AbstractThe multidrug resistant (MDR) opportunistic pathogen Klebsiella pneumoniae has previously been shown to adapt to chlorhexidine by increasing expression of the MFS efflux pump smvA. Here we show that loss of the regulator SmvR, through adaptation to chlorhexidine, results in increased resistance to a number of cationic biocides in K. pneumoniae and other members of the Enterobacteriaceae. Clinical Enterobacteriaceae isolates which lack smvA and smvR also have an increased susceptibility to chlorhexidine. When smvA from Salmonella and K. pneumoniae are expressed in Escherichia coli, which lacks a homologue to SmvAR, resistance to chlorhexidine increased (4-fold) but plasmid carriage of smvA alone was detrimental to the cell. Challenge of K. pneumoniae with chlorhexidine and another cationic biocide, octenidine, resulted in increased expression of smvA (approx. 70 fold). Adaptation to octenidine was achieved through mutating key residues in SmvA (A363V; Y391N) rather than abolishing the function of SmvR, as with chlorhexidine adaptation. Molecular modelling was able to predict that octenidine interacted more strongly with these mutated SmvA forms. These results show that SmvA is a major efflux pump for cationic biocides in several bacterial species and that increased efflux through SmvA can lead to increased chlorhexidine and octenidine tolerance.