Abstract
Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses (e.g., AMPs) and their mimics (e.g., ceragenins). Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa. Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains.
Highlights
Chlorhexidine (Figure 1) is a potent antiseptic agent that has been in use since the 1950s (Davies et al, 1954)
As expected, induction of resistance in Gram-negative bacteria to chlorhexidine resulted in decreased susceptibility to colistin; organisms remained susceptible to AMPs and ceragenins
Previous work has shown that Gram-negative bacteria become resistant to colistin following exposure to a sublethal concentration of colistin; colistin resistance does not correlate with higher Minimum inhibitory concentrations (MICs) levels for AMPs or ceragenins (Hashemi et al, 2017c)
Summary
Chlorhexidine (Figure 1) is a potent antiseptic agent that has been in use since the 1950s (Davies et al, 1954). Cross resistance of bacteria to chlorhexidine and colistin may be due to common features of these antimicrobials Both are cationic (positively charged) with hydrophobic functionality. We recently reported (Hashemi et al, 2017c) that highly colistinresistant bacteria are susceptible to both AMPs and non-peptide mimics of AMPs, termed ceragenins (representative structures are shown in Figure 1) (Hashemi et al, 2017b, 2018b,c,d) This finding led to the question of whether generation of chlorhexidine resistance results in cross resistance to ceragenins and AMPs. Reported are investigations of the means by which Gram-negative bacteria become resistant to chlorhexidine and how this resistance influences susceptibility to colistin, AMPs and ceragenins. Changes to the proteome of chlorhexidine-resistant organisms, relative to a parent strain, were in the outer membrane proteins as well as proteins associated with chaperones, efflux pumps, flagella and cell metabolism
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