Fungicidal Activity of Five Disinfectants on Isolates of Candida Species

Determine fungicidal activity of five disinfectants on Candida auris of clinical and environmental origin. Assess fungicidal efficiency of each disinfectants on the microorganism. Calculate percentage of efficiency (% E) of each disinfectants on Candida strain and determine specific death rate (k) and decimal reduction time for the microorganism. An analytic and experimental research with quantitative methods. We realized a strains workbench by culturing it in corresponding culture media. Analysis was a challenge essay, performed on four surfaces and at different exposure times; 1, 5, 15 and 30 minutes of contact between disinfectant and yeast. Once disinfection process was carried out, what was expected was that after 15 minutes of contact between the yeasts and the disinfectants, a percentage of inhibition of the microorganism between 90 and 95% was presented, thus guaranteeing the duration of elimination of the infectious agent by disinfectants. Results shows that after one minute of contact between Candida auris and the disinfectant in a gloved hand, there was a removal of the yeast, when the clinical and environmental strain were analyzed. Data showing disinfectants efficiency percentages greater than 90% over strains of Candida used in the study. Decimal reduction times in seconds of the strains of Candida against the disinfectants used in the study were between 9.3 and 14.04 s. It was possible to evaluate effectiveness by means inhibition percentage of each disinfectants on the strains of Candida auris of clinical and environmental origin. Similarly, specific death rate and decimal reduction time in seconds were determined for strains under study.


Introduction
Candida genus are a group of yeasts responsible for the majority of fungal infections. In a multicenter study in United States, these yeasts were identified as the seventh most common microorganism related with health-care associated infections, with increasing mortality rates in hospitalized patients [1]; in Brazil it was the sixth most common isolate in intensive care unit infections (ICU) [2] and in German ICU´s it was identified as a common mortality cause among bloodstream infections [3] Candida albicans, C. glabrata, C. krusei, among others, are the main species causing infections, but C. auris has emerged as a microorganism capable of causing outbreaks worldwide [4], exposing major resistance to multiple anti-fungals, such as fluconazole, voriconazole and amphotericin, giving as a result treatment difficulties in managing affected individuals. In order to contain these outbreaks chlorine based products were proposed to disinfect areas or colononized patients with C. auris [5] The aim of this study was to analyze the effect of chlorhexidine and four disinfectants currently used in hospitals and clinics, to assess their anti-fungal activity and determine if they are effective in eliminating the Candida isolates, incluiding C. auris.

Materials and methods
This research was an analytic research with quantitative methods.

Microorganisms:
The microorganisms analyzed were Candida auris, Candida albicans and Candida guillermondii, preserved in Petri dishes with Saboraud medium, at 5°C. A microscopic identification was made using Gram stain. The tests were carried out at the Laboratory of Human and Proteomic Mycoses in Pontificia Universidad Javeriana and the strains were obtained from the Laboratory of the National Institute of Health located in Bogotá-Colombia. The isolate of C. auris used was identified during an outbreak in Valledupar (Colombia) [6] Cell bank. Sabouraud broth was used. The broth was distributed in 4 flasks of 250 mL. Each including 50 mL of a suspension of Candida in question. The flasks were left in a shaker for 24 hours to ensure the growth of the yeasts. Once the time had elapsed, the bank was made using Eppendorf tubes with 500 uL of medium and Candida strain and 500 uL of 85% glycerol. Each strain of Candida species used, had a bank of 50 eppendorf tubes that were frozen at -20 O C.

Analysis of CHG/CTR versus the 4 strains of Candida
To analyze the action of CHG/CTR against the strains of Candida, a gloved hand was immersed into a solution with a known count of each yeast in question(Candida albicans SC5314, Candida guillermondii H025, Candida auris of clinical origin and Candida auris of environmental origin). After 30 seconds, once the solution dried, the glove was exposed to the disinfectants. Counts were made in triplicate at 1, 5, 15 and 30 minutes, to analyze the percentage of inhibition of the microorganism against the disinfectant.

Analysis of GLUT1 and chlorine against the 4 strains of Candida
To analyze the action of GLUT 1 and the chlorine solution against the strains of Candida, different sections of a wall located in a laboratory at the Pontificia Universidad Javeriana in the Faculty of Basic Sciences, were exposed with a known count of each yeast in question (Candida albicans SC5314, Candida guillermondii H025, Candida auris of clinical origin and Candida auris of environmental origin). Cleaning was performed according to the manufacturer´s instructions. After cleaning was executed, counts were made in triplicate at 1, 5, 15 and 30 minutes, to analyze the percentage of inhibition of the microorganism against GLUT 1 and the chlorine solution.

Analysis of GLUT 2 against the 4 strains of Candida
To analyze the action of GLUT 2 against the strains of Candida, a surface of a work bench located in a Laboratory of the Pontificia Universidad Javeriana in the Faculty of Basic Sciences, was exposed with a known count of each yeast in question. (Candida albicans SC5314, Candida guillermondii H025, Candida auris of clinical origin and Candida auris of environmental origin). Cleaning was performed according to the manufacturer´s instructions. Counts were made in triplicate at 1, 5, 15 and 30 minutes, to analyze the percentage of inhibition of the microorganism against the disinfectant.

Analysis of SURF against the 4 strains of Candida
To analyze the action of SURF against the strains of Candida, a surface of a metalwork bench located in a Laboratory of the Pontificia Universidad Javeriana in the Faculty of Basic Sciences, was exposed with a known count of each yeast in question. Where, k is the specific death rate.
The percentage of inhibition is expressed by the formula: Where, R1 is the initial count, R2 is the final count.

RESULTS AND DISCUSSION
Natural logarithm Counts of CFU/ml at the exposure times evaluated are shown in Table 1. The disinfectants used in the study show a significant result regarding elimination of the microorganism within one minute of exposure. Solution C showed elimination of the strain in question, within five minutes of exposure. Percentages of inhibition of microorganisms as a result of the action of the disinfectants is showed in Table 3 Figure 2. shows the decimal reduction times (DRT) in seconds of the strains studied against the disinfectants in question.
The highest DRT was between Candida auris environmental origin and CHG/CTR. Candida auris Clinical origin has the lowest decimal reduction time, so is most sensitive strain in this study. Solution A has the best reduction time. In Candida auris 2 the average was around 13.6 s Using disinfection solutions, there was a complete elimination of the controls used in the study, Candida albicans SC5314 and Candida guillermondii H025 after 1 minute of action of the disinfectant in gloved hand, wall and work bench.
Our results show an important effect of chlorhexidine in combination with cetrimide and some of the conventional disinfectants over clinical isolates of Candida spp., including two isolates of C. auris, identified during an outbreak in Colombia [6]. This study, used two controls of the genus, C. albicans SC5314 and Candida guillermondii H025, due to the ability to form biofilms [7], and the specie in question, Candida auris, a multi-resistant emergent yeast that represents a major risk to the public health [8]. These two isolates were chosen due to the uncertainty related to the effect of this kind of products over such specie. The current recommendation is the use of an Environmental Protection Agency (EPA)-registered hospital-grade disinfectant effective against Clostridium difficile spores, the majority of which correspond to chlorine solutions [9].
In order to guarantee a correct elimination and prevent a dissemination throughout the hospital surfaces and areas, and to prevent patient colonization and infection with the infectious agent, it´s demanding to exercise a correct antisepsis and disinfection.
Analyzing disinfection and survival of Candida on surfaces, a study made by [10], determined how Candida auris and other Candida species are prone to develop on surfaces, especially when they are moist, creating a potential source of transmission of the microorganism. In their study quternary ammonium compounds, acetica acid and ethyl alcohol 29% were unable to eliminate a considerable amount of Candida in the study. In another study conducted by [11], chlorhexidine gluconate was used at different concentrations to control outbreaks of Candida auris in hospitals and clinics. The authors analyzed that exposure times less than two minutes and concentrations below 2% of chlorhexidine gluconate, were not enough to eliminate Candida auris.
In our study, a commercial combination of chlorhexidine 4% plus ceramide 1% was successful to eliminate the isolates in the hand glove model with an effectiveness of 100%. A commonly used strategy is to combine the action of chlorhexidine with isopropyl alcohol, to guarantee antisepsis of skin and hands. A study demonstrated that levels of Candida auris decrease, compared to the initial population of the yeast when exposed to chlorhexidine in combination with isopropyl alcohol, while the use of chlorhexidine alone at 2% was not able to eradicate C. albicans. This study and others, showed also the effectiveness of 10% povidone-iodine to reduce the number of colonies of Candida strains. In our case, the tested product included chlorhexidine at 4% in combination with cetrimide and the observed effect was a clear reduction in the number of colonies.
Worth noting, several studies have shown that Candida auris strains have the ability to form large aggregation of cells, protecting the yeast against the action of chemical disinfectants; reason that can be attributed to the difficult elimination of the microorganism on surfaces. A study conducted by [12], analyses the role of extracellular matrix of Candida species, in biofilm formation leading to drug resistance and difficult elimination of the microorganism on surfaces. A polysaccharide complex of mannan-glucan is found on most of Candida species. This, combined with an abundance of efflux pump transcript and an interestingly high expression of glucan modifying genes, could be considered as signature components involved in antifungal sequestration and drug resistance biofilm in Candida auris.
In order to analyze correctly the effectiveness of the disinfectants in the study, several factors need to be taken into consideration.
According to [13], wet contact time directly affects in the antimicrobial efficacy. When analyzing Candida auris, it is demanding to guarantee a continuous wetness during microbial efficacy testing. The EPA requires this method, when the analysis involves either Candida auris or Clostridium difficile.
The action of quaternary ammonium compounds has been questioned. In the study by Candum et al, these type of products could not reach the same level of activity compared with others. Since there are different types of combinations it is difficult to ascertain if this is a class effect, a difference in the concentrations and products tested or, even technical differences among studies performed. In a study conducted by [14], the action of a quaternary ammonium with potential use in dental care was tested against the genus Candida.
Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations were found in a range that is possible to be reached in clinical or environmental scenarios. In another study conducted by [15], quaternary ammoniums were tested against Candida albicans in combinations with products used for the care of contact lenses. This study showed a limited effect on fungal cells, showing the need for doing different test against this products in the different settings in which they are intended to be used. A study conducted by [16], analyzed the action of glutaraldehyde in combination with a quaternary ammonium against strains of C. albicans. Glutaraldehyde acts by distorting the membrane, affecting its integrity and function, and preventing the formation of biofilms, which are important in the process of colonization and surface infection as mentioned before. Since there are different combinations commercially available, before its use, at least for the disinfection of surfaces potentially containing Candida isolates, a formal test should be done to prove their effectiveness.
Carboxylic acid is a chemical agent used for the elimination of Gram-positive and Gram-negative bacteria including Salmonella sp, Campylobacter sp, Listeria sp and yeasts. In a study carried out by Hai-Nam et al; it was found that carboxylic acid affects the lipid matrix of the cells, facilitating the entry of the chemical agent into the microorganism [17].
In our study, a combination of products including carboxilic acids showed complete elimination of the Candida isolates in the tests done.
The main limitation for our work is that the individual agents were not tested against the Candida isolates, and no conclusion can be drawn from each individual agent.
In conclusion, the present work evaluates the effects of some disinfectant products and chlohexidine in candida isolates over organic free surfaces, providing evidence of the effects of such combination products over isolates of from clinical and environmental samples of C. auris Further experiments are required to establish firm recommendations but our results suggest that other products different from chlorine derivate might be useful in environmental cleaning and clinical antisepsis. Figure 1 Death kinetics of the microorganism vs. the action of the disinfectant in the exposure times evaluated. A single gure is shown because the same behavior was seen throughout the study. Candida albicans SC5314 vs. Solution B y= -0.3931x + 4,5858 R2 = 0.6