SmvA is an important efflux pump for cationic biocides in Klebsiella pneumoniae and other Enterobacteriaceae

Abstract

The 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.

The Transcriptional Repressor SmvR Is Important for Decreased Chlorhexidine Susceptibility in Enterobacter cloacae Complex

“…1 and Table 1). Recently, it has been shown that heterologous expression of smvA from K. pneumoniae or S. enterica in Escherichia coli (which does not possess smvAR homologs) modestly increased the MICs of CHX (from 0.5 to 2 to 4 mg/liter) (6). It is noteworthy that, in contrast to observations in these E. coli strains, we did not observe any growth defect for our ECC transconjugants (data not shown).…”

“…These data contrast with the absence of SmvR mutations in Enterobacter sp. upon CHX exposure while mutations have been observed in Klebsiella, Citrobacter, and Salmonella (6). Because one substitution (G52A) was present into one of the two highly conserved regions of the SmvR sequence in clinical isolates, it is tempting to speculate that it could play a role in high CHX MICs.…”

“…For example, some efflux pumps, such as AceI in Acinetobacter baumannii and CepA in Klebsiella pneumoniae, have been described to be involved in decreased susceptibility to CHX (4,5). Moreover, Wand et al demonstrated that increased tolerance to CHX in K. pneumoniae was associated with overexpression of the smvA gene encoding an efflux pump belonging to the major facilitator superfamily (MFS) and that this gene was under the control of the TetR family transcriptional regulator SmvR (6).…”

Derepression of the smvA Efflux System Arises in Clinical Isolates of Proteus mirabilis and Reduces Susceptibility to Chlorhexidine and Other Biocides

“…Biocides play an important role in infection control and are often used to remove microbes from equipment, surfaces, and skin prior to medical procedures (1–3). The rising incidence of multidrug resistance in nosocomial pathogens has further magnified the clinical value of biocides, and there is increasing reliance on these antimicrobials to reduce antibiotic usage through more effective infection control strategies (1, 4–6). However, despite their clinical significance, biocides are subject to far fewer regulations on their use than therapeutic agents, such as antibiotics (2).…”

Overcoming Intrinsic and Acquired Resistance Mechanisms Associated with the Cell Wall of Gram-Negative Bacteria

“…The latter is evident in E. coli with the multiple antibiotic operon, Mar [ 106 , 123 ]. Furthermore, either direct mutations or mutations in the Tet-R family regulator of the MFS family efflux pump smvA in K. pneumoniae , P. mirabilis, E. cloacae and S. enterica serotype Typhimurium are associated with increased biocide resistance, including to chlorhexidine [ 124 , 125 , 126 ] and acriflavine [ 127 ]. Efflux pump regulatory pathways have been noted to be interwoven into many other regulatory processes that promote bacterial virulence such as biofilm formation, quorum sensing and membrane permeability [ 110 , 128 , 129 ].…”