Effect of ozone gas on cultures of Candida albicans and Aspergillus fumigatus : evaluation of two ozonation devices

Introduction: Candida albicans and Aspergillus fumigatus are two important agents of Healthcare-associated infections. This study aimed to evaluate the antifungal activity of ozone (O 3 ) gas produced by two commercial devices against cultures of these two species. Methodology: Sterile plastic plates were inoculated with C. albicans and A. fumigatus and placed on a countertop at three distances (30 cm, 1 m, and 2 m) and three positions in relation to the wall (near, middle, and away), considering the source of O 3 . Plates were exposed to O 3 for one hour and incubated. After incubation, the counting of colony-forming units was performed. As a control, an inoculated plate was incubated, without being exposed to O 3 . Tests were carried out with two different devices (namely, Mod.I and Mod.II), with the air conditioner on and off, in triplicate. Results: Both devices showed antifungal activity. Mod. I presented better results, due to a higher flow rate. The best activity was on plates at 30 cm, middle position. Contrarily, on plates at 2m, near the wall, the inhibition activity was lower. The best results were obtained with the air conditioner off. Candida albicans was more sensitive to O 3 than A. fumigatus . Conclusions: This method of decontamination by O 3 gas shows potential due to its fast and easy execution. The establishment of new protocols for hygiene and hospital disinfection using this approach should be considered, which may reduce environmental contamination by fungi and, consequently, the burden of fungal infections.


Introduction
Fungal infections are of enormous importance in the scenario of nosocomial infections at Health Care Institutions, with increasing morbidity and mortality rates.Microorganisms transmitted by air, water, and/or food can contaminate body surfaces and remain in the hospital environment, increasing the risk of infectious diseases.Several fungi may act as opportunistic or primary pathogens.In this context, Candida spp.and Aspergillus spp.are especially relevant [1][2][3][4].
Candida albicans is part of the normal microbiota.However, it can cause infections by rupturing the biological balance, and predisposing pathological, mechanical, physiological, or immunological conditions [5][6][7].Aspergillus fumigatus is a ubiquitous saprophytic fungus that releases millions of conidia into the environment.It is a common cause of opportunistic invasive infections when inhaled, along with being an allergen [8][9].Due to its high oxidant power ozone (O3) is used for antimicrobial activity during disinfection processes and/or sterilization [10].Although the mechanisms of its action are not fully understood, O 3 is known to act on cell walls of microorganisms causing the oxidation of glycopeptides, glycoproteins, and amino acids, modifying permeability, and leading to cell lysis.Ozone is recombined with cytoplasmic elements when it reaches inside the cell, causing oxidation of amino acids and nucleic acids, resulting in cleavage and consequent cell death.Ozone can also collapse cellular enzymatic activity due to its action on the sulfhydryl compounds of enzymes and alter the purine and pyrimidine bases of nucleic acids [10][11][12].
This study aimed to compare the antifungal activity of O 3 gas produced by two commercial devices against cultures of C. albicans and A. fumigatus.

Ozone generators -technical specifications
We used two devices from OZON® company (Cuiabá, Brazil), the GEO 20000/AR-TD (Mod.I) and GEO 20000/AR (Mod.II).Their main specifications are shown in Table 1.

Microbial inoculum and experiment site
The isolates tested were C. albicans (ATCC 90028) and A. fumigatus (environmental origin, characterized phenotypically by macromorphology and micromorphology and genotypically by PCR) [13], from the fungal culture collection of the Laboratory of Microbiology of the Department of Dermatological, Infectious, and Parasitic Diseases of the Faculty of Medicine of São José do Rio Preto (FAMERP), São Paulo, Brazil, where experiments were carried out.
The inoculums of C. albicans and A. fumigatus were prepared and adjusted, by spectrophotometry, in correspondence to the 0.5 McFarland scale of turbidity (1 × 10 6 -5 × 10 6 cells/mL).For each species, ten sterile plastic plates containing Brain Heart Infusion Agar (BHI) (Oxoid ® , Basingstoke, Hants, UK) were inoculated with 100 μL of the inoculum, which was spread with a Drigalski spatula.

Ozonation procedure -generators Mod.I and Mod.II
The experiments were carried out on a 2.72 m long and 0.58 m wide granite countertop, in a room 9.0 m 2 in size.After inoculation, nine plates were placed on the surface of the countertop at three different distances from the O 3 gas generator: 30 cm, 1 m, and 2 m.At each distance, the flow targeting of O 3 was evaluated in three different positions, namely A (plates near the wall), B (in the middle), and C (plates away from the wall), as shown in Figure 1.
Plates were kept without a lid during O 3 gas exposure, which occurred in a closed environment for one hour.Subsequently, plates were closed and incubated at 35 °C.As a control, an inoculated plate was incubated according to the same criteria, without receiving O 3 treatment.C. albicans and A. fumigatus, were incubated for 24 hours and 48 hours, respectively.After incubation the number of colony-forming unit (CFU/0.1 mL) in each plate was observed and compared with the control group.The experiments were carried out in triplicate, including the control.Percentages of inhibition were calculated considering the mean CFU counting of the treatment plates in relation to the control.The procedure described above was carried out at two distinct times: with air conditioning turned on and off.When turned on, air conditioning was set to 20 °C and maximum fan power.Temperature and humidity were monitored.
The data were subjected to statistical analysis, according to the arithmetic mean of fungal growth of triplicate tests.Then, the chi-squared test (χ 2 ) was applied to verify the associability and dependence between the variables: positions (A, B, and C) and distances (30 cm, 1 m, and 2 m), with the air conditioning turned on and off.

Results
The O 3 generators (Mod.I and Mod.II) showed antifungal activity in the two species studied.Plates showed a reduction in the number of CFU when compared with the control.
The Mod.I generator showed greater efficiency in the reduction of microbial load, considering distance and targeting parameters (p < 0.05).The results of the Mod.II devices also showed significant reductions (p < 0.05), except for C. albicans at 2 m (p = 0.3581) and A. fumigatus at 1 m (p = 0.5985) and 2 m (p = 0.4874), proving the associability between the variables.
Considering only the parameters of distances and positions, plates at 2 m showed the worst antifungal result for A. fumigatus, and plates B, at 30 cm, showed the best antifungal result for C. albicans, as shown in Figures 2 and 3.
Table 2 (Mod.Ι) and Table 3 (Mod.Π) show the percentage values of inhibition for all variables.The highest inhibition values occurred for C. albicans for both devices, with significant statistical differences (p < 0.05).The inhibition values of A. fumigatus cultures were less expressive, around 50%.
Antifungal activity of O3 was better for the tests performed in the room with air conditioning turned off, for both fungal species.The percentages of inhibition were 83% and 78% for Mod.I and Mod.II, respectively.When the air conditioning was on, the values were lower, 28% and 67%, respectively.
Considering the environmental factors during the experiments, the average temperatures recorded were 21 °C and 25 °C, and the average humidity was 58% and 53%, with the air conditioning turned on and off, respectively.

Discussion
Considering the relevance of C. albicans and A. fumigatus as agents of nosocomial infections, our study evaluated the antifungal activity of O 3 gas on the surface of culture media contaminated by these two species.Our experiments considered different distances and positions of the fungi related to the source of O 3 and tested two devices with different specifications.In addition, we observed the interference of air conditioner flow on the antifungal activity of the O 3 .
Our data showed significantly lower numbers of CFU on the plates exposed to O 3 , as compared to the control, for both devices (Mod.I and Mod.II), and all variables (species, distance, position, air conditioning on and off).Statistically, Mod.I device showed greater efficiency in microbial load reduction, regardless of the fungal species tested, which could be explained by the different flow rates of the devices.Although O 3 concentrations in part per million (ppm) are virtually the same, Mod.I O 3 generator has twice the flow when compared with Mod.II.Consequently, more ozone comes in contact with the samples being tested.
In terms of experimental conditions suitable for microbial load reduction, the most promising results observed for C. albicans was from samples at 30 cm, position B (central), while in case of A. fumigatus, samples at 2 m and position A (plates near the wall), represented the least favorable condition.This may be related to the way the gas flows.In laminar flow, particles move orderly, always maintaining the same relative position.When there is a turbulent flow, these particles move randomly and irregularly [14].Therefore, the wall may have caused a disorder of the O 3 molecules, which could have interfered with the flow, decreasing the antifungal activity when compared with the dishes near the wall.
Regarding the use of an air conditioner, the best antifungal activity for both devices was observed when the air conditioner was turned off, for both fungal species.Here, the airflow may have interfered again.The prolonged exposure to the gas resulted in more affected surfaces.However, when the air conditioning was turned on, it captured room air and filtered it before throwing it again into the environment [15], resulting in a variation in the flow of O 3 gas upon reaching the surface, thereby decreasing its antifungal activity.
According to the literature, factors such as ozone concentration, microbial species, and time of exposure, may lead to different antimicrobial effects of O 3 [16,17].In our study, these factors were kept constant in all experimental conditions, minimizing possible biases.According to the observed values, cultures of C. albicans were more sensitive than A. fumigatus, which is in concordance with existing literature [16,18].A. fumigatus can grow and survive in humid environments and extreme temperatures, by profuse dispersion of conidia [9].According to Santana et al. [7], C. albicans is less adapted to diverse environmental conditions outside the human body than A. fumigatus.
Several other studies have shown the antimicrobial activity of O 3 gas against fungi, with different times of exposure and concentrations.Variables were analyzed to inhibit yeasts and filamentous fungi, germination tube formation, biofilm, and fungus spores, proving that O 3 gas in well-established protocols shows excellent antimicrobial activity [5,12,[19][20][21][22][23].Zotti et al. [24] observed phenotypic changes in the colony of A. flavus and A. niger after exposure to O 3 gas, with a decrease in growth rates for both fungi and a change in their natural pigmentation.Consequently, O 3 gas can be expected to inhibit pigments and protein synthesis, with possible impairment of virulence factors of pathogenic fungal species.
It is worth mentioning that, although the two devices tested here generate low concentrations of O 3 , people should not remain in the environment while the devices are on.Ozone can be toxic when inhaled at high concentrations or longer periods, thus impairing respiratory health [25].
Surface cleaning is crucial for the control of healthcare-associated infections (HAI) [26][27][28][29].Unfortunately, health services proper surface cleaning and disinfection is often overlooked.These practices should be discussed and implemented by the Hospital Infection Commissions, together with nursing and cleaning services, developing activities related to environmental hygiene protocols, supervision, and training [4,[30][31][32].Finally, it is important the search for new products, methods, and practices for surface disinfection, for which O 3 gas appears as a promising alternative.

Conclusions
Our study proved the antifungal potential of O 3 gas produced by two different devices, according to the described criteria.This is a satisfactory procedure for decontamination purposes due to its fast and easy execution.This procedure provides a new perspective for the disinfection of surfaces aiming at better control of microbial dispersion.The incorporation of this procedure into hospital hygiene and disinfection protocols may decrease contamination rates and, consequently, fungal infections in health facilities.The action of O 3 on fungal control is important and further studies should be conducted to optimize the best protocols against this group of microorganisms.

Figure 1 .
Figure 1.Illustrative photographic image of the positions and distances of the plates in relation to the source of O3.

Figure 2 .
Figure 2. Arithmetic means and standard deviations of CFU counting of C. albicans, at 30 cm, 1 m, and 2 m; and positions A, B, and C. D: air conditioning on, Mod.I; E: air conditioning off, Mod.I; F: air conditioning on, Mod.II; G: air conditioning off, Mod.II.Ct. = Control.

Figure 3 .
Figure 3. Arithmetic means and standard deviations of CFU counting of A. fumigatus, at 30 cm, 1 m, and 2 m; and positions A, B, and C. D: air conditioning on, Mod.I; E: air conditioning off, Mod.I; F: air conditioning on, Mod.II; G: air conditioning off, Mod.II.Ct. = Control.

Table 2 .
Percentage of inhibition obtained by the arithmetic mean of the CFU, at 30 cm, 1 m, and 2 m of distances; and positions A, B and C; with the air conditioner on and off; in comparison with the control (Mod.Ι).

Table 3 .
Percentage of inhibition obtained by the arithmetic mean of the CFU, at 30 cm, 1 m, and 2 m of distances; and positions A, B and C; with the air conditioner on and off; in comparison with the control (Mod.IΙ).