Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach

doi:10.21203/rs.3.rs-4546226/v1

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Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach

https://doi.org/10.21203/rs.3.rs-4546226/v1

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published 20 Aug, 2024

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Candida albicans has been listed in critical priority group by the WHO in 2022 depending upon its contribution in invasive candidiasis and increased resistance to conventional drugs. Drug repurposing is an efficient and cost-effective solution to develop alternative therapeutics where alexidine dihydrochloride (AXD) and hexachlorophene (HCP) are FDA approved anti-cancer and anti-septic drugs, respectively. In this study, we have shown antifungal properties of AXD and HCP against C. albicans and clinical isolates. The minimum inhibitory concentrations (MIC₅₀)of AXD and HCP against C. albicans ranged between 0.2-0.4 µg/ml and 8-10 µg/ml, respectively. The biofilm inhibitory and eradication concentration of AXD and HCP also ranged in permissible range for C. albicans biofilm. Further investigations were performed to understand the antifungal mode of action of AXD and HCP by studying virulence features like cell surface hydrophobicity, adhesion, and yeast to hyphae transition, were also reduced upon exposure to both the drugs. Ergosterol content in cell membrane of the wild type strain was upregulated on exposure to AXD and HCP both. Biochemical analyses of the exposed biofilm indicated reduced contents of carbohydrate, protein, and e-DNA in the extracellular matrix of the biofilm when compared to the untreated control biofilm. AXD exposure downregulated activity of tissue invading enzyme, phospholipase in the reference strain. In wild type strain, ROS level, and activities of antioxidant enzymes were found elevated upon exposure to both drugs. FESEM analysis of the drug treated biofilms revealed degraded biofilm. This study has indicated mode of action of antifungal potential of alexidine dihydrochloride and hexachlorophene in C. albicans.

Alexidine dihydrochloride

Biofilm

C. albicans

ECM

Hexachlorophene

ROS

In late 2022, WHO has listed four Candida spp. under critical fungal pathogen category in its first ever fungal pathogen priority list (Fisher & Denning, 2023). Invasive fungal infections associated with C. albicans, are the primary cause of the rising death rate in the immunocompromised population (Low & Rotstein, 2011; Rayens & Norris, 2022). Over the last few decades, invasive candidiasis, caused by C. albicans has become the most prevalent illness among hospitalised patients, giving rise to a critical condition known as candidemia which has been listed as the fourth most frequent cause of bloodstream infection (Bongomin et al., 2017; Pahwa et al., 2014; Weiner-Lastinger et al., 2020). Due to the long-term usage of antifungals, isolates of C. albicans are developing antifungal resistance against available antimycotics (Costa-de-oliveira & Rodrigues, 2020; Dabas et al., 2022). The limited availability of antifungals has made the scenario more complicated (Vandeputte et al., 2012). Drug repurposing has provided a time saving and cost-effective approach and is an alternative to new drug discovery to address the rising issue of antimicrobial resistance. Accordingly, extensive screening of several chemical libraries consisting of FDA-approved and off-patent medications, is being conducted in various parts of the world to find novel hits that have initial indications of being anti-inflammatory, anti-cancer, anti-septic, and anti-depressant (H. C. De Oliveira et al., 2019; Eldesouky et al., 2020; Kim et al., 2020; A. S. Oliveira et al., 2018; Peyclit et al., 2021; Siles et al., 2013a; Wall et al., 2018; Yousfi et al., 2020).

Alexidine dihydrochloride (AXD), a bis-biguanide, is a well-known antibacterial drug with anti-inflammatory and anticancer properties that causes apoptosis by inhibiting mitochondrial tyrosine phosphatase, PTPM1 (Doughty-Shenton et al., 2010; Yip et al., 2006). Pan antifungal properties of AXD against Candida sp. and, other dermatophytes along with filamentous fungi have been documented previously by various groups in vitro and in vivo (Mamouei et al., 2018a; Nabeela et al., 2022; Siles et al., 2013a; Yousfi et al., 2020). AXD is being used in mouthwash as an antiplaque agent for its application in endodontic treatment to remove biofilms (Mamouei et al., 2018b; Ruiz-Linares et al., 2017; Silveira et al., 2013). It exhibited effectiveness against C. albicans and E. faecalis at very low concentrations (Kermeoglu et al., 2018; Siles et al., 2013b). In recent reports, AXD exhibited growth inhibitory properties against multi drug resistant strain of C. auris (Cheng et al., 2021). On the other hand, hexachlorophene (HCP) is extremely lipophilic chlorinated bisphenol with antiseptic properties against several gram-positive bacteria and pathogenic fungi (Gibson 1969.Joswick et al. 1971; Siles et al. 2013a; Yousfi et al. 2020). The antimicrobial, and antifungal activities of HCP alone or in combination with miconazole have been reported against pathogenic bacteria and fungi (De Cremer, Staes, et al., 2015). In recent studies, both the drugs have shown pan-antifungal properties against multidrug resistant filamentous fungi, and Trichophyton (Nabeela et al., 2022; Yousfi et al., 2020).

There are reports indicating antibiofilm activity of AXD and HCP, but the underlying mechanism is still need to be explored (De Cremer, Lanckacker, et al., 2015; Mamouei et al., 2018b; Ruiz-Linares et al., 2017; Silveira et al., 2013), therefore to broaden the understanding of pronounced antibiofilm activities of the two drugs, preluding factors like cell surface hydrophobicity (CSH), germ tube formation and adhesion to the surfaces, were investigated. We further investigated the impact of AXD and HCP on biochemical composition, hydrolytic enzymes, and ROS and antioxidant profile of C. albicans biofilm cells. Moreover, impact of drugs on ergosterol content of C. albicans was investigated. To the best of our knowledge, there is hardly any report on antifungal mode of action of AXD and HCP in C. albicans with especial emphasis on structural and biochemical attributes of the biofilm. In this study, other than including a broad spectrum of the virulence parameters, an effort has been made to explore the antibiofilm mechanism of action of two FDA approved drugs in C. albicans.

Strains and Chemicals

All stains were routinely maintained on yeast peptone dextrose (YPD) broth or agar. Sabouraud dextrose broth (SDB), YPD, and RPMI-1640 (Rosewell Park Memorial Institute) buffered with MOPS (pH-7.0) were used in the experiments. In general, cells were grown at 37°C or as mentioned otherwise. The chemicals, drugs and all the media components were procured from Himedia, and SRL, India. The stock solution drugs were prepared in dimethyl sulfoxide (DMSO). Wild type strain of C. albicans (SC5314) was procured from CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Four C. albicans clinical isolates of invasive candidiasis (CCA1-CCA4) were previously described elsewhere (Gupta et al., 2016).

Antifungal susceptibility test

CLSI guidelines were followed for yeast broth microdilution assay to obtain growth inhibitory and fungicidal concentrations of AXD and HCP against C. albicans and clinical isolates in 96 well MTP (Gupta & Poluri, 2022). Briefly, log phase cells were diluted to adjust the cell density of 2.5 × 10³ cells/ml in RPMI-1640, were exposed to different concentrations of drugs ranging 0.1–0.6 µg/ml for AXD, and 2–25 µg/ml for HCP. The MTP was incubated at 37°C for 48 h. The fungicidal concentration of drug was determined by spotting 5 µl of drug treated cultures on YPD agar plates followed by incubation of the agar plates for 18 h at 37°C before being photographed. Dose points of no growth were considered as MFC.

Time kill assay

For determining the time dependent effects of different doses of the drugs, time kill assay was performed against wild type strain (Priya et al., 2021). Briefly, log phase cells were exposed to MIC₅₀, 2× MIC₅₀, 4× MIC₅₀ concentrations of drugs upto 5 h in YPD broth in different tubes. Average MIC₅₀ doses were used i.e. 0.3 µg/ml and 9 µg/ml for AXD and HCP, respectively. Afterwards, 5 µL of cells suspension from each tube was spotted onto YPD agar plate after different exposures (0, 1, 2, 3, 4, 5 h). The images of plates were taken after 18-hour incubation period at 37°C.

Growth kinetics

The growth kinetics of C. albicans and its clinical isolates in the presence and absence of AXD and HCP by growing them in YPD broth at 37°C (Nordin et al., 2013). Briefly, log phase cells were exposed to average MIC₅₀ doses of AXD and HCP (0.3 µg/ml and 9 µg/ml, respectively for the reference strain) followed by measuring OD₆₀₀nm for 7 h with an interval of 1 h through UV-Vis spectrophotometer (Agilent). Sub lethal doses of AXD and HCP were used in growth kinetic studies.

Ergosterol content

Cell membrane ergosterol content of planktonic cells of wild type and clinical isolates of C. albicans were estimated after exposing them to MIC₅₀ of AXD (0.3 µg/ml) and HCP (9 µg/ml) for 24 h at 37°C (Gupta, Gupta, and Poluri 2021; De Oliveira Pereira, Mendes, and De Oliveira Lima 2013). After the drug treatment in SDB, cells were centrifuged washed with sterile water. Wet weight of pellet was estimated followed by resuspending in lysing solution (25% alcoholic KOH) and vigorous vertexing. The sterols were extracted by mixing water and n- heptane in 1:3 ratio. The organic layer of n- heptane was carefully pipetted out and mixed with absolute ethanol, and scanned using spectrophotometer in the range of 230–300 nm. Percentage ergosterol was calculated using following formula.

\(\varvec{\%}ERG=\frac{\left[\left(\frac{A281}{290}\right)\times F\right]}{pellet weight}-\frac{\left[\left(\frac{A230}{518}\right)\times F\right]}{pellet weight}\) (Eq. 1)

Cell surface hydrophobicity

Log phase cells of overnight grown cultures were diluted to OD_600nm 0.1 and were exposed to minimum inhibitory concentration of the drugs followed by incubation for 24 h at 37°C (AXD: 0.3 µg/ml; HCP: 9 µg/ml) (Gupta et al., 2022). Cells were harvested and washed followed by cell suspension preparation in 3ml sodium phosphate buffer. Octane was then added to this suspension and vortexed for 1 min. After carefully extracting the aqueous phase, the absorbance at 600 nm was used to quantify the cells that were present in the aqueous layer using spectrophotometry. the hydrophobicity index was determined by using the following equation

\(\text{H}\text{I}=\frac{A1-A2}{A1}\times 100\%\) (Eq. 2)

where A₁ is the absorbance of initial inoculum and A₂ is the absorbance of aqueous phase. CSH index of wild type strain as well as selected isolates were analyzed in absence of the drugs with the controls.

Germ tube formation assay

Effect of the drugs on germ tube formation (yeast to hyphae transition) was studied in wild type strain (CA) and selected clinical isolates (CCA2 and CCA4) following a previous study (Bernardes et al., 2012). Briefly, overnight cultures of all strains were diluted in YPD broth (OD_600nm ranging 0.2–0.4) with and without 10% fetal calf serum in presence and absence of drugs at MIC₅₀ doses of reference strain (CA) i.e. 0.3 µg/ml and 9 µg/ml for AXD and HCP, respectively. All tubes were incubated at 37°C with shaking for 4 h. Cultures were examined and photographed microscopically after nigrosine staining at 40X in light microscope (Olympus).

Adhesion assay

Effect of the drug on adhesion of C. albicans and its clinical isolates was studied in 96-well flat bottom MTP (Raut et al., 2013). Briefly, phosphate buffered saline (PBS) of pH 7.0 was used to dilute log phase cultures of the stains to a concentration of 1× 10⁷ cells ml^-1. Fifty microliters of cell suspension were added to each well following the addition of 50 µl of drug dilutions in PBS (AXD: 1–10 µg/ml & HCP 1–12 µg/ml). Control well was kept without drug. The plate was incubated at 37°C for 90 min at 100 rpm. Afterward, wells were washed with sterile PBS, and adhered cells were quantified using XTT reduction assay by measuring OD at 492nm.

Biofilm formation assay

Effects of the drugs on biofilm formation were analyzed on flat bottom MTP followed by XTT reduction assay (Gupta et al., 2018). Briefly, log phase cells were diluted to density of 1× 10⁷ cells ml^-1 in PBS, and 100 µl of cell suspension was added to each well followed by incubation for 90 min at 37°C. After washing with PBS, 200 µl drug dilutions in RPMI were added to different wells (AXD: 1–8 µg/ml & HCP: 1–14 µg/ml) with a control sample without any drug. The MTP was incubated at 37°C for 48 h. After the incubation, wells were washed with sterile PBS, and the developed biofilm was quantified using XTT reduction assay by measuring OD at 492nm.

Biofilm eradication assay

Biofilm eradication effects of AXD and HCP were studied by seeding and maturing biofilm for 24 h as mentioned above (Gupta et al., 2018). After the incubation, the biofilm was washed twice with sterile PBS followed by addition of drug dilutions in RPMI (AXD: 1–8 µg/ml & HCP: 1–16 µg/ml). Control sample was kept without any drug and plate was again incubated for another 24 h at 37°C followed by quantification using XTT reduction assay by measuring OD at 492nm.

Biochemical composition of extra cellular matrix (ECM) of biofilm

ECM is a gelling material of cells and other components of biofilm. The biochemical composition of ECM of the wild type C. albicans was determined in presence of sub-lethal concentrations of the drugs (Gupta et al., 2018). Briefly, C. albicans biofilm were developed in the presence AXD (6 µg/ml) and HCP (8 µg/ml). After drug treatment for 24 h, the biofilm was scrapped out from MTP using a sterile scrapper and PBS (pH 7.0). ECM from the biofilm was isolated by sonicating (GT-Sonic D9) at 35W in an ice bath for five cycles of 30 seconds each. The ECM suspension was centrifuged at high speed to separate out pellet and supernatant. The supernatant with ECM components was examined for biochemical and enzymatic assays including total carbohydrate, protein, eDNA, proteinase, phospholipase, and SOD activity whereas cell pellet was employed for catalase activity measurement.

Carbohydrate, protein and eDNA

Total carbohydrate was estimated by phenol-sulfuric acid method using glucose as a standard. Briefly, 100 µL of supernatant was mixed with 1 ml of sulfuric acid and 200 µL of phenol (5% w/v) in glass tubes followed by incubating at 30°C for 30 min. The tubes were then cooled down and the absorbance was taken at 485 nm.

Total protein was measured using Bradford regent, and bovine serum albumin as standard. The samples were incubated for five min in dark for color development. After the incubation, absorbance of samples was measured at 595 nm.

Total eDNA in samples was quantified by precipitating it by adding one-tenth the volume of sodium acetate (3M) in sample followed by the addition of phenol, chloroform and isoamyl alcohol (25:24:1). The aqueous layer was transferred to fresh tube and 2.5 volume of absolute ethanol was added. The purity of precipitated eDNA was measured using Nanodrop at 260/280 ratio.

Tissue invading enzyme activity

Phospholipase activity in ECM was measured as described in previous study (Gupta et al., n.d.). Briefly, substrate was prepared which consisted of 50 mM Tris-HCL buffer (pH 7.5), 0.25% Triton X-100, 1.6 mM phosphatidylcholine, 20 mM AlCl₃ and 0.124% bromothymol blue followed by filtration and storage of solution at 4°C. For the determination of phospholipase activities, 100 µL of sample was mixed with 900 µL of substrate (pH 7.5) followed by measuring absorbance at 630 nm. The specific phospholipase activity was documented as the absorbance shift per min of the reaction.

For the proteinase activity determination, 1% w/v azocasein substrate was mixed with the supernatant and the sample solution was incubated for 1 h at 37°C. After the incubation, 10% trichloroacetic acid was used to terminate the reaction. The mixture was centrifuged at high speed for 5 min. The obtained supernatant was mixed with 0.5 M NaOH and again incubated for 15 min followed by absorbance measurement at 440 nm. The amount of proteinase activity is defined as the amount of enzyme that caused the absorbance to rise by 0.001 units per minute during the process.

ROS & Antioxidant enzyme activity

Mitochondrial reactive oxygen species

Effect of sub-lethal concentrations of AXD (6 µg/ml) and HCP (8 µg/ml) on reactive oxygen species (ROS) level was studied in cells of C. albicans biofilm. For ROS estimation, DCFDA (10 µM) was added into black well multi well plate (MTP) and incubated at 37°C for 30 min in dark (Gupta et al., n.d.). The fluorescence of DCFDA was documented at the emission and excitation wavelength of 520 nm and 485 nm, respectively (BIOTEK, Agilent).

Catalase activity

For the determination of catalase activity, cell free extract was prepared as mentioned in (Jethwaney et al., n.d.). Glass beads were used for lysing the drug treated C. albicans sessile cells which were collected after ECM isolation and followed by centrifugation at 10,000 rpm. The supernatant obtained was transfer to fresh tube and was used for determining catalase activity. Briefly, 333 µL of 50 mM H₂O₂ and 567 µL of PBS (pH 7.0) was mixed with 100 µL of supernatant flowed by absorbance measurement at 240 nm. One unit catalase activity is the amount of enzyme that decompose 1 µM of H₂O₂ per min of reaction at 37°C (Gupta et al., 2018). Following formula was employed for calculating the catalase activity (U/mg)

\(\frac{\text{U}}{\text{m}\text{g}}=\frac{(\text{A}0-\text{A}2)\times \text{V}\text{t}}{\sum 240 \times d\times Vs\times Ct\times 0.001}\) (Eq. 3)

A0-A2 is the difference in absorbance; Vt is total volume of sample; 240 is the molar coefficient of H₂O₂; d is optical pathlength of cuvette; Ct is the protein concentration in sample

Superoxide dismutase activity

SOD is an antioxidant enzyme which elevates during ROS or free radical generation. SOD activity was determined in the ECM upon exposure to the drugs (Gupta et al., 2022). The intracellular (cell free) extract was mixed with 2 mM EDTA, 55 µM nitroblue tetrazolium, 9.9 mM L-methionine, 1 µM riboflavin, 0.025% Triton ×100 and 50 mM PBS to final volume of 200 µl. The plate was incubated in light as well as in dark for 10 min at room temperature. The U/mg SOD is the amount of protein required to prevent the reduction of NTB by 50%.

FESEM analysis

Morphological changes in wild type C. albicans biofilm cells upon the exposure to sublethal concentrations of AXD (6 µg/ml) and HCP (8 µg/ml), were analyzed using FESEM as per the previous study (Gupta & Poluri, 2022). Briefly, small pieces of polystyrene discs (1 cm²) were preconditioned and 1mL of cell suspension was added onto it after placing in 24 well plate and incubated for 48 h. Media containing sub lethal dose of AXD and HCP was added after 48 hours and again incubated for 24 h at 37°C. The disc was fixed overnight in 2.5% glutaraldehyde, and dehydrated. The air-dried discs were mounted on stubs followed by gold sputtering. Finally, the images were captured using FESEM at magnification ranging from 1000X to 5000X and voltage of 15kV.

Statistical analysis

Mean of three values of OD₆₀₀ nm along with standard deviation is represented in each assay of this study. The significant differences between the values of antifungal activities of drug was analyzed using student’s t-test with P value < 0.05 was considered as significant.

Antifungal Susceptibility Test

Minimum inhibitory concentration

Antifungal susceptibility assay of AXD and HCP was performed in RPMI broth, following CLSI guidelines. Both drugs have shown low MIC values against wild type and clinical isolates. As given in table 1, the MIC₅₀ value of AXD in CA was found to be 0.2–0.4 µg/ml, whereas MIC₅₀ values of AXD in clinical isolates were surprisingly low, ranging from 0.1–0.4 µg/ml. The clinical isolates, CCA1, CCA2, and CCA3 were found sensitive to AXD when compared to wild type strain.

The MIC₅₀ of HCP in CA was 8–10 µg/ml while for the isolates, CCA1, CCA3, and CCA4 MIC₅₀ ranged between 12–20 µg/ml except for a clinical isolate CCA2 which appeared to be the most sensitive with a MIC value less than 2 µg/ml (Table-1). So, the isolates, were found to be resistant to HCP when compared to the wild type strain with an exception of CCA2 which was found sensitive.

Table 1

MIC and MFC of Alexidine dihydrochloride and Hexachlorophene.
Strains	Alexidine dihydrochloride (µg/ml)		Hexachlorophene (µg/ml)
Strains	MIC₅₀	MFC	MIC₅₀	MFC
CA	0.2–0.4	0.6	8–10	25
CCA1	0.1–0.2	0.2	18–20	> 25
CCA2	0.1–0.2	> 0.6	< 2	8
CCA3	0.1–0.2	0.2	12–14	> 25
CCA4	0.2–0.4	0.4	18–20	> 25

*MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration

Minimum fungicidal concentration

All strains of C. albicans were analyzed to determine the fungicidal effect of AXD and HCP. In the given range of AXD used in this study, MFC of CA was observed at 0.6 µg/ml, whereas MFC of AXD for clinical isolates CCA1, CCA3, and CCA4 ranged between 0.2–0.4 µg/ml except for CCA2. Up to 0.6 µg/ml AXD did not show any lethality against CCA2 (Fig. 1A). MFC of HCP was found to be 25 µg/ml and 8 µg/ml in CA and CCA2, respectively. While for other clinical isolates (CCA1, CCA3 and CCA4), MFC was not observed upto a dose of 25 µg/ml (Fig. 1B).

Time kill assay

Wild type strain (CA) was exposed to three concentrations, MIC₅₀, 2 × MIC₅₀, 4 × MIC₅₀ concentrations of AXD and HCP in YPD broth. As shown in Fig. 1C, exposure to MIC₅₀ concentration of AXD caused no sensitivity in any time point when compared to respective control spots upto 5 h. But, exposure to 2 × MIC₅₀ drug concentration, rendered cells sensitive in 1 h followed by increased sensitivity in ascending time points. Exposure to 4 × MIC₅₀ concentration turned out to be lethal dose exhibited by no growth in any time point (Fig. 1C).

In Fig. 1D, it was observed that exposure to MIC₅₀ concentration of HCP caused sensitivity from 1 h exposure, whereas exposures to 2 × MIC₅₀ and 4 × MIC₅₀ concentrations of HCP were observed to be lethal even in 1 h exposure.

Growth kinetics

Up to 7 h, all strains exhibited almost alike growth pattern in YPD broth without any drug (Fig. 1E). Effect of AXD on growth kinetics of C. albicans and its clinical isolates was studied in YPD broth at a concentration of 0.3 µg/ml. AXD exposure caused sensitivity to all strains except the isolate, CCA2 (Fig. 1F). Wild type strain, CA also did not show much sensitivity to AXD in YPD broth. HCP has compromised the growth of all strains significantly in YPD broth at concentration of 9 µg/ml (Fig. 1G). Both drugs were able to reduce the growth of wild type strain along with its clinical isolates in YPD broth except CCA2.

Effect of drugs on ergosterol content

Alterations in the ergosterol contents in wild type strain (CA) and clinical isolates (CCA2 and CCA4) have shown different patterns upon treatment with AXD and HCP (Fig. 2A). AXD exposure has increased ergosterol contents by 71.2% and 19.37% in CA and CCA2, respectively. On the contrary, other isolate, CCA4 has shown reduction of 78.8% when exposed to AXD. Further, HCP treatment of CA caused an increase of 13.25% in the ergosterol content. But, in the isolates, HCP exposure has decreased ergosterol contents by 99.5% and 88.5% in CCA2 and CCA4, respectively.

AXD and HCP reduced Cell Surface Hydrophobicity

Cell surface hydrophobicity (CSH) is an important virulence trait of fungi required for adhesion to the surfaces (Danchik & Casadevall, 2021). MIC₅₀ of both drugs reduced CSH of all strains including wild type remarkably well even at low concentrations (Fig. 2B). In the presence of AXD hydrophobicity indices of CA, CCA2 and CCA4 were significantly reduced to 82.3%, 87.7 % and 99.9%, respectively. SH of HCP treated CA, CCA2 and CCA4 was also significantly reduced to 25%, 30.6% and 80%, respectively.

AXD and HCP reduced germ tube formation of C. albicans and clinical isolates

Germ tube formation assay was studied in wild type strain (CA) and two clinical isolates (CCA2 and CCA4) in the presence of average MIC₅₀ values of AXD and HCP (in reference strain). All strains have shown germ tube formation after 4 h exposure to fetal calf serum. Both the drugs inhibited germ tube formation even in presence of fetal calf serum. A representative field is shown in Fig. 2C.

AXD & HCP reduced adhesion & biofilm formation, and eradicated preformed biofilm

Biofilm is one of the most important virulence traits of pathogen which not only protect the cells from antifungals but also offers an appropriate survival condition for its survival (Ayesha et al., 2021). Both the drugs (AXD and HCP) were reported to reduce in vitro adhesion and biofilm formation in all strains in dose-dependent manner. Likewise, both drugs exhibited pronounced biofilm eradication properties.

Anti-adhesion activity

AXD has been reported to reduce in vitro adhesion of wild type strain C. albicans (CA) by 50% at a concentration of 1–2 µg/ml (AIC₅₀), whereas clinical isolates showed similar extent of reduction in adhesion at a concentration < 1 µg/ml. On the other hand, HCP exhibited AIC₅₀ value ranging 6–8 µg/ml for CA, followed by 2–4 µg/ml for the clinical isolates (CCA1, CCA2, CCA3 and CCA4) (Table 2). It is noteworthy that AXD at 2 µg/ml concentration was found to reduce over 85% adhesion in all strains (Fig. 3A). Similarly, HCP at a concentration of 8 µg/ml has reduced adhesion by 51.5% in CA, and over 85% in clinical isolates (Fig. 3B). Both drugs were more effective against adhesion of isolates when compared with wild type strain.

Anti-biofilm activity

AXD and HCP have shown concentration-dependent decrease in biofilm formation ability of wild type strain and clinical isolates of C. albicans (Table 2). BIC₅₀ of AXD was found to be 1–2 µg/ml for the CA, and < 1 µg/ml for clinical isolates. On the other hand, HCP exhibited BIC₅₀ ranging between 2–4 µg/ml for CA, followed by 4–6 µg/ml for the isolates. AXD at 2 µg/ml was found to reduce over 65% biofilm formation in all strains (Fig. 3C). Similarly, HCP at a concentration of 6 µg/ml has reduced biofilm activity by 54.8–71.9% for the strains used in the study (Fig. 3D). Biofilm formation by the clinical isolates appeared to be more sensitive to the drugs than that of wild type strain.

Biofilm eradication activity

In the presence of drugs, the strains have shown concentration-dependent increase in eradication of mature (preformed) biofilm evidenced by decreasing biofilm activity on increasing drug concentration, AXD (Fig. 3E) and HCP (Fig. 3F). BEC₅₀ of AXD was reported in the range of 4–6 µg/ml for CA, and 1–4 µg/ml for the clinical isolates (Table 2). On the other hand, HCP exhibited 4–8 µg/ml and 12–16 µg/ml BEC₅₀ values for CA and the isolates, respectively.

Table 2

Inhibitory concentrations (IC₅₀) of AXD and HCP against adhesion, biofilm formation- and mature biofilm of *C. albicans* and clinical isolates.
Type of strain	Adhesion Inhibitory Concentration		Biofilm Inhibitory Concentration		Biofilm Eradication Concentration
Type of strain	AIC₅₀ (AXD) (µg/ml)	AIC₅₀ (HCP) (µg/ml)	BIC₅₀ (AXD)µg/ml)	BIC₅₀ (HCP) (µg/ml)	BEC₅₀ (AXD) (µg/ml)	BEC₅₀ (HCP) (µg/ml)
CA	1–2	6–8	1–2	2–4	4–6	4–8
CCA1	< 1	2–4	< 1	4–6	2–4	12–14
CCA2	< 1	2–4	< 1	4–6	1–2	12–14
CCA3	< 1	2–4	< 1	4–6	1–2	14–16
CCA4	< 1	2–4	< 1	4–6	1–2	12–14

Effect of the drugs on ECM components of biofilm:

Carbohydrate, total protein and eDNA

The effects of AXD and HCP on biochemical composition of extracellular matrix were studied in the wild type strain in the presence of sublethal dose of the drugs. As shown in Fig. 4A, the ECM carbohydrate was significantly reduced by 70.7% and 83% after the treatment with AXD and HCP, respectively (Fig. 4B). The protein content was also significantly reduced by 60% in presence of AXD, whereas HCP treatment did not cause any significant reduction in protein content. The eDNA content were significantly reduced to 90.6% and 86.3% in the presence of AXD and HCP, respectively (Fig. 4C).

Phospholipase and Proteinase Activity

The enzymatic activities in the ECM of C. albicans biofilm after exposure to sublethal dose of each drug were analysed. Proteinase belongs to a family of enzymes that are responsible to degrade substrate which are physiologically important, such as albumin, immunoglobulin, and skin proteins. In this study, proteinase activity was found increased significantly after the treatment of HCP in CA, whereas no significant change was recorded in case of AXD treatment (Fig. 4D). Phospholipase are the lipolytic enzymes that are responsible to degrade phospholipids of cell membrane and facilitates invasion of yeast cells. AXD and HCP have shown opposite effect on phospholipase activity. HCP has increased while AXD has decreased phospholipase activity significantly (Fig. 4E).

Mitochondrial reactive oxygen species (mROS) generation

Effect of drugs on mROS was evaluated in the presence and absence of AXD and HCP in wild type strain of C. albicans. (DCFDA) 2′,7′- dichlorodihydrofluorescein diacetate is fluorogenic dye which measures reactive oxygen species inside the cell. Significant increase of 76.7% and 49.7% in intracellular ROS accumulation was recorded in the presence of AXD and HCP treated C. albicans, respectively (Fig. 4F).

Anti-oxidant enzyme activities

Catalase and Superoxide dismutase are important antioxidant enzymes known to protect cell from ROS damage. As shown in (Fig. 4G), catalase activity was reported decreasing by 62% on the exposure of AXD, whereas HCP treatment increased catalase activity by 72% in cells of HCP treated biofilm. As shown in (Fig. 4H), SOD activity was found increased significantly by 96.3% upon exposure to AXD, while HCP treatment did not modify the SOD activity in the cells of the biofilm. Upregulation of antioxidant enzyme activity results through a feedback mechanism on increasing intracellular ROS level.

Effect of the drugs on morphology of the biofilm by FESEM analysis

Results of FESEM clearly indicated that both the drugs have affected the morphology of C. albicans. As illustrated in (Fig. 5), cell density and ECM content of C. albicans biofilm is observed to be reduced with AXD treatment. The untreated hyphae appeared healthier than HCP treated hyphae, but biofilm eradicating activity of AXD was more pronounced than HCP.

Severity of Candida infections is rising with increasing antimicrobial resistance, and limited availability of therapeutic alternatives. Drug repurposing is one of the alternative approaches based on finding new indication of the FDA approved drug. Present study is an effort of unravelling antifungal properties of two approved drugs, alexidine dihydrochloride and hexachlorophene against C. albicans wild type strain and clinical isolates. In the present investigation, the effect of AXD and HCP has been established on growth and virulence related attributes like ergosterol content, CSH, germ tube induction, adhesion and biofilm formation, composition of extracellular matrix of biofilm, mitochondrial ROS, and activities of antioxidant and host tissue invading enzymes.

Since the drug repurposing approach is followed here in pursuit of antifungal molecules where the drugs, AXD and HCP considered for study were previously known for some other indications. Chemically, AXD is an alkyl bisguanide that belong to bisguanide class of antimicrobial where two biguanide are held together by aliphatic hexamethylene. While is an HCP organochlorine compound that has function of disinfection and present in many antiseptics. Due to safety, pharmacological, and well-established biomedical application, these drugs have selected for antifungal studies. Interestingly they have shown positive sign of antifungal activity in in silico studies (unpublished data). To begin with in vitro investigations, the effectiveness of the drugs was established against planktonic and biofilm growth forms of C. albicans along with its clinical isolates (Table 1,2). It is noteworthy that in initial studies AXD has established its superiority over HCP but that was limited to planktonic and biofilm formation studies, as no considerable differences were marked in their biofilm eradication potency of the C. albicans.

To further gain insights into the impact of AXD and HCP on the overall cell topology and physiology, other biochemical investigations were done. Firstly, the change in hydrophobicity of C. albicans cells upon AXD and HCP treatment as compared to control was estimated and a significant decrease in HI was marked (Fig. 2B). CSH is an important surface feature that facilitate cell adhesion to solid surfaces which initiate the process of biofilm formation. Moreover, CSH also have role in cell aggregation and antibiotic resistance (R. Goswami et al., 2017). The reduction in HI could be due to superficial interaction of drug with Csh1p or modulation in its translation in C. albicans (Singleton et al., 2001).

Morphological transition is the prime virulence attribute involved in pathogenesis of C. albicans and is crucial to investigate the effectiveness in restricting this switching. Here, both drugs, AXD and HCP circumvented C. albicans germ tube formation and the clinical isolates (Fig. 2C). The observations are in an extended implication of reduced previous finding have mentioned about interplay between hydrophobicity and germ tube formation; hydrophobicity favours germ tube formation (Hazen’ And & Hazen2, 1988; A. G. Rodrigues et al., 1999). The reflection of this reduced hydrophobicity and germ tube inhibition also reflected in adhesion, and biofilm formation data as AXD and HCP impacted these virulence attributes. Adhesion and biofilm formation are the initial steps in ensuring biofilm development as well as maturation. The drugs, AXD and HCP have effectively remediated these traits in C. albicans including clinical isolates (Table 2).

The previous literature has enough data to provide insights into the antibacterial activity of AXD, where due to its hydrophobic nature, it gets attracted towards negative membrane and slip in between membrane lipids. This facilitates pore formation and killing of bacterial cell (Serrano et al., 2015). In cancer cells, it is known to target PTPMT1, a mitochondrial tyrosine phosphatase that facilitates apoptosis (Doughty-Shenton et al., 2010). Whereas, HCP broadly has bacteriostatic activity where it inhibits the action of respiratory D-lactate dehydrogenase (Joswick et al., 1971). Different groups have established antifungal activity of AXD but the information on its interaction with C. albicans biofilm cells remain delusive. Therefore, to give a detailed insight into AXD and HCP antibiofilm activity, the present study has examined the modulation into biochemical composition of ECM of C. albicans biofilm. The amount of carbohydrate, protein and eDNA were significantly reduced in AXD and HCP treated ECM compared to control (Fig. 4 A, B, C). Also, the activity of proteinase and phospholipase in ECM was selectively impacted in AXD and HCP ECM samples. The biomolecules and enzymes are the arsenals present in ECM of biofilm that extend protection to residing cells from invaders/drugs as well as facilitate epithelial degeneration for tissue invasion (Massey et al, 2023). Previously Ganendren et al., (2004) has also reported inhibition of phospholipase B by AXD in Cryptococcus neoformans (Ganendren et al., 2004). Conceivably, composition variation in the ECM of AXD and HCP exposed C. albicans biofilm is indication of weakening of biofilm structure and its disintegration. The inferences are further supported by the observations of FESEM micrographs that clearly depicts reduced ECM and disintegrated biofilm structure of C. albicans (Fig. 5). Additionally, the ergosterol is one of the crucial components of C. albicans cell membrane that serve as drug target and site of interaction with hydrophobic molecule to regulate their entry into the cell (M. L. Rodrigues, 2018). The ergosterol content has been considerably increased in the AXD and HCP treated C. albicans samples suggesting they both were capable in breaching the biofilm and successfully reached the biofilm cells surface.

To further extend our understanding on antifungal activity of AXD and HCP into intracellular events, the study has investigated its ability in generation of ROS in C. albicans biofilm cells. A measurable difference in the ROS level of C. albicans biofilm cells treated with AXD and HCP was recorded in comparison to control, that convinced us with the fact that AXD and HCP have more complicated role that is not just limited to physical interaction with cell wall/membrane. This study would incrementally broaden the understanding of antifungal mode of action of alexidine dihydrochloride and hexachlorophene in C. albicans with a focus on structure and biochemical function of biofilm.

In conclusion, alexidine dihydrochloride and hexachlorophene have shown noticeable anti-Candida activities against planktonic and biofilm forms of C. albicans and the clinical isolates. AXD showed better inhibition of C. albicans than HCP. Both the drugs were effective against growth and various virulence properties of the pathogenic fungi like CSH, germ tube formation, adhesion, biofilm formation and eradication of mature biofilm etc. Indicative antifungal mode of action of the drugs may include, interfering with ergosterol pathway, reducing CSH, germ tube formation and surface adherence, increasing intracellular ROS levels, and selectively affecting invading enzyme activities etc. Due to their remarkable antibiofilm activities and no toxicity at the lower concentrations in mammals and cell lines favors their extended application into therapeutic formulation development (Lokanatha et al., 1999; Mamouei et al., 2018b).

Author Contributions: A.A.- Conducting experiments, writing first draft of the manuscript and result analyses; D.K.- Conducting experiments; P.G.- Conducting experiments and manuscript proof reading; K.M.P.- Conducting SEM and ROS experiments and review of the manuscript; N.R.- Planning and manuscript review; F.A.- Review, proof reading; N.K.- Conceptualization, planning, result analysis, editing and overall supervision.

Funding: This work was financially supported by Graphic Era Deemed to be University, Dehradun, India.

Institutional Review Board Statement: Not applicable

Informed Consent Statement: Not applicable

Data Availability Statement: The data that support the findings of this study was provided by the corresponding author.

Acknowledgments: The authors thank Prof. (Dr.) Kamal Ghanshala, founder of Graphic Era Deemed to be University, Dehradun (India) for his constant support and encouragement. Finally, the authors acknowledge the services of the ROS and SEM imaging of Department of Biosciences and Bioengineering, Indian Institute of Technology Roorkee, Uttarakhand.

Ethical approval Not applicable

Consent for publication Not applicable

Consent for participate Not applicable

Competing interests Author declare no competing interests

Conflicts of Interest: Authors declare no conflicts of interest.

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Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and Methods

Strains and Chemicals

Antifungal susceptibility test

Time kill assay

Growth kinetics

Ergosterol content

Cell surface hydrophobicity

Germ tube formation assay

Adhesion assay

Biofilm formation assay

Biofilm eradication assay

Biochemical composition of extra cellular matrix (ECM) of biofilm

Carbohydrate, protein and eDNA

Tissue invading enzyme activity

ROS & Antioxidant enzyme activity

Mitochondrial reactive oxygen species

Catalase activity

Superoxide dismutase activity

FESEM analysis

Statistical analysis

Results

Antifungal Susceptibility Test

Minimum inhibitory concentration

Minimum fungicidal concentration

Time kill assay

Growth kinetics

Effect of drugs on ergosterol content

AXD and HCP reduced Cell Surface Hydrophobicity

AXD and HCP reduced germ tube formation of C. albicans and clinical isolates

AXD & HCP reduced adhesion & biofilm formation, and eradicated preformed biofilm

Anti-adhesion activity

Anti-biofilm activity

Biofilm eradication activity

Effect of the drugs on ECM components of biofilm:

Carbohydrate, total protein and eDNA

Phospholipase and Proteinase Activity

Mitochondrial reactive oxygen species (mROS) generation

Anti-oxidant enzyme activities

Effect of the drugs on morphology of the biofilm by FESEM analysis

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

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