, this study generates a new, rapid, sensitive fluorescent assay capable of detecting QAC-susceptible Gram-negative bacteria phenotypes and cell membrane perturbations.