Resistance mechanisms and molecular epidemiology of chlorhexidine acetate-resistant Klebsiella pneumoniae clinical isolates

Abstract

Objective To investigate the chlorhexidine acetate-resistance in Klebsiella pneumoniae (K.pneumoniae) clinical isolates and to analyze the possible mechanisms and molecular epidemiology of resistant isolates. Methods A total of 332 K. pneumoniae clinical isolates were collected in the First Affiliated Hospital of Wenzhou Medical University in 2015. Standard agar dilution was used to screen chlorhexidine acetate-resistant isolates. The minimum inhibition concentrations (MIC) of chlorhexidine acetate to resistant isolates with and without the presence of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which was an efflux pump inhibitor, were analyzed. Efflux pump genes of cepA, qacE and qacΔE1 that carried by and expressed in those isolates were detected by polymerase chain reaction (PCR) and quantitative real-time PCR (RT-qPCR), respectively. The biofilm formation ability was measured by crystal violet staining. The homology among the chlorhexidine acetate-resistant isolates was investigated with multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Results Twenty-five K. pneumoniae strains were resistant to chlorhexidine acetate. The MIC values of chlorhexidine acetate for them were reduced by at least four-fold in the presence of CCCP. Strains carrying the genes of cepA, qacE and qacΔE1 accounted for 100%, 40% and 40%, respectively. The expression of the efflux pump genes in the chlorhexidine acetate-resistant isolates was higher than that in the susceptible isolates. The biofilm formation ability of the chlorhexidine acetate-resistant isolates was better than that of the susceptible isolates. Furthermore, negative, weak-positive and positive biofilm formation ability was observed in four (16%), 20 (80%) and one (4%) strains, respectively. The results of MLST and PFGE showed that the 25 chlorhexidine acetate-resistant isolates belonged to 19 different sequence types (ST) with diverse PFGE patterns. Conclusions This study suggested that active efflux was the main mechanism of chlorhexidine acetate resistance in K. pneumoniae. The 25 chlorhexidine acetate-resistant K. pneumoniae strains possessed different biofilm formation ability and shared low homology. Key words: Chlorhexidine acetate; Klebsiella pneumoniae; Resistance mechanism; Molecular epidemiology