Clinical characteristics, tolerance mechanisms, and molecular epidemiology of reduced susceptibility to chlorhexidine among Pseudomonas aeruginosa isolated from a teaching hospital in China

Chlorhexidine is used widely to prevent the spread of bacteria in the hospital environment. However, bacteria are increasingly becoming tolerant to chlorhexidine. Here we investigated clinical characteristics, tolerance mechanisms, and molecular epidemiology of chlorhexidine-tolerant Pseudomonas aeruginosa. According to the proposed epidemiological cut-off value to determine chlorhexidine tolerance (50 µg/mL) in P. aeruginosa, 32 chlorhexidine-tolerant isolates were detected from 294 P. aeruginosa isolates, which accounted for 10.9%. Our results indicated MICs of chlorhexidine-tolerant strains were 64 µg/mL. Patient's data showed chlorhexidine tolerance was associated with following factors: hospital length of stay, ICU admission, length of stay in ICU, invasive procedure, duration of mechanical ventilation, chlorhexidine usage, and occurrence of nosocomial pneumonia. Tolerance mechanisms were analyzed by efflux pump inhibition test, qRT-PCR, and serial passage experiment. Increased expression of efflux pump genes mexA, mexC, mexE and mexX, and decreased expression of oprD were observed in chlorhexidine-tolerant and chlorhexidine-induced strains, which suggested that hyperexpression of Mex-Opr efflux pump was the main mechanism. Moreover, serial passage experiment found chlorhexidine-induced strains showed decreased susceptibility to tested antibiotics, which illustrated that long-term exposure of P. aeruginosa to chlorhexidine could result in multidrug-resistant (MDR) or cross-resistance phenotypes. MLST and PFGE analysis demonstrated the homology of 32 chlorhexidine-tolerant strains was low and no obvious clonal transmission was observed. We comprehensively investigated the development and molecular mechanisms of chlorhexidine-tolerant P. aeruginosa, which revealed that the control and surveillance of chlorhexidine tolerance should be more strict. Moreover, it seems to make sense to avoid the continuous or unreasonable application of chlorhexidine in hospital settings.