Diutina (Candida) rugosa complex: The biofilm ultrastructure, extracellular matrix, cell wall component and antifungal susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole

The genus Candida is the most common etiological factor of opportunistic fungal infections in humans. The virulence of Candida species is due to a wide repertoire of factors, specifically, the ability to form biofilms. Medical devices such as intravenous catheters, prosthetic heart valves and surgical interventions provide pathogenic microorganisms with a surface to adhere to form biofilm. The objectives of this study were to investigate the biofilm ultrastructure of Diutina (Candida) rugosa (D. rugosa) at different developmental phases using Confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM), quantify β-glucan, total carbohydrate and total protein in the extracellular matrix (ECM) using enzymatic β-glucan kit, phenol-sulfuric acid method and Bradford’s method, respectively, and to identify Sessile Minimum Inhibition Concentrations (SMICs) of amphotericin B, caspofungin, fluconazole, and voriconazole using serial doubling dilution. From the SEM micrographs, D. rugosa biofilms were composed of adherent yeast cells and blastospores with hyphal elements. The ultrastructure of the yeast cells was collapsed and disfigured upon exposure to amphotericin B, fluconazole and voriconazole and the biofilms presented with punctured yeast morphology upon exposure to caspofungin at their respective SMICs. The matrix thickness of embedded yeast cells from CLSM micrographs was 3.9µm at 48h. However, there was reduction in the thickness of the biofilms upon antifungal exposure. The antifungal exposed biofilms exhibit bright, diffuse, green-yellow fluorescence that were not seen in the control. D. rugosa biofilm matrices revealed 172.57µg/mL of carbohydrate, and 27.11µg/mL of protein content. The β-glucan yield in D. rugosa complex planktonic cells were in the range of 2.5 to 4.38%, on the contrary, β-glucan was not detected in the ECM. The SMICs of Diutina biofilm for amphotericin B is 1024μg/mL, caspofungin is 512 μg/mL, whereas fluconazole and voriconazole is 2048 μg/mL, respectively.


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The DNA of the isolates were extracted using GeneAll DNA extraction kit (GeneAll 142 Biotechnology, Korea). The primers used were ITS1 and ITS4, which amplify the internal 143 transcribed spacer regions (ITS) of the ribosomal DNA. The amplicons were sequenced in both 144 directions using primer pair ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-145 TCCTCCGCTTATTGATATGC-3´). The sequences of each isolates were compared to the 146 NCBI GenBank database using BLAST tool.

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Quantification of -glucan 148 The procedures for -glucan measurement in the cell wall planktonic cells and ECM of biofilm 149 were performed using enzymatic yeast β-glucan kit purchased from Megazyme International 150 (Ireland), following the manufacturer's guidelines.

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Antifungal susceptibility testing of biofilm 152 The antifungal susceptibility test for sessile (biofilm) cells was performed using broth serial 153 doubling dilution method according to previously described protocol [24]. The biofilm was 154 tested against four different antifungal agents which were amphotericin B (Nacalai Tesque, 155 Inc., Japan), caspofungin (Sigma Aldrich, USA), fluconazole (Liofilchem, Italy), and 156 voriconazole (Liofilchem, Italy). All antifungal agents were prepared using dimethyl sulfoxide biofilm was grown over 48 hours according to previously described protocol [25], using 96-159 well microtiter plates. It was washed thrice with PBS buffer before the addition of the   171 For the analysis of matrix composition of biofilm, total carbohydrate and total protein were 172 performed. The biofilm growth condition and extracellular matrix (ECM) extraction were 173 adapted from previously described protocols [25]. Total carbohydrate was estimated according 174 previously described protocols with a slight modification, using glucose as a standard [27]. 175 Total protein was estimated according to previously described protocol using bovine serum 176 albumin (BSA) [28]. C. albicans ATCC 90028 was used as a positive control for this study. 177 The assay was carried out in triplicate on three different occasions. The ECM supernatant was 178 stored at -20°C until used for FT-IR spectral measurement. 180 The spectra were recorded with FTIR-Fourier Transform Infrared Spectroscopy (Spectrum 100 181 FT-IR, Perkin Elmer, UK) ranging between wave numbers 4000 and 400 cm −1 . Spectra from 182 glucose and BSA were used as reference to compare the spectra from samples. The sample preparation for CLSM imaging was performed according to previously described 186 protocol with slight modifications [29]. The biofilm was grown using 2-well cell culture 187 chamber slide (SPL Life Sciences Co., Ltd., Korea). For the control (untreated) samples, 1mL 188 of the standardised cell suspension (1x10 6 cells/mL) were pipetted into each chamber of the 2-189 well cell culture chamber slide and incubated at 37ºC at 1.5, 6, 12, 24, 48 and 72 h, respectively. 190 For the treated samples, 48 h biofilm were grown as described above using 2-well cell culture  The sample preparation for SEM imaging was performed according to previously described 201 protocol with slight modifications using 22mm sterile cell culture coverslips (ThermoFisher 202 Scientific, USA) [30]. The coverslips were carefully placed on the bottom of a 6-well plate.              and Z-stack images (lower side of each set, respectively) of preformed 48 h D. rugosa biofilm 396 exposed to antifungal agents at SMIC 50 for another 48 h and stained with Live/Dead Yeast 397 Viability kit with FUN 1. The antifungal exposed biofilm exhibits bright, diffuse, green-yellow 398 fluorescence that were not seen in the control. There was reduction in the thickness of the 399 biofilm in the antifungal exposed biofilm (Fig 10).  (Fig 12). In this study, SMIC 50 from the antifungal susceptibility test using XTT assay for the 426 antifungal agents were tested on 48 h D. rugosa biofilm. C. parapsilosis commonly grows as blastospores, and, it can sometimes produce pseudohyphae 460 [35]. In contrary, C. glabrata is not a polymorphic organism, growing as blastospores only 461 [25,35]. The ability of Candida species to switch from yeast to hyphal and pseudohyphal is 462 strongly associated with its virulence, both the former and latter structures assist in tissue 463 penetration during Candida infections and plays a vital role in organ metastasizing [36]. From 464 this study, according to the SEM micrographs, D. rugosa is a polymorphic yeast, although, 465 blastospore growth is prevalent at all time points, pseudohyphae are generated in the early 466 stages of biofilm formation (Fig 13). candidiasis and denture biofilm model [37]. Nevertheless, in vitro and in vivo C. albicans 477 biofilm models have presented with distinct morphology and characteristics [37][38][39][40][41]. In in vitro

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Biofilm are notable to be resistant to antifungal agents up to 1000-fold more than their 490 planktonic counter parts [15,16]. The particular interest of this study was to assess the

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The findings of this study showed that biofilm of D. rugosa complex isolates have 515 elevated SMICs for all the four antifungal agents tested (Table 1)   binding to β-1,3-glucans prevents the antifungal agents reaching their cellular target [40]. In 26 560 addition, it is notable that echinocandins such as caspofungin binds to β-1,3-glucan synthase 561 to block the synthesis of β-1,3-glucans and causing disruption of the fungal cell wall integrity 562 and eventually leads to cell death. β-1,3-glucans are essential components of Candida cell wall 563 and elevated β-1,3-glucans were observed in the ECM of biofilm both in vitro and in vivo [62].

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A study by Nett J et al. revealed that biofilm treated with β-1,3-glucanase are more susceptible 565 to fluconazole [40]. In this study, β-glucan were detected in the cell wall of D. rugosa complex 566 (2.5-4.2%), however, β-glucan were not detected in the ECM of D. rugosa complex biofilm.

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The ECM extraction method could be one of the reasons for the undetected β-glucan. This