Phenotypic and genotypic changes of Staphylococcus aureus in the presence of the inappropriate concentration of chlorhexidine gluconate

Abstract

Background: Chlorhexidine gluconate (CHG) is a disinfectant agent with different applications in health care. Improper use of CHG causes antimicrobial resistance in bacteria as a public health threat. Since Staphylococcus aureus is a common bacteria, it is expected usually exposed to CHG in the hospital and community. The present study aimed to correlate the phenotypic and genotypic changes in a S. aureus strain upon serial adaptation with supra-inhibitory CHG concentration for 50 days. Results: After in vitro serial culture of 5 × 105 CFU/ml of a clinical vancomycin-susceptible S. aureus strain (VAN-S) into brain heart infusion (BHI) broth containing CHG 1/4, 1/2, 1, and 2 × minimal inhibitory concentration (MIC) values of VAN-S in 37 °C during 50 days, we isolated a S. aureus strain (CHGVan-I) with a ≥ twofold decrease in susceptibility to CHG and vancomycin. CHG-induced CHGVan-I strain was considered as a vancomycin-intermediate S. aureus (VISA) strain with a VAN MIC of 4 μg/ml using the broth macro dilution method. However, reduced resistance was observed to tetracycline family antibiotics (doxycycline and tetracycline) using a modified Kirby-Bauer disk diffusion test. Moreover, a remarkable reduction was detected in growth rate, hemolysis activity (the lysis of human red blood cells by alpha-hemolysin), and colony pigmentation (on BHI agar plates). Biofilm formation (using the Microtiter plate method and crystal violet staining) was significantly increased upon CHG treatment. Adaptive changes in the expression of a set of common genes related to the development of VISA phenotype (graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and fmtA) were analyzed by Reverse Transcription quantitative PCR (RT-qPCR) experiment. Significant changes in vraTSR, agr RNAIII, sceD, and pbpB expression were observed. However, gene sequencing of the two-component system vraTSR using the Sanger sequencing method did not detect any non-synonymous substitution in CHGVan-I compared to wild-type. The clonality of VAN-S and CHGVan-I strains was verified using the pulsed-field gel electrophoresis (PFGE) method. Conclusions: The importance of the present study should be stated in new detected mechanisms underlying VISA development. We found a link between the improper CHX use and the development of phenotypic and genotypic features, typical of VISA clinical isolates, in a CHG-induced strain. Since disruption of the cell wall biosynthesis occurs in VISA isolates, our CHG-induced VISA strain proved new insights into the role of CHG in the stimulation of the S. aureus cell wall.