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. 2022 Jun 22;27(13):3998.
doi: 10.3390/molecules27133998.

Potent Activity of a High Concentration of Chemical Ozone against Antibiotic-Resistant Bacteria

Karyne Rangel  1   2 , Fellipe O Cabral  3 , Guilherme C Lechuga  1   2 , João P R S Carvalho  1   2   4 , Maria H S Villas-Bôas  3 , Victor Midlej  5 , Salvatore G De-Simone  1   2   4
Affiliations

Potent Activity of a High Concentration of Chemical Ozone against Antibiotic-Resistant Bacteria

Karyne Rangel et al. Molecules. .

Abstract

Background: Health care-associated infections (HAIs) are a significant public health problem worldwide, favoring multidrug-resistant (MDR) microorganisms. The SARS-CoV-2 infection was negatively associated with the increase in antimicrobial resistance, and the ESKAPE group had the most significant impact on HAIs. The study evaluated the bactericidal effect of a high concentration of O3 gas on some reference and ESKAPE bacteria.

Material and methods: Four standard strains and four clinical or environmental MDR strains were exposed to elevated ozone doses at different concentrations and times. Bacterial inactivation (growth and cultivability) was investigated using colony counts and resazurin as metabolic indicators. Scanning electron microscopy (SEM) was performed.

Results: The culture exposure to a high level of O3 inhibited the growth of all bacterial strains tested with a statistically significant reduction in colony count compared to the control group. The cell viability of S. aureus (MRSA) (99.6%) and P. aeruginosa (XDR) (29.2%) was reduced considerably, and SEM showed damage to bacteria after O3 treatment Conclusion: The impact of HAIs can be easily dampened by the widespread use of ozone in ICUs. This product usually degrades into molecular oxygen and has a low toxicity compared to other sanitization products. However, high doses of ozone were able to interfere with the growth of all strains studied, evidencing that ozone-based decontamination approaches may represent the future of hospital cleaning methods.

Keywords: ESKAPE pathogens; SEM; antimicrobial activity; antimicrobial resistance; ozone; pathogenic bacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The number of colony-forming units (CFU) in different bacterial strains (S. aureus (ATCC 6538), P. aeruginosa (ATCC 15442), S. enterica (ATCC 10708), and E. coli (ATCC 25922)) was counted. CFU counting was performed in the control group (no treatment) and bacterial suspensions (105 CFU/mL) after exposure to ozone for 1, 10, 20, 30, and 40 min.
Figure 2
Figure 2
Average temperature, relative humidity, and ozone concentration at times of 1, 10, 20, 30, and 40 min with ATCC (S. aureus (ATCC 6538), P. aeruginosa (ATCC 15442), S. enterica (ATCC) strains 10708), and E. coli (ATCC 25922)) and multidrug-resistant S. aureus (MRSA), P. aeruginosa (XDR), A. baumannii (PDR), and K. pneumoniae (KPC+).
Figure 3
Figure 3
Analysis of cell viability after ozone treatment in different bacterial strains (S. aureus (ATCC 6538), S. enterica (ATCC 10708), E. coli (ATCC 25922), and P. aeruginosa (ATCC 15442)). The measurement of fluorescence intensity (relative fluorescence units, RFU) after the conversion of resazurin to resofurin by viable bacteria was performed in the control group (no treatment) and bacterial suspensions (105 CFU/mL) after exposure to ozone for 40 min. Results represent values from 3 randomly chosen colonies in the control group (no treatment) and after treatment with ozone. The black dots represent the values of fluorescence emission after addition of resazurin.
Figure 4
Figure 4
(A) The number of colony-forming units (CFU) in different bacterial strains (S. aureus (MRSA), P. aeruginosa (XDR), A. baumannii (PDR), and K. pneumoniae (KPC+)) were counted. The number of CFU in the control group (no treatment) and bacterial suspensions (105 CFU/mL) after exposure to ozone for 40 min was quantified. The black dots represent the number of CFU count of the different strains. (B) Analysis of cell viability after ozone treatment in different bacterial strains (S. aureus (MRSA), P. aeruginosa (XDR), A. baumannii (PDR), and K. pneumoniae (KPC+)). The measurement of fluorescence intensity (Relative fluorescence units, RFU) after the conversion of resazurin to resofurin by viable bacteria was performed in the control group (no treatment) and bacterial suspensions (105 CFU/mL) after exposure to ozone for 40 min. Results represent three randomly chosen colonies in the control group (no treatment) and after ozone treatment. The black dots show cell viability values through fluorescence emission after the addition of resazurin. ** Statistically significant (p < 0.01); **** statistically significant (p < 0.001).
Figure 5
Figure 5
Morphological analysis of O3 treatment by electron microscopy. S. aureus (MRSA) (a,b) and P. aeruginosa (XDR) (c,d) are seen without (a,c) and under O3 treatment (b,d). An alteration in S. aureus (MRSA) shape is seen (arrowhead) in b. Damage in bacteria is observed after treatment (arrows) (b). Note that control cells are rounded and present in a homogeneous surface (a). Damaged cells are observed after treatment in P. aeruginosa (XDR) (arrows) (d). Some cell wall protrusions are observed in treated cells (arrowhead) (d). These aspects were not verified in control cells (c).
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