Disinfecting Action of Gaseous Ozone on OXA-48-Producing Klebsiella pneumoniae Biofilm In Vitro

Abstract

Klebsiella pneumoniae is an emerging multidrug-resistant pathogen that can contaminate hospital surfaces in the form of a biofilm which is hard to remove with standard disinfectants. Because of biofilm resistance to conservative disinfectants, the application of new disinfection technologies is becoming more frequent. Ozone gas has antimicrobial activity but there is lack of data on its action against K. pneumoniae biofilm. The aim of this study was to investigate the effects and mechanisms of action of gaseous ozone on the OXA-48-procuding K. pneumoniae biofilm. A 24 h biofilm of K. pneumoniae formed on ceramic tiles was subsequently exposed to different concentrations of ozone during one and two hours to determine the optimal ozone concentration. Afterwards, the total bacteria count, total biomass and oxidative stress levels were monitored. A total of 25 ppm of gaseous ozone was determined to be optimal ozone concentration and caused reduction in total bacteria number in all strains of K. pneumoniae for 2.0 log₁₀ CFU/cm², followed by reduction in total biomass up to 88.15%. Reactive oxygen species levels significantly increased after the ozone treatment at 182% for the representative K. pneumoniae NCTC 13442 strain. Ozone gas in the concentration of 25 ppm caused significant biofilm reduction but did not completely eradicate the K. pneumoniae biofilm formed on ceramics. In conclusion, ozone gas has great potential to be used as an additional hygiene measure in joint combat against biofilm in hospital environments.

Keywords: K. pneumoniae; OXA-48 disinfection; biofilm; gaseous disinfection; hospital-acquired infections; multidrug-resistant microorganisms; ozone.

Figure 1

Ozonation in vitro model scheme.

Figure 2

Antimicrobial effect on 24 h biofilm of K. pneumoniae NCTC 13442 (Kp NCTC 13442) and K. pneumoniae ATCC 700603 (Kp ATCC 700603) of different ozone concentrations (ppm) for 1 h exposure time (A) and 2 h exposure time (B). Results are presented with average (·) and standard deviation.

Figure 3

Total bacteria count expressed as log10 CFU/cm² of 7 tested strains of Klebsiella pneumoniae (Kp). Results shown with median value (-) and minimum and maximum value. The lowercase letters, a–g, express the statistically significant difference between the treated and control group for the 7 tested strains (Mann–Whitney U test, p < 0.05). The capital letter A marks the statistically significant difference between Kp ATCC 700603 and OXA-48-producing strains Kp strain 14, Kp strain 15, Kp strain 16 and Kp strain 33, and vice versa (Kruskal–Wallis test, p < 0.05).

Figure 4

Biomass determination (CV staining) for control and treated K. pneumoniae strains (Kp) after treatment of 24 h biofilm with 25 ppm of ozone for 1 h. Results shown with median value (-) and minimum and maximum value. Lowercase letters, a–g, express the statistically significant difference between the treated and control group for the 7 tested strains (Mann–Whitney U test, p < 0.05). The capital letter A marks the statistically significant difference between strains Kp ATCC 700603, Kp strain 14, Kp strain 33, Kp strain 34 and Kp NCTC 13442, and vice versa (Kruskal–Wallis test, p < 0.05).

Figure 5

ATP bioluminescence method for control and treated K. pneumoniae strains after treatment of 24 h biofilm with 25 ppm of ozone for 1 h. Lowercase letters, a–g, express the statistically significant difference between the treated and control group for the 7 tested strains (Mann–Whitney U test, p < 0.05). The capital letter A marks the statistically significant difference between Kp NCTC 13442 and strains Kp strain 14, Kp strain 15, Kp strain 33 and Kp strain 34, and the capital letter B marks the significant difference between Kp strain 14, Kp strain 33 and Kp NCTC 13442, while C marks the statistically significant difference between Kp strain 16 and Kp strain 33 (Kruskal–Wallis test, p < 0.05).

Figure 6

The three-dimensional (above) and two-dimensional (below) images of ceramic tiles surfaces inoculated with representative strain K. pneumoniae NCTC 13442 biofilm: control sample (A) and ozone-treated sample (B). The dark blue stains present the bacteria stained with crystal violet.

Figure 7

Atomic force microscopy of representative strain K. pneumoniae NCTC 13442 biofilm on ceramic tiles. Topology of biofilm created after 24 h (A–C) and topology of biofilm treated with 25 ppm of ozone for 1 h (D–F). Scanning area for (B,E) is 10 µm × 10 µm and for (C,F) is 1 µm × 1 µm.

Figure 8

Morphological changes in representative strain K. pneumoniae NCTC 13442 biofilm biomass and bacterial cells after ozone treatment. Recess areas in the biofilm topology were observed (A–C), as well as bacterial cell surface alteration (D) (arrowhead).

Figure 9

Viability of the representative strain K. pneumoniae NCTC 13442 cells in biofilm. Viability of nontreated control group, and 25 ppm of ozone/1h treated group using Dead/Live staining. Green fluorescence is representing viable cells with intact membrane, and red fluorescence indicates dead cells with permeable membrane.

Figure 10

Measured fluorescence intensity for control and treated representative strain K. pneumoniae NCTC 13442 biofilm. Results shown with median, minimum and maximum values. The lowercase letter a marks the statistically significant difference between the control and treated group of K. pneumoniae NCTC 13442 strain. On the right are fluorescence microscopy images magnified 1000× of the control (A) and ozone-treated (B) representative strain K. pneumoniae NCTC 13442 biofilm. Excitation wavelength was 488 nm and emission were detected in the range of 500–600 nm.