Effect of ozone gas on cultures of candida albicans and aspergillus fumigatus : evaluation of two ozonation equipment

The use of O 3 as antimicrobial has stood out as a useful chemical compound for disinfection processes and/or sterilization due to its high oxidant power. Antimicrobial effects of ozone gas (O 3 ), produced by two commercial equipment on cultures of two fungi were studied.


Background
Fungal infections are of enormous importance in the scenario of nosocomial diseases in 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, favoring the risk of infectious diseases. Several fungi such as opportunists or primary pathogens are frequent agents, especially the species Candida spp. and Aspergillus spp. [1][2][3][4].
Candida albicans ( C. albicans) is part of the normal microbiota; however, it can cause infections by ruptured biological balance, by predisposing pathological, mechanical, physiological or immunological conditions [5][6][7]. The Aspergillus fumigatus ( A. fumigatus) is a ubiquitous saprophytic fungus that releases millions of conidia into the environment which is a common cause of invasive infections when inhaled, as well as being an allergen agent [8][9].
The use of O 3 as antimicrobial has stood out as a useful chemical compound for disinfection processes and/or sterilization due to its high oxidant power [10]. Although the mechanisms of its action are not fully understood, O 3 is known to act on cell walls given the oxidation of glycopeptides, glycoproteins and amino acids, modifying permeability and causing lysis. O 3 is recombined with cytoplasmic elements when reaches the inside of the cell, causing the oxidation of amino acids and nucleic acids, causing cleavage and consequent cell death. O 3 also causes the collapse of cellular enzymatic activity due to its action in the sulfhydryl compounds of enzymes, in addition to altering the purine and pyrimidine bases of nucleic acids [10][11][12].
Our study sought to compare the antifungal activity of gas O 3 produced by two commercial equipment, against cultures of C. albicans and A. fumigatus.

O 3 generators -technical specifications
We used two equipment from OZON® company, called GEO 20000/AR-TD (Mod.I) and GEO 20000/AR (Mod.II), whose specifications are indicated in Table 1.

Discussion
Our study is a pioneer in the evaluation of the antifungal action of O 3 gas on the surface In this context, the best experimental condition occurred to C. albicans, at 30 cm of distance, direction B (central), while A. fumigatus, at 2 m of distance and direction A (dishes near the wall), represented the less favorable condition. This may be related to the gas flow rates. In laminar flows, particles move orderly, always maintaining the same relative position. In the turbulent regime, these particles move randomly and irregularly [13], that is, the flow of O 3 gas when reaches the wall causes disorder, thus decreasing antifungal activity when compared with the dishes near the wall, which may have interfered with the O 3 flow, thus justifying the variation in the number of CFU in all distances and directions.
The best antifungal activity in the ozonation process for both devices when the air conditioner was turned off, for both fungal species, may have been observed due to the lack of interference with the O 3 flow, resulting in surfaces more affected by O 3 gas. Unlike when the air conditioning was turned on, with the function of capturing air and filter before throwing it again into the environment [14], the flow of O 3 gas varies when reaching the surface, thus decreasing its antifungal activity.
According to the literature, temperature, relative humidity variation and treatment time lead to a modified O 3 antimicrobial effect [15]. In our study, these factors were maintained equal in all experimental conditions, minimizing possible biases. According to the values we detected, the cultures of C. albicans were more sensitive than A. fumigatus, which corroborates the literature [15]. In fact, A. fumigatus has can grow and survive humidity environments and extreme temperatures, with dispersion of conidia [9].
According to Santana et al [7], C. albicans is less adapted to the diversity of environments outside the human body than A. fumigatus.
Several studies have been conducted aiming at investigating the antimicrobial activity of shows excellent antimicrobial activity [5,12,17,18]. Thus, it is evident that our study corroborates other studies in the literature.
Zotti et al. [19] observed phenotypic changes in the colony of A. flavus and A. niger after exposure to O 3 gas, with sharp decrease in growth of both fungi, and change in their natural pigmentation. In addition to the antimicrobial activity, O 3 gas can be expected to inhibit pigments and protein synthesis, with future impairment to virulence factors of pathogenic fungal species.
Surface cleaning is crucial for the control of Healthcare-Associated Infections (HAI) [20].
Unfortunately, health services surface cleaning and disinfection is often overlooked.
Cleaning and disinfection practices of the environment, equipment, and surfaces should be implemented and discussed by the Hospital Infection Commissions, together with nursing and cleaning services, developing activities related to environmental hygiene protocols, supervision, and training [4,21,22,23,24].
Finally, it is important the search for new products, methods, and practices for surface disinfection, and the O 3 gas appears as a promising compound.

Conclusion
Our study proved the antifungal potential of O 3 produced by two different devices, The present study met national and international ethical precepts. The research did not involve humans, and for this reason received ethical waivers by the Human Research Ethics Committee.

Consent to publish
Not applicable.

Availability of data and materials
All data and materials are available upon request to the corresponding author (Alvaro Francisco Lopes Sousa).

Competing Interests
One of the authors of the manuscript (Alvaro Francisco Lopes Sousa) is on the editorial board of BMC Infectious Diseases, as Associate Editor.

Funding
The study was supported by the Coordenação