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

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.

Applying fluorescent dye assays to discriminate Escherichia coli chlorhexidine resistance phenotypes from porin and mlaA deletions and efflux pumps

“…To verify CHX resistance phenotypes of the various E. coli strains prior to RFDMIA analysis, AST was performed with E. coli gene deletion mutants, plasmid transformants, and CHX-adapted isolates to determine their CHX MIC values (Table 1 ). As previously reported 22 , AST of CHX-adapted E. coli CHXR1 demonstrated a fourfold increase in CHX MIC values when compared to the unadapted E. coli BW25113 strain (Table 1 ). Prior genetic analyses of CHXR1 in a recent study revealed that this adapted isolate possesses numerous gene mutations, including deleterious mutations to mlaA and its upstream promoter 22 .…”

“…As previously reported 22 , AST of CHX-adapted E. coli CHXR1 demonstrated a fourfold increase in CHX MIC values when compared to the unadapted E. coli BW25113 strain (Table 1 ). Prior genetic analyses of CHXR1 in a recent study revealed that this adapted isolate possesses numerous gene mutations, including deleterious mutations to mlaA and its upstream promoter 22 . As shown in Table 1 , only plasmid complementation with a wildtype copy of mlaA reverted this isolate back to CHX susceptible phenotypes observed for the unadapted WT 22 .…”

“…MlaA forms a complex with OmpC/OmpF 23 and is part of the Mla intermembrane phospholipid transport system that maintains lipopolysaccharide asymmetry in the outer membrane 24 . Deletion of mlaA was shown to decrease CHX resistance by fourfold in CHX-adapted E. coli 22 and other in vitro CHX adaption studies in E. coli 25 . The mechanism of Δ mlaA resistance is hypothesized to reduce CHX binding to lipopolysaccharides enriched in the outer membrane by increasing phospholipid accumulation.…”

“…In addition to efflux, CHX resistance mechanisms are speculated to alter outer membrane drug permeability caused by the loss or down-regulation of general diffusion porins such as OmpC/F 5 , 21 , however, porins have never been directly examined for CHX resistance. A recent multi-omics study of in vitro CHX-adapted E. coli K-12 identified that ompF was significantly down-regulated in the transcriptomes and proteomes of CHX-adapted E. coli isolates (CHXR1) 22 . In the same multi-omics study, the loss of an outer membrane lipoprotein MlaA was also implicated 22 .…”

“…A recent multi-omics study of in vitro CHX-adapted E. coli K-12 identified that ompF was significantly down-regulated in the transcriptomes and proteomes of CHX-adapted E. coli isolates (CHXR1) 22 . In the same multi-omics study, the loss of an outer membrane lipoprotein MlaA was also implicated 22 . MlaA forms a complex with OmpC/OmpF 23 and is part of the Mla intermembrane phospholipid transport system that maintains lipopolysaccharide asymmetry in the outer membrane 24 .…”