Long-term exposure to octenidine in a simulated sink-trap environment results in selection of Pseudomonas aeruginosa, Citrobacter and Enterobacter isolates with mutations in efflux pump regulators.

Octenidine based disinfection products are becoming increasingly popular for infection control of multidrug resistant (MDR) Gram-negative isolates. When a waste trap was removed from a hospital and allowed to acclimatise in a standard tap rig in our laboratory, it was shown that Klebsiella pneumoniae, Pseudomonas aeruginosa, Citrobacter and Enterobacter spp. were readily isolated. This study aimed to understand the potential impact of prolonged exposure to low doses of a commercial product containing octenidine on these bacteria. Phenotypic and genotypic analysis showed that P. aeruginosa strains had increased tolerance to octenidine which was characterised by mutations in the Tet-repressor SmvR. Enterobacter species demonstrated increased tolerance to many other cationic biocides, though not octenidine, as well as the antibiotics ciprofloxacin, chloramphenicol and ceftazidime through mutations in another Tet-repressor RamR. Citrobacter species with mutations in RamR and MarR were identified following octenidine exposure and this is linked to development of resistance to ampicillin, piperacillin and chloramphenicol as well as an increased MIC level for ciprofloxacin. Isolates were able to retain fitness as characterised by growth, biofilm formation and virulence in Galleria mellonella, after prolonged contact with octenidine, although there were strain to strain differences. These results demonstrate that continued low-level octenidine exposure in a simulated sink-trap environment selects for mutations which affect smvR. It may also promote microbial adaptation to other cationic biocides and cross-resistance to antibiotics, whilst not incurring a fitness cost. This suggests that hospital sink traps may act as a reservoir for more biocide tolerant organisms. Importance Multi-drug resistant (MDR) strains of bacteria are a major clinical problem and several reports have linked outbreaks of MDR bacteria with bacterial populations in hospital sinks. Biocides such as octenidine are used clinically in body washes and other products such as wound dressings for infection control. Therefore, increased tolerance to these biocides would be detrimental to infection control processes. Here, we exposed bacterial populations originally from hospital sink traps to repeated dosing with an octenidine containing product over several weeks and observed how particular species adapted. We found mutations in genes related to biocide and antibiotic susceptibility which resulted in increased tolerance, although this was species dependent. Bacteria which became more tolerant to octenidine also showed no loss of fitness. This shows that prolonged octenidine exposure has the potential to promote microbial adaptation in the environment and that hospital sink traps may act as a reservoir for increased biocide and antibiotic tolerant organisms.