Viability of Candida albicans in different fomites and hospital surfaces under disinfectants and biological fluids influence

Hospital environment has factors that promote increased infections caused by microorganisms allowing them to remain viable for long periods on surfaces and fomites. The aim of our study was to identify the viability of Candida albicans in hospital environment common surfaces with biological fluids presence and disinfectants. Four surfaces (cotton fabric, synthetic fabric, egg crate foam mattress and, ceramic floor) were contaminated with a strain of Candida albicans ATCC 26790 and with an association of distilled water, blood, saliva and urine. Also, combinations of surfaces and fluids with addition of 70% alcohol and 2% sodium hypochlorite were tested. Viability was identified at 1, 3, 7, 14 and 21 days by Colony Forming Units counting. C. albicans presented 14 days viability in hospitals fabrics with absence of liquids and maintained this persistence under influence of distilled water and blood. It also showed low growth with urine addition and there was growth inhibition with 2% sodium hypochlorite while with 70% alcohol and blood there was persistence for up to 7 days in synthetic fabric and mattress. The understanding of these yeasts persistence in hospital surfaces and fomites is essential for a better delineation of protocols control the spread and these infections prevention in healthcare environments.


Introduction
Candida albicans adhesins secretion and ability to form hyphae contributes to the formation of fungal biofilm (Chen et al., 2020;Mccall et al., 2019, Ponde et al., 2021. It allows the yeast attachment, maintenance and propagation on abiotic surfaces for long periods and thus can make these environments sources for cross-infection in the healthcare environment (Safdar et al., 2019;Viela et al., 2020). This fungi is able to modulate the metabolism in different environmental conditions as variable temperature, pH and osmolarity, and availability of nutrients (Santos et al., 2018). Those characteristics leads Candida species as the fourth pathogen most commonly isolated in Healthcare-associated infections (HAI) and the second related to Urinary Tract Infections (UTI) associated with catheters (Enoch et al., 2017;Ture and Alp 2018;Chmielarczyk et al., 2021).
Despite information about C. albicans survival on nosocomial microenvironment and its correlation with infection processes have been studied over the years, influence of body fluids and disinfectants efficacy remains unknown. The Brazilian Health Regulatory Agency (ANVISA, 2012), recommended strict procedures to surface disinfection which aim to decrease the counting microorganisms cells number. Facing that, we evaluated the Candida albicans viability on surfaces and fomites common to the hospital environment, under body fluids and disinfectants influence.

Methodology
Yeast strain, media and culture conditions: strain C. albicans ATCC26790 used in this study belongs to Microbiology and Immunology Research Laboratory stock of University of Western Sao Paulo (UNOESTE). It was kept frozen at -20ºC in Nutrient Broth supplemented with 25% glycerol and 2% glucose. Reactivation of frozen strain was done by inoculum in Brain Heart Infusion (BHI) broth incubated for 24-48 hours at 35ºC, followed by seeding agar Sabouraud Dextrose and incubation for 48 hours at 35ºC. Inoculum for surfaces contamination was prepared from microorganism culture in sterile distilled water at a concentration equivalent to 1.0 on McFarland scale (3.0x108 Colony Forming Units / mL).
Cell viability assay: yeast viability and persistence on different surfaces were identified from immersion of the contaminated surfaces in flasks (urine collection bottles) containing BHI broth and then incubated for 48 hours at 35°C. Then, after homogenization, 100uL of broth was transferred to tubes with sterile saline solution for serial dilution standardized at 10-3. Colony Forming Units (CFU) was counted from the lowest dilution (1:1000)

Results
Candida albicans viability and persistence can be altered (p <0.05) by the surface composition and modified over the days through the nutrient's availability from the medium (biological fluid). We observed that, in the absence of fluids, C.
albicans showed better persistent on surfaces with higher moisture retention capacity (egg crate foam mattress, cotton fabric and synthetic fabric) than on those with lower moisture retention capacity (ceramic floor). Viability was observed until 14 days in cotton fiber and synthetic fiber fabrics, 7 days in egg carton mattress and up to 3 days in ceramic tile (Figure 1). 4 floors with distilled water for this yeast. Regarding manipulated saliva, C. albicans was able to survive for 7 days on synthetic fabric, ceramic floor and mattress, however, in cotton fabric; its survival rate was observed for three days.
In urine, yeast growth was identified for seven days on cotton fiber fabric, three days on synthetic fiber fabric and one day on egg crate foam mattress. Nevertheless, C. albicans showed greater viability with blood and persistence for 14 days in both fabrics and, for seven days on ceramic floor and mattress.
We observed decreased fungal viability on surfaces with less moisture retention (ceramic floor) under influence of all fluids (Figure 2). In comparison between groups with addition and groups without of fluids, we found higher viability of C. albicans (49.65CFU/mL) in groups without body fluids (p = 0.0001). Followed by blood (23.80 CFU/mL), saliva (14.20 CFU/mL), urine (13.35 CFU/mL) and distilled water (13.10 CFU/mL). Use of alcohol 70% associated with blood fluid allowed C. albicans survival for up seven days in the synthetic fiber tissue and egg crate foam mattress, against 3 days in the ceramic floor.

Discussion
We identified C. albicans survival for 14 days in all fabrics. Previous studies have shown that C. albicans can survive and persist for months on abiotic surfaces (Kramer et al., 2006;Prada et al., 2019). Neely and Orloff, 2001, studied fungi survival of medical importance in fabrics and plastics from hospital environment and identified four days an average survival of in all materials for C. albicans, C. tropicalis and C. krusei species. Slightly differing from our data, Patojenlerin et al., 2012 reported persistence of C. albicans for up to six days on hospital fabrics with cotton fibers and synthetic fabric (60% cotton and 40% polyester) and 12 days in wool and silk. Prolonged natural persistence of C. albicans on abiotic surfaces leads it to become reservoirs and continuous sources for cross infection transmission if there is no efficacy in regular preventive cleaning.
Porosity of surface influenced C. albicans survival in this study. In accordance to our findings, Springthorpe et al., 2002 reported Candida species better growth and maintenance of viability on porous tissues. In addition, we observed increasing in colonies number on 7th day in comparison with the survival rates after firsts 24 hours, followed by colony decrease until the 14th day. Porosity might be also related to moisture retention capacity of surface. We found that surfaces with lower moisture retention capacity supplemented with urine presented lower survival rates. While surfaces with higher moisture retention capacity presented a better environment permitting C. albicans to survive longer periods. The ability to retain moisture inside creates, observed in egg crate foam mattress for example, makes the microenvironment viable for maintenance of fungal cells. Mitchell et al., 2015 reported that physical characteristics of fabric, surfaces electron charges and, also bacterial cell wall electron charges influence microorganism's fixation. The data found by them are similar to our findings concerning yeast persistence, besides the aid in the yeast fixation caused by its own hyphae production, typical of C. albicans.
As an important hospital surface, our study brought ceramic floor and egg crate foam mattress to the evaluation of C. albicans viability. No scientific data has presented C. albicans ability to survive on those surfaces. Piedrahita et al., 2017 has detected C. albicans persistence for at least seven days on dry (steel discs) and wet (non-nutrient agar) surfaces. These data are similar ours for egg crate foam mattress and higher in comparison with ceramic floor. Traoré et al., 2002 reported survival period of three days in glass and metal, similar results we found on our study concerning ceramic floor. Those data agree with C. albicans persistence in different surfaces and, our data, support the hypothesis of hospital surfaces are a relevant transmission source.
Our results indicated that saliva addition decreased C. albicans survival in comparison with distilled water. On the other hand, using similar methodology with medical interest bacteria, Esteves et al., 2015 found bacteria viability on surface containing saliva after eight weeks. We observed low persistence of C. albicans in urine sample. This might be related to urine pH which avoids microbial survival for long periods which decreased in the presence of urine (Esteves et al., 2015). Blood showed greater yeast viability due to its high ability to provide a rich nutritional environment for microorganism (Esteves et al., 2015). This characteristic might allow yeast to persist long periods on abiotic surfaces as showed in our study.
Disinfectants recommended for hospital surfaces disinfection used in this study led to the expected effect on total growth inhibition, mainly sodium hypochlorite 2% and also on most surfaces in association of biological fluids with 70% alcohol. Our data corroborate with Pereira et al., 2014 who showed inhibition growth of C. albicans and non-albicans species after 30 seconds of sodium hypochlorite contact. No data were found in current scientific publications regarding the identification of yeast viability in association with alcohol 70% then Peters et al., 2012 evaluated ethanol efficacy against microorganism biofilms. They found that incubation with ethanol 30% for 4 hours is sufficient to kill and completely inhibit C.
albicans growth. Our data may has shown different pattern using alcohol 70% due to absence of cleaning movements, which are recommended by ANVISA. However, even growth persistence using alcohol 70%, we identified a decreasing cell counting in synthetic fabrics and, ceramic floor when compared with blood treatment.

Conclusion
C. albicans strains significant persist viable on surfaces with higher moisture retention capacity. Use of 2% sodium hypochlorite may still be an effective control strategy for these yeasts in nosocomial environments. By using 70% alcohol in association of blood in the ceramic floor, egg crate foam mattress and synthetic fabric, the expected chemical agent action was not immediate. The understanding of these yeast persistence in hospital surfaces and fomites is essential for a better definition of protocols to control viability of C. albicans on abiotic surfaces, leading to infections prevention in healthcare environments.
We suggest controlling the spread of nosocomial pathogens through routine surface disinfection using 2% sodium hypochlorite, including to control cross-contamination caused by C. albicans. In addition, it is necessary to conduct further research on different surfaces with different sanitizers including nanoparticles in the search for hospital environments free of pathogenic microorganisms such as yeasts of the genus Candida.