P75 Mechanisms of increased resistance to chlorhexidine in <i>Klebsiella pneumoniae</i>

Abstract Objectives Biocides are important antimicrobial agents used extensively in healthcare settings as antiseptics or disinfectants. The increasing use of biocides has been driven by efforts to reduce antibiotic use and the Covid-19 pandemic. However, evidence is accumulating that biocides can select for undesirable traits in bacterial pathogens, including antibiotic resistance. Despite this, the mechanisms underpinning changes in biocide susceptibility in important bacterial pathogens are not well understood. It is also unclear how biocide exposure and associated mutations impact clinically relevant microbiomes and biofilms. The aim of this study was to identify mechanisms involved in reduced chlorhexidine susceptibility in Klebsiella pneumoniae. Methods A library of mini-Tn5 mutants was generated from the WT K. pneumoniae pre-antibiotic Murry isolate M109 and screened to identify those with reduced susceptibility to chlorhexidine (CHD). Whole genome sequencing and comparison to the WT genome were used to identify genes disrupted by Tn insertions and any spontaneous secondary mutations. Tn-mutants exhibiting at least a four-fold increase in CHD MBC and lacking any secondary point mutations were selected for further study and characterized to assess polar effects of Tn insertion, and evaluate a range of relevant phenotypes (growth, biofilm formation). Results 2 mutants out of 1584 mini-Tn5 K. pneumoniae M109 mutants screened exhibited a 4-fold increase in CHD MBC compared to the parental WT, with no secondary mutations. Tn insertions in these mutants were mapped to the promotor region of RcnA (encoding a nickel/cobalt efflux system), and the M109 plasmid upstream of FecA (encoding a TonB-dependent Fe(3) dicitrate receptor). Biofilm formation in these mutants was significantly increased in LB medium, but not artificial urine medium compared to the WT. Conclusions These findings indicate the disruption in of RcnA and FecA can reduce CHD susceptibility and modulate biofilm formation in K. pneumoniae M109. However, changes in biofilm formation were only observed in LB media suggesting these may not be relevant to CAUTI.