Evaluation of the role of ATP-binding cassette (ABC) transporters on chlorhexidine digluconate biocide activity in nosocomial pathogens

Abstract

The use of biocides against bacterial infections over the years has brought numerous benefits to the health sector, but a disturbing prospect of biocide resistance to nosocomial pathogens is emergent. The evolution of biocide resistant microorganisms increases the frequency of nosocomial infections. Nosocomial infections are a cause of elevated morbidity and mortality and impose extra expenses upon the health service. Nosocomial pathogens have been reported to show signs of resistance towards the commonly used biocide chlorhexidine digluconate. Efflux is a common mechanism liable for bacterial resistance to biocides. The aim of the study was to evaluate the role of ATP-binding cassette (ABC) transporters on accumulation of chlorhexidine in bacterial cells. In this study, it was hypothesized that the effectiveness of chlorhexidine against bacteria could be reduced by ABC transporters. Understanding the role of the ABC transporter in the effluxing of chlorhexidine digluconate from bacterial cells would be a basis for rationale development of efflux pump inhibitor (EPI) to counteract the pumping out of the biocide. The inhibition of efflux pump activity by an EPI would maintain or increase the effectiveness of the biocide. A clinical strain each of P. aeruginosa and S. aureus and their respective laboratory strains ATCC 27853 and ATCC 9144 were used in the study. All four isolates were tested by the broth microdilution method to compare the changes in the minimum inhibitory concentration (MIC) values in the absence and presence of an EPI. The comparison served to indicate if the isolates exhibited any efflux pump activity. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine were the EPIs utilised. A spectrophotometric method to quantify the amount of chlorhexidine digluconate accumulated within bacterial cells was developed. The suitability of using the UV/VIS spectrophotometer in quantifying chlorhexidine at a wavelength of 255 nm was validated based on the linear regression value obtained from the calibration curve for the absorbance of a series of chlorhexidine concentrations (12 μM to 0.75 μM). The spectrophotometric method developed was used to measure the amount of chlorhexidine accumulated in the presence of EPIs: reserpine or CCCP. Sub-inhibitory concentrations of CCCP and reserpine pre-determined in the study using the MICs of the respective EPIs were used in the accumulation assays. The MIC values of chlorhexidine digluconate against the clinical strains of P. aeruginosa and S. aureus showed a two-fold reduction (from 6.3 μg/ml to 3.2 μg/ml) in the presence of reserpine. The presence of reserpine failed to show any change on the MIC value of chlorhexidine digluconate against ATCC isolates of both P. aeruginosa and S. aureus. The MIC values of chlorhexidine digluconate in the presence of CCCP for the clinical and ATCC strain of P. aeruginosa remained unchanged at 6.3 μg/ml and 3.2 μg/ml respectively. In the presence of CCCP, the two isolates of S. aureus showed a magnified reduction in their MIC value of chlorhexidine digluconate. A linear regression value of 0.99 was obtained from the calibration curve for the absorbance of a series of chlorhexidine concentrations. The linear regression value validating the suitability of using the spectrophotometric method developed to quantify the amount of chlorhexidine accumulated in cells. The bioassay may be used to find novel inhibitors of efflux of chlorhexidine. At P˂ 0.05, only the clinical strain of P. aeruginosa showed a significant active efflux of chlorhexidine, while the ATCC strain of P. aeruginosa, the clinical and ATCC strains of S. aureus exhibited no signifying active efflux. The findings of this study suggest the participation of ABC transporters in pumping out chlorhexidine digluconate from clinical strain cells of P. aeruginosa.