Effects of common disinfectants on biofilm formation and eradication in Klebsiella pneumoniae with different resistance phenotypes.

OBJECTIVE

Biofilm formation is a critical virulence factor that facilitates the persistence and transmission of Klebsiella pneumoniae (KP) in healthcare settings, complicating infection control efforts. This study aimed to characterize the biofilm-forming capacity of KP isolates with different resistance phenotypes and to evaluate the antibacterial, biofilm-inhibitory, and biofilm-eradicating activities of commonly used disinfectants.

METHODS

Forty-five clinical KP isolates were enrolled, comprising 15 susceptible isolates, 15 extended-spectrum β-lactamase (ESBL)-producing isolates, and 15 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of povidone-iodine, chlorhexidine, glutaraldehyde, benzalkonium bromide, and sodium hypochlorite were determined by broth microdilution. Biofilm formation was quantified over 7 days using crystal violet staining (OD590). The inhibitory and eradicating effects of chlorhexidine, benzalkonium bromide, and sodium hypochlorite on biofilms were further assessed by crystal violet staining and confocal laser scanning microscopy (CLSM).

RESULTS

MIC and MBC values of the five disinfectants were identical across the three KP groups. Chlorhexidine exhibited the most potent antibacterial activity (MIC: 16 µg/mL; MBC: 32 µg/mL). Biofilm biomass increased progressively in all groups, peaking on day 5. In the inhibition assay, OD590 values in disinfectant-treated groups remained consistently lower than those in the positive control group, with 16 µg/mL chlorhexidine, 32 µg/mL benzalkonium bromide, and 1000 µg/mL sodium hypochlorite all significantly reducing biofilm biomass by day 5 (P < 0.05). In the eradication assay, 2000 µg/mL chlorhexidine and 5000 µg/mL sodium hypochlorite significantly reduced OD590 values compared with the untreated control (P < 0.05), whereas 2000 µg/mL sodium hypochlorite did not.

CONCLUSION

Chlorhexidine, benzalkonium bromide, and sodium hypochlorite effectively inhibited biofilm formation in KP isolates regardless of resistance phenotype. Chlorhexidine and benzalkonium bromide exhibited stronger inhibitory activity than sodium hypochlorite. Chlorhexidine and sodium hypochlorite also demonstrated biofilm-eradicating activity, with chlorhexidine showing the greatest overall efficacy.