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

Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach Ayesha Ansari, Darshan Kumar, Payal Gupta, Krishna Mohan Poluri, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4546226/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Aug, 2024 Read the published version in Archives of Microbiology → Version 1 posted 4 You are reading this latest preprint version Abstract 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 (MIC 50 ) 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction 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., 2013 a; 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., 2018 a; Nabeela et al., 2022 ; Siles et al., 2013 a; 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., 2018 b; 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., 2013 b). 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. 2013 a; 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., 2018 b; 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 . Materials and Methods 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 × 10 3 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 MIC 50 , 2× MIC 50 , 4× MIC 50 concentrations of drugs upto 5 h in YPD broth in different tubes. Average MIC 50 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 MIC 50 doses of AXD and HCP (0.3 µg/ml and 9 µg/ml, respectively for the reference strain) followed by measuring OD 600 nm 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 MIC 50 of AXD (0.3 µg /ml) and HCP (9 µ g/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 µ g/ml; HCP: 9 µ g/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 A 1 is the absorbance of initial inoculum and A 2 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 MIC 50 doses of reference strain (CA) i.e. 0.3 µ g/ml and 9 µ g/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× 10 7 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× 10 7 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 µ L of supernatant was mixed with 1 ml of sulfuric acid and 200 µ L 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 AlCl 3 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 ; 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 µ g/ml), were analyzed using FESEM as per the previous study (Gupta & Poluri, 2022 ). Briefly, small pieces of polystyrene discs (1 cm 2 ) 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 OD 600 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. Results 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 MIC 50 value of AXD in CA was found to be 0.2–0.4 µg/ml, whereas MIC 50 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 MIC 50 of HCP in CA was 8–10 µg/ml while for the isolates, CCA1, CCA3, and CCA4 MIC 50 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) MIC 50 MFC MIC 50 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 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, MIC 50 , 2 × MIC 50 , 4 × MIC 50 concentrations of AXD and HCP in YPD broth. As shown in Fig. 1C, exposure to MIC 50 concentration of AXD caused no sensitivity in any time point when compared to respective control spots upto 5 h. But, exposure to 2 × MIC 50 drug concentration, rendered cells sensitive in 1 h followed by increased sensitivity in ascending time points. Exposure to 4 × MIC 50 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 MIC 50 concentration of HCP caused sensitivity from 1 h exposure, whereas exposures to 2 × MIC 50 and 4 × MIC 50 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 ). MIC 50 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 MIC 50 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 (AIC 50 ), whereas clinical isolates showed similar extent of reduction in adhesion at a concentration < 1 µg/ml. On the other hand, HCP exhibited AIC 50 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). BIC 50 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 BIC 50 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). BEC 50 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 BEC 50 values for CA and the isolates, respectively. Table 2 Inhibitory concentrations (IC 50 ) 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 AIC 50 (AXD) ( µg /ml) AIC 50 (HCP) ( µg /ml) BIC 50 (AXD) µg /ml) BIC 50 (HCP) ( µg /ml) BEC 50 (AXD) ( µg /ml) BEC 50 (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. Discussion 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. Conclusions 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., 2018 b). Declarations 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4546226","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":312185502,"identity":"6bbe63f0-1e00-4640-ac7e-9cb0ac8836e3","order_by":0,"name":"Ayesha Ansari","email":"","orcid":"","institution":"Graphic Era University","correspondingAuthor":false,"prefix":"","firstName":"Ayesha","middleName":"","lastName":"Ansari","suffix":""},{"id":312185503,"identity":"24135e45-c9ef-4e27-b9b3-5ceab0fd4d95","order_by":1,"name":"Darshan Kumar","email":"","orcid":"","institution":"Graphic Era University","correspondingAuthor":false,"prefix":"","firstName":"Darshan","middleName":"","lastName":"Kumar","suffix":""},{"id":312185504,"identity":"55e11883-7364-4508-874e-643499746d91","order_by":2,"name":"Payal Gupta","email":"","orcid":"","institution":"Graphic Era University","correspondingAuthor":false,"prefix":"","firstName":"Payal","middleName":"","lastName":"Gupta","suffix":""},{"id":312185505,"identity":"fd16e246-e649-43db-a8b5-8b21465e7ff9","order_by":3,"name":"Krishna Mohan Poluri","email":"","orcid":"","institution":"Indian Institute of Technology Roorkee","correspondingAuthor":false,"prefix":"","firstName":"Krishna","middleName":"Mohan","lastName":"Poluri","suffix":""},{"id":312185507,"identity":"ffb7d621-a5ce-4bbf-a322-277e25fb5c86","order_by":4,"name":"Nishant Rai","email":"","orcid":"","institution":"Graphic Era University","correspondingAuthor":false,"prefix":"","firstName":"Nishant","middleName":"","lastName":"Rai","suffix":""},{"id":312185508,"identity":"ddfb4ff2-e5bd-40ae-a5f8-5a7c36f725f8","order_by":5,"name":"Faud Ameen","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Faud","middleName":"","lastName":"Ameen","suffix":""},{"id":312185511,"identity":"1dce4211-85e8-4c29-b5ac-8684a21af5f9","order_by":6,"name":"Navin Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9UlEQVRIiWNgGAWjYJCCA0AIAmwMDBUwMQNitRw4Q6QWBriWg21EOMqc/Yzh4YIzh6P5Zx9ge/xxXq2cwQHmhx8YCu7g1GLZk2NweMaNw7kzziWwGxzcdtzY4ACbsQSDwTOcWgwOpCUc5vlwOLfhDAObxMFtxxI3HGAwA4ofxq3l/DOIlvlgLXOO1W84wP4Nv5YbyQcO8wAdtgGspaEmweAADwFbbjwGajmTnrvxDGO7wZljBwxnHuYplkjA67DE5s88x6xz551hPvagoqZOnu94+8YPH/7g1oIEGBuABFAlM5BKIEYDFNSRoHYUjIJRMApGCgAABLRgrJfwpuQAAAAASUVORK5CYII=","orcid":"","institution":"Graphic Era University","correspondingAuthor":true,"prefix":"","firstName":"Navin","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2024-06-07 12:56:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4546226/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4546226/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00203-024-04103-3","type":"published","date":"2024-08-20T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58928297,"identity":"8831944c-779c-4e25-88b4-00cc0ab0dc7b","added_by":"auto","created_at":"2024-06-24 08:42:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":205986,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of alexidine dihydrochloride (AXD) and hexachlorophene (HCP) on growth and sensitivity of wild type \u003cem\u003eC. albicans\u003c/em\u003e and/or clinical isolates. \u003cstrong\u003e(A)\u003c/strong\u003e Determining minimum fungicidal concentrations of AXD, and \u003cstrong\u003e(B)\u003c/strong\u003e HCP for all strains as described in methods; \u003cstrong\u003e(C)\u003c/strong\u003e time dependent lethality of the drugs in reference strain (SC5314) up to 5 h exposures to MIC\u003csub\u003e50\u003c/sub\u003e, 2 × MIC\u003csub\u003e50\u003c/sub\u003e, 4 × MIC\u003csub\u003e50\u003c/sub\u003e concentrations of AXD, and \u003cstrong\u003e(D) \u003c/strong\u003eHCP in YPD broth; \u003cstrong\u003e(E) \u003c/strong\u003eAnalyses of growth curves of all strains in YPD broth without drug, and with MIC\u003csub\u003e50\u003c/sub\u003e (for ref. strain) concentrations of \u003cstrong\u003e(F)\u003c/strong\u003e AXD\u0026nbsp; and \u003cstrong\u003e(G)\u003c/strong\u003e HCP by measuring OD\u003csub\u003e600 nm\u003c/sub\u003e on different time intervals up to 7 h. In graphs, data represents mean values ± SDs of three independent experiments.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/286ab2afc00d8569bf58148a.png"},{"id":58928303,"identity":"f42ec197-3838-4f02-9d71-5e86a59565a1","added_by":"auto","created_at":"2024-06-24 08:42:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":157094,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of alexidine dihydrochloride and hexachlorophene on ergosterol content, cell surface hydrophobicity, and germ tube formation in C. albicans wild type and clinical isolates (CCA2 and CCA4). \u003cstrong\u003e(A)\u003c/strong\u003e Bar diagram showing percent ergosterol contents in presence and absence of AXD and HCP; \u003cstrong\u003e(B)\u003c/strong\u003e Bar diagram displaying effect on percent hydrophobicity indices upon exposure to AXD and HCP; \u003cstrong\u003e(C)\u003c/strong\u003e Microscopic images displaying impact of the drugs on germ tube induction in wild type \u003cstrong\u003e(a)\u003c/strong\u003e, CCA1 \u003cstrong\u003e(b)\u003c/strong\u003e, and CCA4 \u003cstrong\u003e(c) \u003c/strong\u003eafter negative staining at 40X. In bar diagrams, mean value ± SDs of three independent experiments has been shown. *P value \u0026lt;0.050 considered significant with respect to control.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/c19527f149d32588877c2bb1.png"},{"id":58930143,"identity":"37887abb-76c5-4430-ad20-f929be9169a0","added_by":"auto","created_at":"2024-06-24 08:58:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":231994,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of alexidine dihydrochloride and hexachlorophene exposure on sessile forms of wild type C. albicans and clinical isolates. \u003cstrong\u003e(A)\u003c/strong\u003e Impact of different concentrations of drugs on in vitro adhesion on flat bottom MTP upon exposure to AXD, and \u003cstrong\u003e(B)\u003c/strong\u003e HCP with a no drug control sample; \u003cstrong\u003e(C)\u003c/strong\u003eDifferences in biofilm formation on flat bottom MTP in presence of different concentrations of AXD, and \u003cstrong\u003e(D)\u003c/strong\u003e HCP; \u003cstrong\u003e(E)\u003c/strong\u003e Extent of eradication of preformed biofilm on flat bottom MTP by different concentrations of AXD, and \u003cstrong\u003e(F)\u003c/strong\u003e HCP. In bar diagrams, mean value ± SDs of three independent experiments has been shown. *P value \u0026lt;0.050 considered significant when compared with respective control sample.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/552262425d82ab89f058b882.png"},{"id":58928298,"identity":"f65ad0a3-439b-4be5-9235-d9d58e5f2d00","added_by":"auto","created_at":"2024-06-24 08:42:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268502,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of alexidine dihydrochloride and hexachlorophene on biochemical composition, enzyme activity and oxidation state in biofilm of wild type strain. \u003cstrong\u003e(A) \u003c/strong\u003eGraphs showing effect of drugs on carbohydrate, \u003cstrong\u003e(B)\u003c/strong\u003e protein, \u003cstrong\u003e(C)\u003c/strong\u003e eDNA, \u003cstrong\u003e(D)\u003c/strong\u003eproteinase enzyme activity, and \u003cstrong\u003e(E)\u003c/strong\u003e phospholipase activity in extracellular matrix; \u003cstrong\u003e(F) \u003c/strong\u003eGraphs depicting intracellular oxidation state of cells in biofilm upon drug treatment, mROS level, \u003cstrong\u003e(G)\u003c/strong\u003e catalase activity, and \u003cstrong\u003e(H)\u003c/strong\u003e SOD activity. Treatments: \u003cstrong\u003eUT\u003c/strong\u003e-Untreated; \u003cstrong\u003eAXD\u003c/strong\u003e-Alexidine dihydrochloride (6 µg/ml); \u003cstrong\u003eHCP\u003c/strong\u003e-Hexachlorophene (8 µg/ml). Data represent means ± SDs of three independent experiments. *P value \u0026lt;0.050 considered significant when compared with control.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/b30024c0329110abf35d5f01.png"},{"id":58929113,"identity":"14a6bab8-6d76-4897-b577-537c0862276f","added_by":"auto","created_at":"2024-06-24 08:50:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":204084,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of alexidine dihydrochloride and hexachlorophene on mature biofilm of C. albicans by FESEM analysis. Biofilms were grown on polystyrene disc for 48 h followed by drug or saline treatment for 24 h; \u003cstrong\u003e(A)\u003c/strong\u003e FESEM image of control biofilm treated with saline; \u003cstrong\u003e(B)\u003c/strong\u003e AXD (6 µg/ml) exposure;\u003cstrong\u003e (C)\u003c/strong\u003eHCP (8 µg/ml) exposure at 5000X.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/b683ac4ed255035682f2a46a.png"},{"id":63300678,"identity":"2b59fa6e-9e5b-4e84-86b9-b9507197d1a6","added_by":"auto","created_at":"2024-08-26 16:16:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2109202,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/8587a365-1b43-4fd8-b080-aa0ca027f45f.pdf"},{"id":58929115,"identity":"2915de7a-7770-417b-af11-4ac808a947c6","added_by":"auto","created_at":"2024-06-24 08:50:41","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":128470,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4546226/v1/edbcac6459f227ff2417b14c.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn late 2022, WHO has listed four \u003cem\u003eCandida spp.\u003c/em\u003e under critical fungal pathogen category in its first ever fungal pathogen priority list (Fisher \u0026amp; Denning, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Invasive fungal infections associated with \u003cem\u003eC. albicans\u003c/em\u003e, are the primary cause of the rising death rate in the immunocompromised population (Low \u0026amp; Rotstein, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rayens \u0026amp; Norris, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Over the last few decades, invasive candidiasis, caused by \u003cem\u003eC. albicans\u003c/em\u003e 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., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Pahwa et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Weiner-Lastinger et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to the long-term usage of antifungals, isolates of \u003cem\u003eC. albicans\u003c/em\u003e are developing antifungal resistance against available antimycotics (Costa-de-oliveira \u0026amp; Rodrigues, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dabas et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The limited availability of antifungals has made the scenario more complicated (Vandeputte et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). 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., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Eldesouky et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; A. S. Oliveira et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Peyclit et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Siles et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003ea; Wall et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yousfi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlexidine 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., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Yip et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Pan antifungal properties of AXD against \u003cem\u003eCandida sp.\u003c/em\u003e and, other dermatophytes along with filamentous fungi have been documented previously by various groups \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e (Mamouei et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003ea; Nabeela et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Siles et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003ea; Yousfi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). AXD is being used in mouthwash as an antiplaque agent for its application in endodontic treatment to remove biofilms (Mamouei et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb; Ruiz-Linares et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silveira et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It exhibited effectiveness against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eE. faecalis\u003c/em\u003e at very low concentrations (Kermeoglu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siles et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003eb). In recent reports, AXD exhibited growth inhibitory properties against multi drug resistant strain of \u003cem\u003eC. auris\u003c/em\u003e (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. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Siles et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003ea; Yousfi et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 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., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In recent studies, both the drugs have shown pan-antifungal properties against multidrug resistant filamentous fungi, and \u003cem\u003eTrichophyton\u003c/em\u003e (Nabeela et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yousfi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere are reports indicating antibiofilm activity of AXD and HCP, but the underlying mechanism is still need to be explored (De Cremer, Lanckacker, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mamouei et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb; Ruiz-Linares et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Silveira et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), 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 \u003cem\u003eC. albicans\u003c/em\u003e biofilm cells. Moreover, impact of drugs on ergosterol content of \u003cem\u003eC. albicans\u003c/em\u003e was investigated. To the best of our knowledge, there is hardly any report on antifungal mode of action of AXD and HCP in \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and Chemicals\u003c/h2\u003e \u003cp\u003eAll 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\u0026deg;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 \u003cem\u003eC. albicans\u003c/em\u003e (SC5314) was procured from CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Four \u003cem\u003eC. albicans\u003c/em\u003e clinical isolates of invasive candidiasis (CCA1-CCA4) were previously described elsewhere (Gupta et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal susceptibility test\u003c/h2\u003e \u003cp\u003eCLSI guidelines were followed for yeast broth microdilution assay to obtain growth inhibitory and fungicidal concentrations of AXD and HCP against \u003cem\u003eC. albicans\u003c/em\u003e and clinical isolates in 96 well MTP (Gupta \u0026amp; Poluri, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, log phase cells were diluted to adjust the cell density of 2.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/ml in RPMI-1640, were exposed to different concentrations of drugs ranging 0.1\u0026ndash;0.6 \u0026micro;g/ml for AXD, and 2\u0026ndash;25 \u0026micro;g/ml for HCP. The MTP was incubated at 37\u0026deg;C for 48 h. The fungicidal concentration of drug was determined by spotting 5 \u0026micro;l of drug treated cultures on YPD agar plates followed by incubation of the agar plates for 18 h at 37\u0026deg;C before being photographed. Dose points of no growth were considered as MFC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTime kill assay\u003c/h2\u003e \u003cp\u003eFor determining the time dependent effects of different doses of the drugs, time kill assay was performed against wild type strain (Priya et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Briefly, log phase cells were exposed to MIC\u003csub\u003e50\u003c/sub\u003e, 2\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 4\u0026times; MIC\u003csub\u003e50\u003c/sub\u003e concentrations of drugs upto 5 h in YPD broth in different tubes. Average MIC\u003csub\u003e50\u003c/sub\u003e doses were used i.e. 0.3 \u0026micro;g/ml and 9 \u0026micro;g/ml for AXD and HCP, respectively. Afterwards, 5 \u0026micro;L 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\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eGrowth kinetics\u003c/h2\u003e \u003cp\u003eThe growth kinetics of \u003cem\u003eC. albicans\u003c/em\u003e and its clinical isolates in the presence and absence of AXD and HCP by growing them in YPD broth at 37\u0026deg;C (Nordin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Briefly, log phase cells were exposed to average MIC\u003csub\u003e50\u003c/sub\u003e doses of AXD and HCP (0.3 \u0026micro;g/ml and 9 \u0026micro;g/ml, respectively for the reference strain) followed by measuring OD\u003csub\u003e600\u003c/sub\u003enm 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eErgosterol content\u003c/h2\u003e \u003cp\u003eCell membrane ergosterol content of planktonic cells of wild type and clinical isolates of \u003cem\u003eC. albicans\u003c/em\u003e were estimated after exposing them to MIC\u003csub\u003e50\u003c/sub\u003e of AXD (0.3 \u003cem\u003e\u0026micro;g\u003c/em\u003e/ml) and HCP (9 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml) for 24 h at 37\u0026deg;C (Gupta, Gupta, and Poluri 2021; De Oliveira Pereira, Mendes, and De Oliveira Lima \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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 \u003cem\u003en\u003c/em\u003e- heptane in 1:3 ratio. The organic layer of \u003cem\u003en-\u003c/em\u003e heptane was carefully pipetted out and mixed with absolute ethanol, and scanned using spectrophotometer in the range of 230\u0026ndash;300 nm. Percentage ergosterol was calculated using following formula.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\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}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;1)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell surface hydrophobicity\u003c/h2\u003e \u003cp\u003eLog phase cells of overnight grown cultures were diluted to OD\u003csub\u003e600nm\u003c/sub\u003e 0.1 and were exposed to minimum inhibitory concentration of the drugs followed by incubation for 24 h at 37\u0026deg;C (AXD: 0.3 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml; HCP: 9 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml) (Gupta et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{H}\\text{I}=\\frac{A1-A2}{A1}\\times 100\\%\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;2)\u003c/p\u003e \u003cp\u003ewhere A\u003csub\u003e1\u003c/sub\u003e is the absorbance of initial inoculum and A\u003csub\u003e2\u003c/sub\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGerm tube formation assay\u003c/h2\u003e \u003cp\u003eEffect 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., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Briefly, overnight cultures of all strains were diluted in YPD broth (OD\u003csub\u003e600nm\u003c/sub\u003e ranging 0.2\u0026ndash;0.4) with and without 10% fetal calf serum in presence and absence of drugs at MIC\u003csub\u003e50\u003c/sub\u003e doses of reference strain (CA) i.e. 0.3 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml and 9 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml for AXD and HCP, respectively. All tubes were incubated at 37\u0026deg;C with shaking for 4 h. Cultures were examined and photographed microscopically after nigrosine staining at 40X in light microscope (Olympus).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion assay\u003c/h2\u003e \u003cp\u003eEffect of the drug on adhesion of \u003cem\u003eC. albicans\u003c/em\u003e and its clinical isolates was studied in 96-well flat bottom MTP (Raut et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Briefly, phosphate buffered saline (PBS) of pH 7.0 was used to dilute log phase cultures of the stains to a concentration of 1\u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e-1\u003c/sup\u003e. Fifty microliters of cell suspension were added to each well following the addition of 50 \u0026micro;l of drug dilutions in PBS (AXD: 1\u0026ndash;10 \u0026micro;g/ml \u0026amp; HCP 1\u0026ndash;12 \u0026micro;g/ml). Control well was kept without drug. The plate was incubated at 37\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm formation assay\u003c/h2\u003e \u003cp\u003eEffects of the drugs on biofilm formation were analyzed on flat bottom MTP followed by XTT reduction assay (Gupta et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Briefly, log phase cells were diluted to density of 1\u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells ml\u003csup\u003e-1\u003c/sup\u003e in PBS, and 100 \u0026micro;l of cell suspension was added to each well followed by incubation for 90 min at 37\u0026deg;C. After washing with PBS, 200 \u0026micro;l drug dilutions in RPMI were added to different wells (AXD: 1\u0026ndash;8 \u0026micro;g/ml \u0026amp; HCP: 1\u0026ndash;14 \u0026micro;g/ml) with a control sample without any drug. The MTP was incubated at 37\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBiofilm eradication assay\u003c/h2\u003e \u003cp\u003eBiofilm eradication effects of AXD and HCP were studied by seeding and maturing biofilm for 24 h as mentioned above (Gupta et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). After the incubation, the biofilm was washed twice with sterile PBS followed by addition of drug dilutions in RPMI (AXD: 1\u0026ndash;8 \u0026micro;g/ml \u0026amp; HCP: 1\u0026ndash;16 \u0026micro;g/ml). Control sample was kept without any drug and plate was again incubated for another 24 h at 37\u0026deg;C followed by quantification using XTT reduction assay by measuring OD at 492nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical composition of extra cellular matrix (ECM) of biofilm\u003c/h2\u003e \u003cp\u003eECM is a gelling material of cells and other components of biofilm. The biochemical composition of ECM of the wild type \u003cem\u003eC. albicans\u003c/em\u003e was determined in presence of sub-lethal concentrations of the drugs (Gupta et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Briefly, \u003cem\u003eC. albicans\u003c/em\u003e biofilm were developed in the presence AXD (6 \u0026micro;g/ml) and HCP (8 \u0026micro;g/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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCarbohydrate, protein and eDNA\u003c/h2\u003e \u003cp\u003eTotal \u003cb\u003ecarbohydrate\u003c/b\u003e was estimated by phenol-sulfuric acid method using glucose as a standard. Briefly, 100 \u003cem\u003e\u0026micro;\u003c/em\u003eL of supernatant was mixed with 1 ml of sulfuric acid and 200 \u003cem\u003e\u0026micro;\u003c/em\u003eL of phenol (5% w/v) in glass tubes followed by incubating at 30\u0026deg;C for 30 min. The tubes were then cooled down and the absorbance was taken at 485 nm.\u003c/p\u003e \u003cp\u003eTotal \u003cb\u003eprotein\u003c/b\u003e 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.\u003c/p\u003e \u003cp\u003eTotal \u003cb\u003eeDNA\u003c/b\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTissue invading enzyme activity\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePhospholipase activity\u003c/b\u003e 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 AlCl\u003csub\u003e3\u003c/sub\u003e and 0.124% bromothymol blue followed by filtration and storage of solution at 4\u0026deg;C. For the determination of phospholipase activities, 100 \u0026micro;L of sample was mixed with 900 \u0026micro;L 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.\u003c/p\u003e \u003cp\u003eFor the \u003cb\u003eproteinase activity\u003c/b\u003e determination, 1% w/v azocasein substrate was mixed with the supernatant and the sample solution was incubated for 1 h at 37\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eROS \u0026amp; Antioxidant enzyme activity\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial reactive oxygen species\u003c/h2\u003e \u003cp\u003eEffect of sub-lethal concentrations of AXD (6 \u0026micro;g/ml) and HCP (8 \u0026micro;g/ml) on reactive oxygen species (ROS) level was studied in cells of \u003cem\u003eC. albicans\u003c/em\u003e biofilm. For ROS estimation, DCFDA (10 \u0026micro;M) was added into black well multi well plate (MTP) and incubated at 37\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCatalase activity\u003c/h2\u003e \u003cp\u003eFor the determination of \u003cb\u003ecatalase activity\u003c/b\u003e, cell free extract was prepared as mentioned in (Jethwaney et al., n.d.). Glass beads were used for lysing the drug treated \u003cem\u003eC. albicans\u003c/em\u003e 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 \u0026micro;L of 50 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 567 \u0026micro;L of PBS (pH 7.0) was mixed with 100 \u0026micro;L of supernatant flowed by absorbance measurement at 240 nm. One unit catalase activity is the amount of enzyme that decompose 1 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e per min of reaction at 37\u0026deg;C (Gupta et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Following formula was employed for calculating the catalase activity (U/mg)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\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}\\)\u003c/span\u003e \u003c/span\u003e (Eq.\u0026nbsp;3)\u003c/p\u003e \u003cp\u003eA0-A2 is the difference in absorbance; Vt is total volume of sample; 240 is the molar coefficient of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e; d is optical pathlength of cuvette; Ct is the protein concentration in sample\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSuperoxide dismutase activity\u003c/h2\u003e \u003cp\u003e \u003cb\u003eSOD\u003c/b\u003e 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., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The intracellular (cell free) extract was mixed with 2 mM EDTA, 55 \u0026micro;M nitroblue tetrazolium, 9.9 mM L-methionine, 1 \u0026micro;M riboflavin, 0.025% Triton \u0026times;100 and 50 mM PBS to final volume of 200 \u0026micro;l. 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%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFESEM analysis\u003c/h2\u003e \u003cp\u003eMorphological changes in wild type \u003cem\u003eC. albicans\u003c/em\u003e biofilm cells upon the exposure to sublethal concentrations of AXD (6 \u003cem\u003e\u0026micro;g\u003c/em\u003e/ml) and HCP (8 \u003cem\u003e\u0026micro;\u003c/em\u003eg/ml), were analyzed using FESEM as per the previous study (Gupta \u0026amp; Poluri, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, small pieces of polystyrene discs (1 cm\u003csup\u003e2\u003c/sup\u003e) 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\u0026deg;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eMean of three values of OD\u003csub\u003e600\u003c/sub\u003e 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\u0026rsquo;s t-test with \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eAntifungal Susceptibility Test\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003eMinimum inhibitory concentration\u003c/h2\u003e \u003cp\u003eAntifungal 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 MIC\u003csub\u003e50\u003c/sub\u003e value of AXD in CA was found to be 0.2\u0026ndash;0.4 \u0026micro;g/ml, whereas MIC\u003csub\u003e50\u003c/sub\u003e values of AXD in clinical isolates were surprisingly low, ranging from 0.1\u0026ndash;0.4 \u0026micro;g/ml. The clinical isolates, CCA1, CCA2, and CCA3 were found sensitive to AXD when compared to wild type strain.\u003c/p\u003e \u003cp\u003eThe MIC\u003csub\u003e50\u003c/sub\u003e of HCP in CA was 8\u0026ndash;10 \u0026micro;g/ml while for the isolates, CCA1, CCA3, and CCA4 MIC\u003csub\u003e50\u003c/sub\u003e ranged between 12\u0026ndash;20 \u0026micro;g/ml except for a clinical isolate CCA2 which appeared to be the most sensitive with a MIC value less than 2 \u0026micro;g/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.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC and MFC of Alexidine dihydrochloride and Hexachlorophene.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrains\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAlexidine dihydrochloride (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHexachlorophene (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMFC\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eMIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eMFC\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026ndash;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026ndash;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026ndash;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.1\u0026ndash;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.2\u0026ndash;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18\u0026ndash;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eMinimum fungicidal concentration\u003c/h2\u003e \u003cp\u003eAll strains of \u003cem\u003eC. albicans\u003c/em\u003e 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 \u0026micro;g/ml, whereas MFC of AXD for clinical isolates CCA1, CCA3, and CCA4 ranged between 0.2\u0026ndash;0.4 \u0026micro;g/ml except for CCA2. Up to 0.6 \u0026micro;g/ml AXD did not show any lethality against CCA2 (Fig.\u0026nbsp;1A). MFC of HCP was found to be 25 \u0026micro;g/ml and 8 \u0026micro;g/ml in CA and CCA2, respectively. While for other clinical isolates (CCA1, CCA3 and CCA4), MFC was not observed upto a dose of 25 \u0026micro;g/ml (Fig.\u0026nbsp;1B).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eTime kill assay\u003c/h2\u003e \u003cp\u003eWild type strain (CA) was exposed to three concentrations, MIC\u003csub\u003e50\u003c/sub\u003e, 2 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e, 4 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e concentrations of AXD and HCP in YPD broth. As shown in Fig.\u0026nbsp;1C, exposure to MIC\u003csub\u003e50\u003c/sub\u003e concentration of AXD caused no sensitivity in any time point when compared to respective control spots upto 5 h. But, exposure to 2 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e drug concentration, rendered cells sensitive in 1 h followed by increased sensitivity in ascending time points. Exposure to 4 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e concentration turned out to be lethal dose exhibited by no growth in any time point (Fig.\u0026nbsp;1C).\u003c/p\u003e \u003cp\u003eIn Fig.\u0026nbsp;1D, it was observed that exposure to MIC\u003csub\u003e50\u003c/sub\u003e concentration of HCP caused sensitivity from 1 h exposure, whereas exposures to 2 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e and 4 \u0026times; MIC\u003csub\u003e50\u003c/sub\u003e concentrations of HCP were observed to be lethal even in 1 h exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eGrowth kinetics\u003c/h2\u003e \u003cp\u003eUp to 7 h, all strains exhibited almost alike growth pattern in YPD broth without any drug (Fig.\u0026nbsp;1E). Effect of AXD on growth kinetics of \u003cem\u003eC. albicans\u003c/em\u003e and its clinical isolates was studied in YPD broth at a concentration of 0.3 \u0026micro;g/ml. AXD exposure caused sensitivity to all strains except the isolate, CCA2 (Fig.\u0026nbsp;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 \u0026micro;g/ml (Fig.\u0026nbsp;1G). Both drugs were able to reduce the growth of wild type strain along with its clinical isolates in YPD broth except CCA2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eEffect of drugs on ergosterol content\u003c/h2\u003e \u003cp\u003eAlterations 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.\u0026nbsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eAXD and HCP reduced Cell Surface Hydrophobicity\u003c/h2\u003e \u003cp\u003eCell surface hydrophobicity (CSH) is an important virulence trait of fungi required for adhesion to the surfaces (Danchik \u0026amp; Casadevall, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MIC\u003csub\u003e50\u003c/sub\u003e of both drugs reduced CSH of all strains including wild type remarkably well even at low concentrations (Fig.\u0026nbsp;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.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAXD and HCP reduced germ tube formation of C. albicans and clinical isolates\u003c/h3\u003e\n\u003cp\u003eGerm tube formation assay was studied in wild type strain (CA) and two clinical isolates (CCA2 and CCA4) in the presence of average MIC\u003csub\u003e50\u003c/sub\u003e 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.\u0026nbsp;2C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eAXD \u0026amp; HCP reduced adhesion \u0026amp; biofilm formation, and eradicated preformed biofilm\u003c/h2\u003e \u003cp\u003eBiofilm 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 \u003cem\u003ein vitro\u003c/em\u003e adhesion and biofilm formation in all strains in dose-dependent manner. Likewise, both drugs exhibited pronounced biofilm eradication properties.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eAnti-adhesion activity\u003c/h2\u003e \u003cp\u003eAXD has been reported to reduce \u003cem\u003ein vitro\u003c/em\u003e adhesion of wild type strain \u003cem\u003eC. albicans\u003c/em\u003e (CA) by 50% at a concentration of 1\u0026ndash;2 \u0026micro;g/ml (AIC\u003csub\u003e50\u003c/sub\u003e), whereas clinical isolates showed similar extent of reduction in adhesion at a concentration\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026micro;g/ml. On the other hand, HCP exhibited AIC\u003csub\u003e50\u003c/sub\u003e value ranging 6\u0026ndash;8 \u0026micro;g/ml for CA, followed by 2\u0026ndash;4 \u0026micro;g/ml for the clinical isolates (CCA1, CCA2, CCA3 and CCA4) (Table\u0026nbsp;2). It is noteworthy that AXD at 2 \u0026micro;g/ml concentration was found to reduce over 85% adhesion in all strains (Fig.\u0026nbsp;3A). Similarly, HCP at a concentration of 8 \u0026micro;g/ml has reduced adhesion by 51.5% in CA, and over 85% in clinical isolates (Fig.\u0026nbsp;3B). Both drugs were more effective against adhesion of isolates when compared with wild type strain.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eAnti-biofilm activity\u003c/h2\u003e \u003cp\u003eAXD and HCP have shown concentration-dependent decrease in biofilm formation ability of wild type strain and clinical isolates of \u003cem\u003eC. albicans\u003c/em\u003e (Table\u0026nbsp;2). BIC\u003csub\u003e50\u003c/sub\u003e of AXD was found to be 1\u0026ndash;2 \u0026micro;g/ml for the CA, and \u0026lt;\u0026thinsp;1 \u0026micro;g/ml for clinical isolates. On the other hand, HCP exhibited BIC\u003csub\u003e50\u003c/sub\u003e ranging between 2\u0026ndash;4 \u0026micro;g/ml for CA, followed by 4\u0026ndash;6 \u0026micro;g/ml for the isolates. AXD at 2 \u0026micro;g/ml was found to reduce over 65% biofilm formation in all strains (Fig.\u0026nbsp;3C). Similarly, HCP at a concentration of 6 \u0026micro;g/ml has reduced biofilm activity by 54.8\u0026ndash;71.9% for the strains used in the study (Fig.\u0026nbsp;3D). Biofilm formation by the clinical isolates appeared to be more sensitive to the drugs than that of wild type strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eBiofilm eradication activity\u003c/h2\u003e \u003cp\u003eIn 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.\u0026nbsp;3E) and HCP (Fig.\u0026nbsp;3F). BEC\u003csub\u003e50\u003c/sub\u003e of AXD was reported in the range of 4\u0026ndash;6 \u0026micro;g/ml for CA, and 1\u0026ndash;4 \u0026micro;g/ml for the clinical isolates (Table\u0026nbsp;2). On the other hand, HCP exhibited 4\u0026ndash;8 \u0026micro;g/ml and 12\u0026ndash;16 \u0026micro;g/ml BEC\u003csub\u003e50\u003c/sub\u003e values for CA and the isolates, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) of AXD and HCP against adhesion, biofilm formation- and mature biofilm of \u003cem\u003eC. albicans\u003c/em\u003e and clinical isolates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eType of strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eAdhesion Inhibitory Concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eBiofilm Inhibitory Concentration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eBiofilm Eradication Concentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(AXD)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eAIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(HCP)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eBIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(AXD)\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eBIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(HCP)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eBEC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(AXD)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eBEC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(HCP)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(\u003c/b\u003e\u003cb\u003e\u0026micro;g\u003c/b\u003e\u003cb\u003e/ml)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u0026ndash;8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u0026ndash;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u0026ndash;16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u0026ndash;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u0026ndash;6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u0026ndash;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u0026ndash;14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eEffect of the drugs on ECM components of biofilm:\u003c/h3\u003e\n\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003eCarbohydrate, total protein and eDNA\u003c/h2\u003e \u003cp\u003eThe 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.\u0026nbsp;4A, the ECM carbohydrate was significantly reduced by 70.7% and 83% after the treatment with AXD and HCP, respectively (Fig.\u0026nbsp;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.\u0026nbsp;4C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003ePhospholipase and Proteinase Activity\u003c/h2\u003e \u003cp\u003eThe enzymatic activities in the ECM of \u003cem\u003eC. albicans\u003c/em\u003e 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.\u0026nbsp;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.\u0026nbsp;4E).\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eMitochondrial reactive oxygen species (mROS) generation\u003c/h2\u003e \u003cp\u003eEffect of drugs on mROS was evaluated in the presence and absence of AXD and HCP in wild type strain of \u003cem\u003eC. albicans\u003c/em\u003e. (DCFDA) 2\u0026prime;,7\u0026prime;- 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 \u003cem\u003eC. albicans\u003c/em\u003e, respectively (Fig.\u0026nbsp;4F).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eAnti-oxidant enzyme activities\u003c/h2\u003e \u003cp\u003eCatalase and Superoxide dismutase are important antioxidant enzymes known to protect cell from ROS damage. As shown in (Fig.\u0026nbsp;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.\u0026nbsp;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eEffect of the drugs on morphology of the biofilm by FESEM analysis\u003c/h2\u003e \u003cp\u003eResults of FESEM clearly indicated that both the drugs have affected the morphology of \u003cem\u003eC. albicans.\u003c/em\u003e As illustrated in (Fig.\u0026nbsp;5), cell density and ECM content of \u003cem\u003eC. albicans\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeverity of \u003cem\u003eCandida\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eSince 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 \u003cem\u003eC. albicans\u003c/em\u003e along with its clinical isolates (Table\u0026nbsp;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 \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003eC. albicans\u003c/em\u003e cells upon AXD and HCP treatment as compared to control was estimated and a significant decrease in HI was marked (Fig.\u0026nbsp;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., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The reduction in HI could be due to superficial interaction of drug with Csh1p or modulation in its translation in \u003cem\u003eC. albicans\u003c/em\u003e (Singleton et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMorphological transition is the prime virulence attribute involved in pathogenesis of \u003cem\u003eC. albicans\u003c/em\u003e and is crucial to investigate the effectiveness in restricting this switching. Here, both drugs, AXD and HCP circumvented \u003cem\u003eC. albicans\u003c/em\u003e germ tube formation and the clinical isolates (Fig.\u0026nbsp;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\u0026rsquo; And \u0026amp; Hazen2, 1988; A. G. Rodrigues et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). 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 \u003cem\u003eC. albicans\u003c/em\u003e including clinical isolates (Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe 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., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In cancer cells, it is known to target PTPMT1, a mitochondrial tyrosine phosphatase that facilitates apoptosis (Doughty-Shenton et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Whereas, HCP broadly has bacteriostatic activity where it inhibits the action of respiratory D-lactate dehydrogenase (Joswick et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1971\u003c/span\u003e). Different groups have established antifungal activity of AXD but the information on its interaction with \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e biofilm. The amount of carbohydrate, protein and eDNA were significantly reduced in AXD and HCP treated ECM compared to control (Fig.\u0026nbsp;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., (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) has also reported inhibition of phospholipase B by AXD in \u003cem\u003eCryptococcus neoformans\u003c/em\u003e (Ganendren et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Conceivably, composition variation in the ECM of AXD and HCP exposed \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e (Fig.\u0026nbsp;5). Additionally, the ergosterol is one of the crucial components of \u003cem\u003eC. albicans\u003c/em\u003e cell membrane that serve as drug target and site of interaction with hydrophobic molecule to regulate their entry into the cell (M. L. Rodrigues, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The ergosterol content has been considerably increased in the AXD and HCP treated \u003cem\u003eC. albicans\u003c/em\u003e samples suggesting they both were capable in breaching the biofilm and successfully reached the biofilm cells surface.\u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003eC. albicans\u003c/em\u003e biofilm cells. A measurable difference in the ROS level of \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e with a focus on structure and biochemical function of biofilm.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, alexidine dihydrochloride and hexachlorophene have shown noticeable anti-\u003cem\u003eCandida\u003c/em\u003e activities against planktonic and biofilm forms of \u003cem\u003eC. albicans\u003c/em\u003e and the clinical isolates. AXD showed better inhibition of \u003cem\u003eC. albicans\u003c/em\u003e 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., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Mamouei et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was financially supported by Graphic Era Deemed to be University, Dehradun, India.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data that support the findings of this study was provided by the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for participate\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eAuthor declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e Authors declare no conflicts of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFisher MC, Denning DW (2023) The WHO fungal priority pathogens list as a game-changer. 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John Wiley \u0026amp; Sons, Inc\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alexidine dihydrochloride, Biofilm, C. albicans, ECM, Hexachlorophene, ROS","lastPublishedDoi":"10.21203/rs.3.rs-4546226/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4546226/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eCandida albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e and clinical isolates. The minimum inhibitory concentrations (MIC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003e \u003c/sub\u003eof AXD and HCP against \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e 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 \u003cem\u003eC. albicans\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into antifungal potential of Alexidine dihydrochloride and Hexachlorophene in Candida albicans: A Drug repurposing approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 08:42:36","doi":"10.21203/rs.3.rs-4546226/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-09T02:28:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-09T02:27:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-08T05:52:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2024-06-07T12:55:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aomi","sideBox":"Learn more about [Archives of Microbiology](https://www.springer.com/journal/203)","snPcode":"203","submissionUrl":"https://submission.nature.com/new-submission/203/3","title":"Archives of Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3b063a3e-b7d8-4c63-a57a-5150128b0e06","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:09:25+00:00","versionOfRecord":{"articleIdentity":"rs-4546226","link":"https://doi.org/10.1007/s00203-024-04103-3","journal":{"identity":"archives-of-microbiology","isVorOnly":false,"title":"Archives of Microbiology"},"publishedOn":"2024-08-20 15:57:37","publishedOnDateReadable":"August 20th, 2024"},"versionCreatedAt":"2024-06-24 08:42:36","video":"","vorDoi":"10.1007/s00203-024-04103-3","vorDoiUrl":"https://doi.org/10.1007/s00203-024-04103-3","workflowStages":[]},"version":"v1","identity":"rs-4546226","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4546226","identity":"rs-4546226","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}