Reduced chlorhexidine susceptibility in multidrug-resistant Klebsiella pneumoniae; genomic and functional analysis of invasive isolates

Objectives Carbapenem-resistant Klebsiella pneumoniae has emerged as a major cause of healthcare-associated infections, driven by the global dissemination of high-risk clones. Since chlorhexidine is widely used for infection prevention in healthcare settings, we investigated whether multidrug-resistant Klebsiella pneumoniae lineages display reduced chlorhexidine susceptibility. Methods Chlorhexidine susceptibility was evaluated in 110 invasive KPC-2-producing K. pneumoniae bloodstream isolates collected from a Brazilian tertiary-care hospital cohort. Chlorhexidine MICs, time-kill assays, whole-genome sequencing, and genetic complementation experiments were performed to identify and validate mechanisms associated with reduced susceptibility. Results Chlorhexidine MICs were higher in the strains belonging to this local ST258 sub-lineage compared to other STs (MIC 50 64 vs. 16 µg/mL). Time-kill assays showed that a representative ST258 isolate survived exposure to 0.02% chlorhexidine, a concentration that achieved ≥3 to 6 log 10 killing in control strains. Whole-genome sequencing revealed that all 42 ST258 isolates carried a 7-nucleotide deletion in smvR , the TetR-like repressor of the SmvA efflux pump. Genetic complementation with wild-type smvR , but not with the frameshift allele, restored lower chlorhexidine MICs, confirming causality. The effect of smvR inactivation was specific to chlorhexidine susceptibility and did not confer cross-resistance to colistin. Conclusion This study identifies smvR inactivation as an important mechanism of reduced chlorhexidine susceptibility in multidrug resistant K. pneumoniae . Although MICs remained below in-use concentrations, survival at low chlorhexidine levels could facilitate environmental persistence and potentially contribute to nosocomial transmission. Our results support the integration of antiseptic susceptibility monitoring into infection-control surveillance frameworks targeting multidrug-resistant pathogens.