Biocide exposure in extended-spectrum β-lactamase-producing Klebsiella pneumoniae: effect on increased biocide and antibiotic MICs, genetic changes, and fitness cost.

OBJECTIVES: To test whether biocide exposure in extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) isolates is associated with (i) decreased susceptibility to biocides and antibiotics (co-resistance), (ii) emergence of mutations, (iii) changes in expression of efflux pump and porin genes, and iv) fitness cost.

METHODS AND RESULTS: The minimum inhibitory concentrations (MICs) of chlorhexidine digluconate (CHX), benzalkonium chloride (BZK), sodium hypochlorite (SOD), propanol-based solution (PRO), povidone-iodine (POV), ethanol (ET), and triclosan (TRI) were determined against 57 isolates. Isolates K27 and K29 were adapted to growth with BZK, CHX, or TRI and subjected to whole genome sequencing (MiSeq). Relative gene expression (RGE) of acrB, oqxB, kpnE, ompK35, and ompK36 was determined by qRT-PCR. Fitness cost was determined by mean generation time (MGT). The MICs of BZK, CHX, and TRI against K27 and K29 adapted to biocides were ≥4-fold higher than those observed before biocide adaptation. Biocide adaptation was associated with increased MICs of imipenem, co-trimoxazole, norfloxacin, and tigecycline. Adaptation to biocides led to mutations in genes involved in metabolism, transcription regulation, efflux pumps, membrane transporters, and stress response. Mean RGE increased from 3.6 to 6.3 (acrB with BZK and CHX) and decreased from 0.03 to 0.4 (ompK35 and/or ompK36 with TRI). MGTs ranged from 23.1 to 32.7 min and were similar before and after biocide adaptation.

CONCLUSIONS: Biocide exposure in ESBL-Kp is associated with (i) acquisition of biocide and antibiotic co-resistance, (ii) emergence of mutations in genes involved in co-resistance, and those related to relevant biological processes, (iii) upregulation of acrB and downregulation of ompK35 and ompK36, and (iv) no significant fitness cost.