Influence of Environmental and Nutritional Conditions on Nakaseomyces glabratus Biofilm Development In Vitro

Influence of Environmental and Nutritional Conditions on Nakaseomyces glabratus Biofilm Development In Vitro | 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 Influence of Environmental and Nutritional Conditions on Nakaseomyces glabratus Biofilm Development In Vitro Gabriely Pereira da Silva, Pedro Filipe Carvalho Valentini, Douglas Roberto Monteiro, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9421212/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Biofilm formation is a major virulence trait in Nakaseomyces glabratus , contributing to antifungal tolerance and persistent infections. Environmental and nutritional parameters influence fungal biofilm development, but standardized experimental frameworks for evaluating N. glabratus biofilm modulation remain limited. This study investigated how defined environmental conditions, including incubation time, agitation, and carbon source supplementation modulate biofilm development under controlled in vitro conditions. Biofilms were formed for 24 or 72 h under static (0 rpm) and dynamic (120 rpm) conditions in Sabouraud dextrose broth supplemented with glucose or galactose as carbon sources. Biofilm formation was assessed using complementary analytical methods, including crystal violet staining for total biomass, XTT reduction assay for metabolic activity, viable cell counts, and scanning electron microscopy for structural characterization. Incubation time and culture medium composition significantly affected biofilm development, whereas agitation exerted a comparatively minor effect. High carbon supplementation acted as a controlled metabolic challenge, enabling the investigation of biofilm adaptability under carbon-excess conditions. Overall, this study establishes a reproducible in vitro framework for investigating environmental and nutritional modulation of N. glabratus biofilms and provides a basis for future functional and antifungal studies. Nakaseomyces glabratus Candida glabrata Fungal biofilm Environmental modulation Carbon metabolism In vitro biofilm model Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Biofilms are structured microbial communities embedded in a self-produced extracellular matrix and attached to biotic or abiotic surfaces [ 1 ]. This mode of growth provides enhanced protection against environmental stress, host immune responses, and antimicrobial agents, playing a central role in the persistence and recurrence of infectious diseases [ 2 ]. In clinical settings, biofilm-associated microorganisms are frequently implicated in device-related infections, mucosal colonization, and chronic inflammatory conditions, representing a major challenge for effective antimicrobial therapy [ 3 ]. Nakaseomyces glabratus (formerly Candida glabrata ) is an opportunistic yeast increasingly associated with healthcare-related infections, particularly in immunocompromised and hospitalized patients [ 4 , 5 ]. Unlike Candida albicans , N. glabratus does not form true hyphae but exhibits remarkable tolerance to environmental stressors and antifungal agents, partly mediated by its ability to form biofilms [ 6 – 8 ]. These biofilms contribute to therapeutic failure and recurrent infections, emphasizing the need to better understand the environmental and nutritional factors that modulate their development [ 9 ]. Biofilm formation in N. glabratus is a dynamic and highly regulated process influenced by multiple physicochemical and nutritional parameters, including pH, oxygen availability, shear forces, and carbon source composition [ 10 – 12 ]. Carbon metabolism, in particular, plays a pivotal role in fungal adhesion, extracellular matrix production, and stress adaptation [ 13 , 14 ]. Previous studies have demonstrated that different carbohydrates may differentially affect growth, virulence, and antifungal susceptibility in Candida species [ 10 , 13 , 15 ]. However, data addressing how defined culture conditions modulate N. glabratus biofilm development remain limited. In vitro biofilm models commonly employ nutrient-rich media and controlled environmental challenges to ensure reproducible biomass formation and sufficient signal-to-noise for quantitative and microscopic analyses [ 16 – 18 ]. Within this framework, high carbon supplementation may serve as a proof-of-principle metabolic challenge to enhance phenotypic contrast and to probe biofilm adaptability under carbon-excess conditions, rather than to reproduce host physiological environments. Such standardized approaches are particularly useful for comparative studies and for developing reliable models to test antifungal agents and environmental influences on biofilm formation. Therefore, the present study aimed to investigate how defined environmental and nutritional parameters—including incubation time, agitation speed, and glucose or galactose supplementation—modulate N. glabratus biofilm development under controlled in vitro conditions. Biofilms were characterized in terms of biomass, metabolic activity, viable cell counts, and ultrastructural organization in order to establish a reproducible framework for environmental modulation studies. The null hypothesis was that these experimental parameters would not significantly influence biofilm development. Materials and Methods Strain and culture media The reference strain Nakaseomyces glabratus ATCC 90030 was used throughout the study. Stock cultures were maintained in Sabouraud dextrose broth (SDB; Difco, Le Pont de Claix, France) supplemented with 20% (v/v) glycerol at − 80°C. Prior to each experiment, the strain was subcultured on Sabouraud dextrose agar (SDA; Difco) and incubated at 37°C for 24 h. Colonies were transferred to SDB and incubated at 37°C for 24 h under orbital agitation (120 rpm). Cells were harvested by centrifugation (8000 × g, 5 min), washed twice with phosphate-buffered saline (PBS; 0.1 mol/L, pH 7.0), and resuspended in fresh SDB. Cell density was adjusted to 1 × 10⁷ cells/mL using a Neubauer hemocytometer. Three culture media were evaluated: SDB alone, SDB supplemented with 500 mmol/L glucose (GLU; Sigma-Aldrich, St. Louis, MO, USA), or SDB supplemented with 500 mmol/L galactose (GAL; Sigma-Aldrich). This concentration was selected to impose a controlled carbon-excess condition and to enhance phenotypic discrimination across complementary assays, rather than to mimic physiological carbohydrate levels. Biofilm formation Biofilms were formed in sterile, flat-bottom 96-well polystyrene microplates (Costar, Corning Inc., Tewksbury, MA, USA). Aliquots of 200 µL of standardized cell suspension were added to each well. Plates were incubated at 37°C for 24 or 72 h under static (0 rpm) or dynamic (120 rpm) conditions in an orbital shaker. The culture medium was gently replaced every 24 h to ensure nutrient availability. After each incubation period, wells were washed once with PBS to remove non-adherent cells. Total biomass quantification (crystal violet assay) Biofilm biomass was quantified using the crystal violet (CV) staining method. After washing, biofilms were fixed with 200 µL of 99% methanol (Sigma-Aldrich) for 15 min and air-dried. Subsequently, 200 µL of 0.1% (w/v) crystal violet solution was added to each well and incubated for 5 min at room temperature. Excess dye was removed by gentle rinsing with distilled water. After drying, bound crystal violet was solubilized with 200 µL of 33% (v/v) acetic acid. The resulting solution was transferred to a new microplate, and absorbance was measured at 540 nm using a microplate spectrophotometer (SpectraMax i3x, Molecular Devices, San Jose, CA, USA) [ 19 ]. Metabolic activity quantification (XTT assay) Metabolic activity was assessed using the XTT reduction assay. A freshly prepared solution containing XTT (0.5 mg/mL) and menadione (1 µmol/L) (Sigma-Aldrich) was used. After washing, 200 µL of XTT-menadione solution was added to each well, followed by incubation at 37°C for 3 h in the dark under gentle agitation (120 rpm). Subsequently, 100 µL of the supernatant was transferred to a new microplate, and absorbance was measured at 490 nm. Background absorbance from cell-free wells was subtracted prior to analysis [ 19 ]. Given the known low tetrazolium-reducing capacity of N. glabratus , XTT results were interpreted as complementary indicators of metabolic activity and not as standalone viability measures. Counting of Colony-forming units (CFU) For viable cell enumeration, biofilms were mechanically disrupted by scraping and vigorous pipetting. The resulting suspensions were serially diluted in PBS, and aliquots were plated on SDA. Plates were incubated at 37°C for 24–48 h, and colonies were counted to determine colony-forming units (CFU). Results were expressed as log₁₀ CFU/cm² [ 19 ]. Scanning electron microscopy (SEM) analysis For morphological analysis, biofilms were formed in 24-well plates under static conditions for 24 or 72 h. After washing with PBS, samples were fixed with 2.5% (v/v) glutaraldehyde in 0.1 mol/L cacodylate buffer for 2 h at room temperature. Specimens were rinsed and dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), followed by critical-point drying. Samples were mounted on aluminum stubs, sputter-coated with gold, and examined using a field-emission scanning electron microscope (FEG-VP Supra 35; Carl Zeiss, Jena, Germany). Representative micrographs were obtained for qualitative analysis. Statistical analysis All experiments were performed in triplicate in three independent assays. Data were normalized by well surface area. Normality and homoscedasticity were assessed using the Shapiro–Wilk and Levene’s tests, respectively. A three-way analysis of variance (ANOVA) was performed to evaluate the effects of incubation time, agitation speed, and culture medium, followed by Tukey’s post hoc test (α = 0.05). Statistical analyses were conducted using R software (R Foundation for Statistical Computing, Vienna, Austria). Results Effects of incubation time, agitation, and culture medium on biofilm biomass Total biomass measurements revealed a significant three-way interaction among incubation time, agitation speed, and culture medium (p < 0.05) (Fig. 1 ). At 24 h under static conditions, biofilms grown in GLU medium exhibited significantly lower biomass than those formed in GAL medium. Under dynamic conditions, SDB alone resulted in the lowest biomass at this time point. At 72 h under static conditions, biofilms formed in SDB and GLU medium showed significantly higher biomass than those grown in GAL medium. Overall, agitation did not markedly affect biomass accumulation, except for 24-h biofilms grown in GLU medium, which exhibited increased biomass under dynamic conditions. Effects of experimental conditions on metabolic activity Metabolic activity, assessed by the XTT assay, was significantly influenced by incubation time and culture medium (p < 0.05) (Fig. 2 ). GAL consistently promoted higher XTT reduction compared with the other media, particularly at 72 h. No statistically significant differences were observed between static and dynamic conditions. Despite the low tetrazolium-reducing capacity of N. glabratus , reproducible trends were observed across independent experiments, supporting the comparative interpretation of metabolic activity under the tested conditions. Influence of growth conditions on viable cell counts Quantification of viable cells demonstrated that incubation time was a major determinant of biofilm development (p < 0.05) (Fig. 3 ). In general, 72-h biofilms exhibited significantly higher CFU counts than 24-h biofilms, regardless of agitation speed. GLU resulted in significantly lower CFU values compared with SDB alone at most time points, whereas GAL did not markedly suppress cultivable cell numbers. Agitation exerted no consistent effect on CFU counts across the evaluated conditions. Ultrastructural organization of biofilms Scanning electron microscopy revealed time-dependent changes in biofilm architecture (Fig. 4 ). At 24 h, biofilms consisted mainly of loosely organized clusters of yeast cells with limited extracellular matrix. After 72 h, more compact cellular aggregates were observed, indicating progressive structural maturation. No consistent morphological differences were detected among the tested culture media. Under all conditions, biofilms were composed predominantly of yeast-form cells partially embedded in an extracellular matrix. Discussion The present study investigated how defined environmental and nutritional conditions influence the development of N. glabratus biofilms under controlled in vitro conditions. The results indicate that incubation time and culture medium composition displayed the strongest influence on biofilm maturation, whereas agitation had only a limited effect under the experimental conditions employed. By applying a carbon-excess framework as a controlled metabolic challenge, this model allowed the assessment of biofilm adaptability under defined environmental conditions rather than attempting to reproduce host physiological conditions. These results highlight the importance of standardized experimental parameters for reproducible biofilm analyses and comparative studies. Incubation time emerged as a key determinant of biofilm maturation. Although 72-h biofilms displayed higher viable cell counts, biomass values at 24 h were comparable or even higher under certain conditions. This apparent discrepancy suggests that extracellular matrix production and early adhesion events may reflect the rapid establishment of adhesion and extracellular matrix production during early stages of biofilm formation in N. glabratus , followed by progressive increases in viable cell density over time. Similar patterns have been reported for other Candida species, where early biofilm consolidation contributes to surface persistence and protection from environmental stress.[ 20 – 22 ]. The limited influence of agitation observed in this study is likely related to the low shear forces generated in microplate-based systems. While moderate fluid flow can enhance nutrient diffusion and oxygen availability, the experimental setup employed here may not have been sufficient to produce biologically relevant mechanical stress. Consequently, static and dynamic conditions yielded largely comparable biofilm phenotypes, consistent with previous in vitro studies using similar platforms [ 18 , 23 , 24 ]. Carbon source availability is widely recognized as a major determinant of fungal metabolism, stress adaptation, and virulence [ 24 – 26 ]. In the present study, glucose supplementation (GLU) was associated with reduced viable cell counts, whereas GAL promoted higher metabolic activity at later stages of biofilm development. These observations suggest differential metabolic responses to distinct carbon sources, reflecting the metabolic flexibility of N. glabratus . Similar responses have been described in other fungal species, where carbon metabolism influences cellular fitness, stress tolerance, and biofilm behavior [ 27 – 29 ]. These metabolic differences are consistent with known regulatory pathways involved in fungal adaptation to carbon availability, including modulation of oxidative stress responses, mitochondrial activity, and carbon sensing mechanisms such as the cAMP–PKA signaling pathway [ 25 – 27 , 29 ]. Although not directly assessed, these mechanisms may contribute to the observed differences in biofilm development under distinct nutritional conditions. Importantly, the high sugar concentration employed in this study was not intended to reproduce physiological conditions but to impose a controlled carbon-excess challenge. This proof-of-principle approach aimed to enhance phenotypic contrast and improve the detection of biofilm responses across complementary assays. Nutrient-rich media are frequently employed in in vitro biofilm research to ensure reproducible biomass formation and to support downstream analyses such as antifungal susceptibility testing [ 16 , 18 ]. Therefore, the present findings should be interpreted within the context of a controlled experimental framework rather than as a direct representation of host conditions. Metabolic activity was assessed using the XTT assay. However, this method has recognized limitations when applied to N. glabratus due to its relatively low tetrazolium-reducing capacity. This phenomenon has been attributed to interspecies variability in tetrazolium metabolism among Candida species [ 30 , 31 ]. For this reason, XTT data were interpreted as complementary indicators of metabolic activity rather than direct measures of viability. The combined assessment of biomass, CFU counts, and ultrastructural characteristics therefore provided a more comprehensive evaluation of biofilm development, and the consistency observed among these parameters supports the robustness of the experimental model [ 32 , 33 ]. Scanning electron microscopy revealed relatively simple biofilm architectures characterized by clusters of yeast cells embedded in a sparse extracellular matrix. These findings are consistent with previous reports indicating that N. glabratus forms less complex biofilms than filamentous Candida species [ 34 ]. The moderate structural organization observed even after prolonged incubation may reflect intrinsic species-specific traits and reinforce the importance of integrating quantitative and structural data when evaluating biofilm maturation in this organism [ 16 , 32 ]. This study has limitations that should be acknowledged. First, no functional assays, antifungal susceptibility tests, or gene expression analyses were performed, which limits the ability to establish direct mechanistic or clinical correlations. Second, the use of a single reference strain may not capture the full phenotypic variability of clinical isolates. Therefore, the findings should be interpreted within the context of a controlled in vitro model designed to assess environmental modulation rather than to infer virulence or antifungal tolerance mechanisms. In conclusion, this study establishes a reproducible in vitro model for evaluating how environmental and nutritional parameters modulate N. glabratus biofilm development. Incubation time and carbon source composition were identified as key factors influencing biofilm maturation, while agitation had a limited effect under the conditions tested. The findings should be interpreted within the context of a controlled experimental framework and provide a basis for future studies incorporating functional and mechanistic analyses. Declarations Author contributions GPS (Investigation, Visualization; Writing – original draft), PFCV (Visualization; Writing – original draft), DRM (Conceptualization, Supervision, Methodology, Validation, Formal analysis, Writing – review & editing), JPP (Resources, Validation, Writing – review & editing), ACBD (Resources, Funding acquisition), RLP (Formal analysis, Software), LSA (Conceptualization, Project administration, Data curation, Supervision, Writing – original draft; Writing – review & editing). Acknowledgments The authors thank Dr. Ana Paula Miranda Vieira for her collaboration in the microbiological assays. Funding This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Funding Code 001. No additional financial support was received. Competing Interests The authors have no competing interests to declare. Ethical Approval This study did not involve human participants, animals, or clinical samples. All experiments were conducted using commercially available microbial strains under standard laboratory conditions. Therefore, ethical approval was not required. Data Availability Statement The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. References Sharma S, Mohler J, Mahajan SD, Schwartz SA, Bruggemann L, Aalinkeel R (2023) Microbial Biofilm: A Review on Formation, Infection, Antibiotic Resistance, Control Measures, and Innovative Treatment. Microorganisms;11(6):1614. 10.3390/microorganisms11061614 Sahoo K, Meshram S (2024) Biofilm Formation in Chronic Infections: A Comprehensive Review of Pathogenesis, Clinical Implications, and Novel Therapeutic Approaches. Cureus;16(10):e70629. 10.7759/cureus.70629 Douglas LJ (2003) Candida biofilms and their role in infection. Trends Microbiol 11(1):30–36. 10.1016/s0966-842x(02)00002-1 Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC (2012) Hidden killers: human fungal infections. Sci Transl Med 4(165):165rv113. 10.1126/scitranslmed.3004404 Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20(1):133–163. 10.1128/cmr.00029-06 Kołaczkowska A, Dyląg M, Kołaczkowski M (2013) Differential expression of the Candida glabrata CgRTA1 and CgRSB1 genes in response to various stress conditions. Biochem Biophys Res Commun 432(1):169–174. 10.1016/j.bbrc.2013.01.047 Hassan Y, Chew SY, Than LTL (2021) Candida glabrata: Pathogenicity and Resistance Mechanisms for Adaptation and Survival. J Fungi 7(8):667. 10.3390/jof7080667 Ksiezopolska E, Schikora-Tamarit MA, Carlos Nunez-Rodriguez J, Gabaldon T (2024) Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata). Front Cell Infect Microbiol 14:1416509. 10.3389/fcimb.2024.1416509 Tumbarello M, Posteraro B, Trecarichi EM, Fiori B, Rossi M, Porta R et al (2007) Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. J Clin Microbiol 45(6):1843–1850. 10.1128/jcm.00131-07 Serrano-Fujarte I, Lopez-Romero E, Reyna-Lopez GE, Martinez-Gamez MA, Vega-Gonzalez A, Cuellar-Cruz M (2015) Influence of culture media on biofilm formation by Candida species and response of sessile cells to antifungals and oxidative stress. Biomed Res Int 2015:783639. 10.1155/2015/783639 Nobile CJ, Nett JE, Andes DR, Mitchell AP (2006) Function of Candida albicans adhesin Hwp1 in biofilm formation. Eukaryot Cell 5(10):1604–1610. 10.1128/ec.00194-06 Nett JE, Marchillo K, Spiegel CA, Andes DR (2010) Development and validation of an in vivo Candida albicans biofilm denture model. Infect Immun 78(9):3650–3659. 10.1128/iai.00480-10 Nikawa H, Nishimura H, Hamada T, Kumagai H, Samaranayake LP (1997) Effects of dietary sugars and, saliva and serum on Candida biofilm formation on acrylic surfaces. Mycopathologia 139(2):87–91. 10.1023/a:1006851418963 Mukherjee PK, Chand DV, Chandra J, Anderson JM, Ghannoum MA (2009) Shear stress modulates the thickness and architecture of Candida albicans biofilms in a phase-dependent manner. Mycoses 52(5):440–446. 10.1111/j.1439-0507.2008.01632.x Sampaio C, Cusicanqui Mendez DA, Buzalaf MAR, Pessan JP, Cruvinel T (2024) Influence of different growth conditions on the composition and acidogenicity of saliva-derived microcosm biofilm and their effects on enamel demineralization. Biofouling 40(10):743–753. 10.1080/08927014.2024.2410781 Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr., Mowat E, Ramage G et al (2008) A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3(9):1494–1500. 10.1038/nport.2008.141 Baillie GS, Douglas LJ (1998) Iron-limited biofilms of Candida albicans and their susceptibility to amphotericin B. Antimicrob Agents Chemother 42(8):2146–2149. 10.1128/aac.42.8.2146 Ramage G, Vande Walle K, Wickes BL, López-Ribot JL (2001) Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45(9):2475–2479. 10.1128/aac.45.9.2475-2479.2001 Arias LS, Pessan JP, de Souza Neto FN, Lima BHR, de Camargo ER, Ramage G et al (2020) Novel nanocarrier of miconazole based on chitosan-coated iron oxide nanoparticles as a nanotherapy to fight Candida biofilms. Colloids Surf B Biointerfaces 192:111080. 10.1016/j.colsurfb.2020.111080 Chandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183(18):5385–5394. 10.1128/jb.183.18.5385-5394.2001 Nobile CJ, Johnson AD (2015) Candida albicans Biofilms and Human Disease. Annu Rev Microbiol 69:71–92. 10.1146/annurev-micro-091014-104330 Seneviratne CJ, Jin L, Samaranayake LP (2008) Biofilm lifestyle of Candida: a mini review. Oral Dis 14(7):582–590. 10.1111/j.1601-0825.2007.01424.x Purevdorj B, Costerton JW, Stoodley P (2002) Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 68(9):4457–4464. 10.1128/aem.68.9.4457-4464.2002 Fernandez CE, Aspiras MB, Dodds MW, Gonzalez-Cabezas C, Rickard AH (2017) The effect of inoculum source and fluid shear force on the development of in vitro oral multispecies biofilms. J Appl Microbiol 122(3):796–808. 10.1111/jam.13376 Ene IV, Adya AK, Wehmeier S, Brand AC, MacCallum DM, Gow NA et al (2012) Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol 14(9):1319–1335. 10.1111/j.1462-5822.2012.01813.x Pellon A, Begum N, Sadeghi Nasab SD, Harzandi A, Shoaie S, Moyes DL (2022) Role of Cellular Metabolism during Candida-Host Interactions. Pathogens;11(2):184. 10.3390/pathogens11020184 Rodaki A, Bohovych IM, Enjalbert B, Young T, Odds FC, Gow NA et al (2009) Glucose promotes stress resistance in the fungal pathogen Candida albicans. Mol Biol Cell 20(22):4845–4855. 10.1091/mbc.e09-01-0002 Brown AJ, Brown GD, Netea MG, Gow NA (2014) Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels. Trends Microbiol 22(11):614–622. 10.1016/j.tim.2014.07.001 Grahl N, Shepardson KM, Chung D, Cramer RA (2012) Hypoxia and fungal pathogenesis: to air or not to air? Eukaryot Cell 11(5):560–570. 10.1128/ec.00031-12 Kuhn DM, Balkis M, Chandra J, Mukherjee PK, Ghannoum MA (2003) Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol 41(1):506–508. 10.1128/jcm.41.1.506-508.2003 Nett JE, Cain MT, Crawford K, Andes DR (2011) Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay. J Clin Microbiol 49(4):1426–1433. 10.1128/jcm.02273-10 Kean R, Delaney C, Rajendran R, Sherry L, Metcalfe R, Thomas R et al (2018) Gaining Insights from Candida Biofilm Heterogeneity: One Size Does Not Fit All. J Fungi (Basel) 4(1). 10.3390/jof4010012 Melo AS, Bizerra FC, Freymüller E, Arthington-Skaggs BA, Colombo AL (2011) Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex. Med Mycol 49(3):253–262. 10.3109/13693786.2010.530032 Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J (2011) Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol 19(5):241–247. 10.1016/j.tim.2011.02.003 Additional Declarations No competing interests reported. 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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-9421212","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":629096357,"identity":"abefefaa-c85a-424d-91ed-f22f68dfd00e","order_by":0,"name":"Gabriely Pereira da Silva","email":"","orcid":"","institution":"Anhanguera-Uniderp University","correspondingAuthor":false,"prefix":"","firstName":"Gabriely","middleName":"Pereira da","lastName":"Silva","suffix":""},{"id":629096358,"identity":"dd0da18d-ee64-4e89-b1ab-e5441d5a7152","order_by":1,"name":"Pedro Filipe Carvalho Valentini","email":"","orcid":"","institution":"Anhanguera-Uniderp University","correspondingAuthor":false,"prefix":"","firstName":"Pedro","middleName":"Filipe Carvalho","lastName":"Valentini","suffix":""},{"id":629096359,"identity":"1fb582a9-4f31-47b1-9d53-aac20496507d","order_by":2,"name":"Douglas Roberto Monteiro","email":"","orcid":"","institution":"São Paulo State University (UNESP), Araçatuba School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Douglas","middleName":"Roberto","lastName":"Monteiro","suffix":""},{"id":629096360,"identity":"2b2918f5-add7-40ed-b9cc-f473c4d64a7a","order_by":3,"name":"Juliano Pelim Pessan","email":"","orcid":"","institution":"São Paulo State University (UNESP), Araçatuba School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Juliano","middleName":"Pelim","lastName":"Pessan","suffix":""},{"id":629096361,"identity":"378a2505-84c9-44c1-b158-85cfaa963e3a","order_by":4,"name":"Alberto Carlos Botazzo Delbem","email":"","orcid":"","institution":"São Paulo State University (UNESP), Araçatuba School of Dentistry","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"Carlos Botazzo","lastName":"Delbem","suffix":""},{"id":629096362,"identity":"4e95c5be-6db7-4c0d-b271-97146d79ca8c","order_by":5,"name":"Rosana Leal do Prado","email":"","orcid":"","institution":"Universidade Federal de Minas Gerais (UFMG)","correspondingAuthor":false,"prefix":"","firstName":"Rosana","middleName":"Leal do","lastName":"Prado","suffix":""},{"id":629096363,"identity":"45b88982-5adb-405d-9881-36a59d2358de","order_by":6,"name":"Laís Salomão Arias","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBACPgbGBhDNw8DAfAAhDBHEDtgQWtgSiNUCBzwGRGphP9z8gaHmngx//5mPHz78uiPPwH746AbGHfdwa+FJbJNgOFbMI3Ejd7PkzL5nhg08aWk3GM8U43FYYhvIGzwMN3i3MfP2HGZskOAxu8HYloBbC/9DoMP+JfDInz/zDKTFnrAWicQGCaACHoMDOWzMPD8OJxKh5WGbRGJfAo/hjTRjyZkNh5PbQH5JPINbCz9/+uMPH74l2MudP/zww4c/h2372Q8fu/FxB24tYACXZmyDxhQBDcjgD/FKR8EoGAWjYOQAALmjUx48RsFIAAAAAElFTkSuQmCC","orcid":"","institution":"Anhanguera-Uniderp University","correspondingAuthor":true,"prefix":"","firstName":"Laís","middleName":"Salomão","lastName":"Arias","suffix":""}],"badges":[],"createdAt":"2026-04-15 03:38:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9421212/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9421212/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107917620,"identity":"296c844c-541f-4ebe-bfbb-1f4ccff79b3c","added_by":"auto","created_at":"2026-04-27 14:26:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":399175,"visible":true,"origin":"","legend":"\u003cp\u003eMean absorbance per cm² for total biomass quantification of \u003cem\u003eNakaseomyces glabratus \u003c/em\u003ebiofilms formed at 0 vs. 120 rpm during 24 or 72 hours in Sabouraud dextrose broth (SDB), SDB supplemented with either 500 mmol/L glucose (GLU) or 500 mmol/L galactose (GAL). Bars represent the standard deviations of the means. Different uppercase letters indicate significant differences among culture media for each biofilm formation period and agitation speed. Different lowercase letters indicate significant differences among biofilm formation periods within each culture medium and agitation speed. A significant difference between agitation speeds was observed for each culture medium and biofilm formation period (three-way ANOVA followed by Tukey’s post hoc test, p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlt text:\u003c/strong\u003e Bar graph showing total biofilm biomass of \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e formed for 24 and 72 h under static (0 rpm) and dynamic (120 rpm) conditions in SDB, GLU, and GAL media. Bars represent mean absorbance values per cm² at 540 nm with standard deviation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9421212/v1/30a3bb9322f5a0d8e557ea87.png"},{"id":107917622,"identity":"6c1f2ba4-b693-42c3-830a-346e5df162a5","added_by":"auto","created_at":"2026-04-27 14:26:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":408811,"visible":true,"origin":"","legend":"\u003cp\u003eMean absorbance per cm² for metabolic activity evaluation of \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e ATCC 90030 biofilms formed at 0 vs. 120 rpm during 24 or 72 hours in Sabouraud dextrose broth (SDB), SDB supplemented with either 500 mmol/L glucose (GLU) or 500 mmol/L galactose (GAL). Bars represent the standard deviations of the means. Different uppercase letters indicate significant differences among culture media for each biofilm formation period. Different lowercase letters indicate significant differences among biofilm formation periods within each culture medium (three-way ANOVA followed by Tukey’s post hoc test, p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlt text:\u003c/strong\u003e Bar graph displaying metabolic activity of \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e ATCC 90030 biofilms measured by XTT reduction at 490 nm after 24 and 72 h under static and dynamic conditions in SDB, GLU, and GAL media, with error bars indicating standard deviation.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9421212/v1/bd8db13686186b5a10bb42ec.png"},{"id":107917646,"identity":"c37147df-e87f-416b-b420-471ad865cb95","added_by":"auto","created_at":"2026-04-27 14:26:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":382344,"visible":true,"origin":"","legend":"\u003cp\u003eMean logarithm of colony-forming units per cm² (log\u003csub\u003e10\u003c/sub\u003e CFU/cm²) for \u003cem\u003eNakaseomyces glabratus \u003c/em\u003ebiofilms formed at 0 vs. 120 rpm during 24 or 72 hours in Sabouraud dextrose broth (SDB), SDB supplemented with either 500 mmol/L glucose (GLU) or 500 mmol/L galactose (GAL). Bars represent the standard deviations of the means. Different uppercase letters indicate significant differences among culture media for each biofilm formation period. Different lowercase letters indicate significant differences among biofilm formation periods within each culture medium (three-way ANOVA followed by Tukey’s post hoc test, p \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlt text:\u003c/strong\u003e Bar graph showing viable cell counts of \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e biofilms expressed as log₁₀ CFU/cm² after 24 and 72 h under static and dynamic conditions in SDB, GLU, and GAL media. Error bars indicate standard deviation.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9421212/v1/1872a513621700ca8050cba3.png"},{"id":107917617,"identity":"58f3a032-6531-4e9c-8397-867a137062b9","added_by":"auto","created_at":"2026-04-27 14:26:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1104636,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy images of \u003cem\u003eNakaseomyces glabratus \u003c/em\u003ebiofilms formed under static conditions for 24 or 72 hours in Sabouraud dextrose broth (SDB), SDB supplemented with 500 mmol/L glucose (GLU), and SDB supplemented with 500 mmol/L galactose (GAL). (Magnification: ×1000; scale bar = 10.0 µm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlt text:\u003c/strong\u003e Scanning electron micrographs of \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e biofilms formed for 24 and 72 h under static conditions in SDB, GLU, and GAL media, showing clusters of yeast cells and sparse extracellular matrix. Scale bar represents 10 µm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9421212/v1/1ea60023b0930f13b49e8660.png"},{"id":108006253,"identity":"91f8a490-4221-47d7-a293-eaf0667ef0f4","added_by":"auto","created_at":"2026-04-28 12:54:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3610670,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9421212/v1/0757842a-1a7d-4b48-a032-cf1cbed206e6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Environmental and Nutritional Conditions on Nakaseomyces glabratus Biofilm Development In Vitro","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiofilms are structured microbial communities embedded in a self-produced extracellular matrix and attached to biotic or abiotic surfaces [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This mode of growth provides enhanced protection against environmental stress, host immune responses, and antimicrobial agents, playing a central role in the persistence and recurrence of infectious diseases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In clinical settings, biofilm-associated microorganisms are frequently implicated in device-related infections, mucosal colonization, and chronic inflammatory conditions, representing a major challenge for effective antimicrobial therapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e (formerly \u003cem\u003eCandida glabrata\u003c/em\u003e) is an opportunistic yeast increasingly associated with healthcare-related infections, particularly in immunocompromised and hospitalized patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Unlike \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003eN. glabratus\u003c/em\u003e does not form true hyphae but exhibits remarkable tolerance to environmental stressors and antifungal agents, partly mediated by its ability to form biofilms [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These biofilms contribute to therapeutic failure and recurrent infections, emphasizing the need to better understand the environmental and nutritional factors that modulate their development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiofilm formation in \u003cem\u003eN. glabratus\u003c/em\u003e is a dynamic and highly regulated process influenced by multiple physicochemical and nutritional parameters, including pH, oxygen availability, shear forces, and carbon source composition [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Carbon metabolism, in particular, plays a pivotal role in fungal adhesion, extracellular matrix production, and stress adaptation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Previous studies have demonstrated that different carbohydrates may differentially affect growth, virulence, and antifungal susceptibility in \u003cem\u003eCandida\u003c/em\u003e species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. However, data addressing how defined culture conditions modulate \u003cem\u003eN. glabratus\u003c/em\u003e biofilm development remain limited.\u003c/p\u003e \u003cp\u003eIn vitro biofilm models commonly employ nutrient-rich media and controlled environmental challenges to ensure reproducible biomass formation and sufficient signal-to-noise for quantitative and microscopic analyses [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Within this framework, high carbon supplementation may serve as a proof-of-principle metabolic challenge to enhance phenotypic contrast and to probe biofilm adaptability under carbon-excess conditions, rather than to reproduce host physiological environments. Such standardized approaches are particularly useful for comparative studies and for developing reliable models to test antifungal agents and environmental influences on biofilm formation.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to investigate how defined environmental and nutritional parameters\u0026mdash;including incubation time, agitation speed, and glucose or galactose supplementation\u0026mdash;modulate \u003cem\u003eN. glabratus\u003c/em\u003e biofilm development under controlled in vitro conditions. Biofilms were characterized in terms of biomass, metabolic activity, viable cell counts, and ultrastructural organization in order to establish a reproducible framework for environmental modulation studies. The null hypothesis was that these experimental parameters would not significantly influence biofilm development.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrain and culture media\u003c/h2\u003e \u003cp\u003eThe reference strain \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e ATCC 90030 was used throughout the study. Stock cultures were maintained in Sabouraud dextrose broth (SDB; Difco, Le Pont de Claix, France) supplemented with 20% (v/v) glycerol at \u0026minus;\u0026thinsp;80\u0026deg;C. Prior to each experiment, the strain was subcultured on Sabouraud dextrose agar (SDA; Difco) and incubated at 37\u0026deg;C for 24 h.\u003c/p\u003e \u003cp\u003eColonies were transferred to SDB and incubated at 37\u0026deg;C for 24 h under orbital agitation (120 rpm). Cells were harvested by centrifugation (8000 \u0026times; g, 5 min), washed twice with phosphate-buffered saline (PBS; 0.1 mol/L, pH 7.0), and resuspended in fresh SDB. Cell density was adjusted to 1 \u0026times; 10⁷ cells/mL using a Neubauer hemocytometer.\u003c/p\u003e \u003cp\u003eThree culture media were evaluated: SDB alone, SDB supplemented with 500 mmol/L glucose (GLU; Sigma-Aldrich, St. Louis, MO, USA), or SDB supplemented with 500 mmol/L galactose (GAL; Sigma-Aldrich). This concentration was selected to impose a controlled carbon-excess condition and to enhance phenotypic discrimination across complementary assays, rather than to mimic physiological carbohydrate levels.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiofilm formation\u003c/h3\u003e\n\u003cp\u003eBiofilms were formed in sterile, flat-bottom 96-well polystyrene microplates (Costar, Corning Inc., Tewksbury, MA, USA). Aliquots of 200 \u0026micro;L of standardized cell suspension were added to each well. Plates were incubated at 37\u0026deg;C for 24 or 72 h under static (0 rpm) or dynamic (120 rpm) conditions in an orbital shaker. The culture medium was gently replaced every 24 h to ensure nutrient availability. After each incubation period, wells were washed once with PBS to remove non-adherent cells.\u003c/p\u003e\n\u003ch3\u003eTotal biomass quantification (crystal violet assay)\u003c/h3\u003e\n\u003cp\u003eBiofilm biomass was quantified using the crystal violet (CV) staining method. After washing, biofilms were fixed with 200 \u0026micro;L of 99% methanol (Sigma-Aldrich) for 15 min and air-dried. Subsequently, 200 \u0026micro;L of 0.1% (w/v) crystal violet solution was added to each well and incubated for 5 min at room temperature. Excess dye was removed by gentle rinsing with distilled water. After drying, bound crystal violet was solubilized with 200 \u0026micro;L of 33% (v/v) acetic acid. The resulting solution was transferred to a new microplate, and absorbance was measured at 540 nm using a microplate spectrophotometer (SpectraMax i3x, Molecular Devices, San Jose, CA, USA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMetabolic activity quantification (XTT assay)\u003c/h3\u003e\n\u003cp\u003eMetabolic activity was assessed using the XTT reduction assay. A freshly prepared solution containing XTT (0.5 mg/mL) and menadione (1 \u0026micro;mol/L) (Sigma-Aldrich) was used. After washing, 200 \u0026micro;L of XTT-menadione solution was added to each well, followed by incubation at 37\u0026deg;C for 3 h in the dark under gentle agitation (120 rpm). Subsequently, 100 \u0026micro;L of the supernatant was transferred to a new microplate, and absorbance was measured at 490 nm. Background absorbance from cell-free wells was subtracted prior to analysis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the known low tetrazolium-reducing capacity of \u003cem\u003eN. glabratus\u003c/em\u003e, XTT results were interpreted as complementary indicators of metabolic activity and not as standalone viability measures.\u003c/p\u003e\n\u003ch3\u003eCounting of Colony-forming units (CFU)\u003c/h3\u003e\n\u003cp\u003eFor viable cell enumeration, biofilms were mechanically disrupted by scraping and vigorous pipetting. The resulting suspensions were serially diluted in PBS, and aliquots were plated on SDA. Plates were incubated at 37\u0026deg;C for 24\u0026ndash;48 h, and colonies were counted to determine colony-forming units (CFU). Results were expressed as log₁₀ CFU/cm\u0026sup2; [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy (SEM) analysis\u003c/h2\u003e \u003cp\u003eFor morphological analysis, biofilms were formed in 24-well plates under static conditions for 24 or 72 h. After washing with PBS, samples were fixed with 2.5% (v/v) glutaraldehyde in 0.1 mol/L cacodylate buffer for 2 h at room temperature. Specimens were rinsed and dehydrated through a graded ethanol series (30%, 50%, 70%, 90%, and 100%), followed by critical-point drying. Samples were mounted on aluminum stubs, sputter-coated with gold, and examined using a field-emission scanning electron microscope (FEG-VP Supra 35; Carl Zeiss, Jena, Germany). Representative micrographs were obtained for qualitative analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed in triplicate in three independent assays. Data were normalized by well surface area. Normality and homoscedasticity were assessed using the Shapiro\u0026ndash;Wilk and Levene\u0026rsquo;s tests, respectively. A three-way analysis of variance (ANOVA) was performed to evaluate the effects of incubation time, agitation speed, and culture medium, followed by Tukey\u0026rsquo;s post hoc test (α\u0026thinsp;=\u0026thinsp;0.05). Statistical analyses were conducted using R software (R Foundation for Statistical Computing, Vienna, Austria).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffects of incubation time, agitation, and culture medium on biofilm biomass\u003c/h2\u003e \u003cp\u003eTotal biomass measurements revealed a significant three-way interaction among incubation time, agitation speed, and culture medium (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At 24 h under static conditions, biofilms grown in GLU medium exhibited significantly lower biomass than those formed in GAL medium. Under dynamic conditions, SDB alone resulted in the lowest biomass at this time point.\u003c/p\u003e\u003cp\u003eAt 72 h under static conditions, biofilms formed in SDB and GLU medium showed significantly higher biomass than those grown in GAL medium. Overall, agitation did not markedly affect biomass accumulation, except for 24-h biofilms grown in GLU medium, which exhibited increased biomass under dynamic conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEffects of experimental conditions on metabolic activity\u003c/h2\u003e \u003cp\u003eMetabolic activity, assessed by the XTT assay, was significantly influenced by incubation time and culture medium (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GAL consistently promoted higher XTT reduction compared with the other media, particularly at 72 h. No statistically significant differences were observed between static and dynamic conditions.\u003c/p\u003e\u003cp\u003eDespite the low tetrazolium-reducing capacity of \u003cem\u003eN. glabratus\u003c/em\u003e, reproducible trends were observed across independent experiments, supporting the comparative interpretation of metabolic activity under the tested conditions.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of growth conditions on viable cell counts\u003c/h2\u003e \u003cp\u003eQuantification of viable cells demonstrated that incubation time was a major determinant of biofilm development (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In general, 72-h biofilms exhibited significantly higher CFU counts than 24-h biofilms, regardless of agitation speed.\u003c/p\u003e \u003cp\u003eGLU resulted in significantly lower CFU values compared with SDB alone at most time points, whereas GAL did not markedly suppress cultivable cell numbers. Agitation exerted no consistent effect on CFU counts across the evaluated conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eUltrastructural organization of biofilms\u003c/h2\u003e \u003cp\u003eScanning electron microscopy revealed time-dependent changes in biofilm architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At 24 h, biofilms consisted mainly of loosely organized clusters of yeast cells with limited extracellular matrix. After 72 h, more compact cellular aggregates were observed, indicating progressive structural maturation.\u003c/p\u003e\u003cp\u003eNo consistent morphological differences were detected among the tested culture media. Under all conditions, biofilms were composed predominantly of yeast-form cells partially embedded in an extracellular matrix.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study investigated how defined environmental and nutritional conditions influence the development of \u003cem\u003eN. glabratus\u003c/em\u003e biofilms under controlled in vitro conditions. The results indicate that incubation time and culture medium composition displayed the strongest influence on biofilm maturation, whereas agitation had only a limited effect under the experimental conditions employed. By applying a carbon-excess framework as a controlled metabolic challenge, this model allowed the assessment of biofilm adaptability under defined environmental conditions rather than attempting to reproduce host physiological conditions. These results highlight the importance of standardized experimental parameters for reproducible biofilm analyses and comparative studies.\u003c/p\u003e \u003cp\u003eIncubation time emerged as a key determinant of biofilm maturation. Although 72-h biofilms displayed higher viable cell counts, biomass values at 24 h were comparable or even higher under certain conditions. This apparent discrepancy suggests that extracellular matrix production and early adhesion events may reflect the rapid establishment of adhesion and extracellular matrix production during early stages of biofilm formation in \u003cem\u003eN. glabratus\u003c/em\u003e, followed by progressive increases in viable cell density over time. Similar patterns have been reported for other \u003cem\u003eCandida\u003c/em\u003e species, where early biofilm consolidation contributes to surface persistence and protection from environmental stress.[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe limited influence of agitation observed in this study is likely related to the low shear forces generated in microplate-based systems. While moderate fluid flow can enhance nutrient diffusion and oxygen availability, the experimental setup employed here may not have been sufficient to produce biologically relevant mechanical stress. Consequently, static and dynamic conditions yielded largely comparable biofilm phenotypes, consistent with previous in vitro studies using similar platforms [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarbon source availability is widely recognized as a major determinant of fungal metabolism, stress adaptation, and virulence [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the present study, glucose supplementation (GLU) was associated with reduced viable cell counts, whereas GAL promoted higher metabolic activity at later stages of biofilm development. These observations suggest differential metabolic responses to distinct carbon sources, reflecting the metabolic flexibility of \u003cem\u003eN. glabratus\u003c/em\u003e. Similar responses have been described in other fungal species, where carbon metabolism influences cellular fitness, stress tolerance, and biofilm behavior [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. These metabolic differences are consistent with known regulatory pathways involved in fungal adaptation to carbon availability, including modulation of oxidative stress responses, mitochondrial activity, and carbon sensing mechanisms such as the cAMP\u0026ndash;PKA signaling pathway [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although not directly assessed, these mechanisms may contribute to the observed differences in biofilm development under distinct nutritional conditions.\u003c/p\u003e \u003cp\u003eImportantly, the high sugar concentration employed in this study was not intended to reproduce physiological conditions but to impose a controlled carbon-excess challenge. This proof-of-principle approach aimed to enhance phenotypic contrast and improve the detection of biofilm responses across complementary assays. Nutrient-rich media are frequently employed in in vitro biofilm research to ensure reproducible biomass formation and to support downstream analyses such as antifungal susceptibility testing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, the present findings should be interpreted within the context of a controlled experimental framework rather than as a direct representation of host conditions.\u003c/p\u003e \u003cp\u003eMetabolic activity was assessed using the XTT assay. However, this method has recognized limitations when applied to \u003cem\u003eN. glabratus\u003c/em\u003e due to its relatively low tetrazolium-reducing capacity. This phenomenon has been attributed to interspecies variability in tetrazolium metabolism among \u003cem\u003eCandida\u003c/em\u003e species [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For this reason, XTT data were interpreted as complementary indicators of metabolic activity rather than direct measures of viability. The combined assessment of biomass, CFU counts, and ultrastructural characteristics therefore provided a more comprehensive evaluation of biofilm development, and the consistency observed among these parameters supports the robustness of the experimental model [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eScanning electron microscopy revealed relatively simple biofilm architectures characterized by clusters of yeast cells embedded in a sparse extracellular matrix. These findings are consistent with previous reports indicating that \u003cem\u003eN. glabratus\u003c/em\u003e forms less complex biofilms than filamentous \u003cem\u003eCandida\u003c/em\u003e species [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The moderate structural organization observed even after prolonged incubation may reflect intrinsic species-specific traits and reinforce the importance of integrating quantitative and structural data when evaluating biofilm maturation in this organism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has limitations that should be acknowledged. First, no functional assays, antifungal susceptibility tests, or gene expression analyses were performed, which limits the ability to establish direct mechanistic or clinical correlations. Second, the use of a single reference strain may not capture the full phenotypic variability of clinical isolates. Therefore, the findings should be interpreted within the context of a controlled in vitro model designed to assess environmental modulation rather than to infer virulence or antifungal tolerance mechanisms.\u003c/p\u003e \u003cp\u003eIn conclusion, this study establishes a reproducible in vitro model for evaluating how environmental and nutritional parameters modulate \u003cem\u003eN. glabratus\u003c/em\u003e biofilm development. Incubation time and carbon source composition were identified as key factors influencing biofilm maturation, while agitation had a limited effect under the conditions tested. The findings should be interpreted within the context of a controlled experimental framework and provide a basis for future studies incorporating functional and mechanistic analyses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGPS (Investigation, Visualization; Writing \u0026ndash; original draft), PFCV (Visualization; Writing \u0026ndash; original draft), DRM (Conceptualization, Supervision, Methodology, Validation, Formal analysis, Writing \u0026ndash; review \u0026amp; editing), JPP (Resources, Validation, Writing \u0026ndash; review \u0026amp; editing), ACBD (Resources, Funding acquisition), RLP (Formal analysis, Software), LSA (Conceptualization, Project administration, Data curation, Supervision, Writing \u0026ndash; original draft; Writing \u0026ndash; review \u0026amp; editing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr. Ana Paula Miranda Vieira for her collaboration in the microbiological assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) \u0026ndash; Funding Code 001. No additional financial support was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants, animals, or clinical samples. All experiments were conducted using commercially available microbial strains under standard laboratory conditions. Therefore, ethical approval was not required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma S, Mohler J, Mahajan SD, Schwartz SA, Bruggemann L, Aalinkeel R (2023) Microbial Biofilm: A Review on Formation, Infection, Antibiotic Resistance, Control Measures, and Innovative Treatment. Microorganisms;11(6):1614. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/microorganisms11061614\u003c/span\u003e\u003cspan address=\"10.3390/microorganisms11061614\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahoo K, Meshram S (2024) Biofilm Formation in Chronic Infections: A Comprehensive Review of Pathogenesis, Clinical Implications, and Novel Therapeutic Approaches. Cureus;16(10):e70629. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.70629\u003c/span\u003e\u003cspan address=\"10.7759/cureus.70629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouglas LJ (2003) Candida biofilms and their role in infection. Trends Microbiol 11(1):30\u0026ndash;36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/s0966-842x(02)00002-1\u003c/span\u003e\u003cspan address=\"10.1016/s0966-842x(02)00002-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC (2012) Hidden killers: human fungal infections. Sci Transl Med 4(165):165rv113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/scitranslmed.3004404\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.3004404\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20(1):133\u0026ndash;163. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/cmr.00029-06\u003c/span\u003e\u003cspan address=\"10.1128/cmr.00029-06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKołaczkowska A, Dyląg M, Kołaczkowski M (2013) Differential expression of the Candida glabrata CgRTA1 and CgRSB1 genes in response to various stress conditions. Biochem Biophys Res Commun 432(1):169\u0026ndash;174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2013.01.047\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2013.01.047\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHassan Y, Chew SY, Than LTL (2021) Candida glabrata: Pathogenicity and Resistance Mechanisms for Adaptation and Survival. J Fungi 7(8):667. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/jof7080667\u003c/span\u003e\u003cspan address=\"10.3390/jof7080667\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKsiezopolska E, Schikora-Tamarit MA, Carlos Nunez-Rodriguez J, Gabaldon T (2024) Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata). Front Cell Infect Microbiol 14:1416509. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcimb.2024.1416509\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2024.1416509\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTumbarello M, Posteraro B, Trecarichi EM, Fiori B, Rossi M, Porta R et al (2007) Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. J Clin Microbiol 45(6):1843\u0026ndash;1850. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jcm.00131-07\u003c/span\u003e\u003cspan address=\"10.1128/jcm.00131-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerrano-Fujarte I, Lopez-Romero E, Reyna-Lopez GE, Martinez-Gamez MA, Vega-Gonzalez A, Cuellar-Cruz M (2015) Influence of culture media on biofilm formation by Candida species and response of sessile cells to antifungals and oxidative stress. Biomed Res Int 2015:783639. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2015/783639\u003c/span\u003e\u003cspan address=\"10.1155/2015/783639\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNobile CJ, Nett JE, Andes DR, Mitchell AP (2006) Function of Candida albicans adhesin Hwp1 in biofilm formation. Eukaryot Cell 5(10):1604\u0026ndash;1610. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/ec.00194-06\u003c/span\u003e\u003cspan address=\"10.1128/ec.00194-06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNett JE, Marchillo K, Spiegel CA, Andes DR (2010) Development and validation of an in vivo Candida albicans biofilm denture model. Infect Immun 78(9):3650\u0026ndash;3659. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/iai.00480-10\u003c/span\u003e\u003cspan address=\"10.1128/iai.00480-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikawa H, Nishimura H, Hamada T, Kumagai H, Samaranayake LP (1997) Effects of dietary sugars and, saliva and serum on Candida biofilm formation on acrylic surfaces. Mycopathologia 139(2):87\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1023/a:1006851418963\u003c/span\u003e\u003cspan address=\"10.1023/a:1006851418963\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMukherjee PK, Chand DV, Chandra J, Anderson JM, Ghannoum MA (2009) Shear stress modulates the thickness and architecture of Candida albicans biofilms in a phase-dependent manner. Mycoses 52(5):440\u0026ndash;446. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1439-0507.2008.01632.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1439-0507.2008.01632.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSampaio C, Cusicanqui Mendez DA, Buzalaf MAR, Pessan JP, Cruvinel T (2024) Influence of different growth conditions on the composition and acidogenicity of saliva-derived microcosm biofilm and their effects on enamel demineralization. Biofouling 40(10):743\u0026ndash;753. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/08927014.2024.2410781\u003c/span\u003e\u003cspan address=\"10.1080/08927014.2024.2410781\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePierce CG, Uppuluri P, Tristan AR, Wormley FL Jr., Mowat E, Ramage G et al (2008) A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3(9):1494\u0026ndash;1500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nport.2008.141\u003c/span\u003e\u003cspan address=\"10.1038/nport.2008.141\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaillie GS, Douglas LJ (1998) Iron-limited biofilms of Candida albicans and their susceptibility to amphotericin B. Antimicrob Agents Chemother 42(8):2146\u0026ndash;2149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.42.8.2146\u003c/span\u003e\u003cspan address=\"10.1128/aac.42.8.2146\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamage G, Vande Walle K, Wickes BL, L\u0026oacute;pez-Ribot JL (2001) Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45(9):2475\u0026ndash;2479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.45.9.2475-2479.2001\u003c/span\u003e\u003cspan address=\"10.1128/aac.45.9.2475-2479.2001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArias LS, Pessan JP, de Souza Neto FN, Lima BHR, de Camargo ER, Ramage G et al (2020) Novel nanocarrier of miconazole based on chitosan-coated iron oxide nanoparticles as a nanotherapy to fight Candida biofilms. Colloids Surf B Biointerfaces 192:111080. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.colsurfb.2020.111080\u003c/span\u003e\u003cspan address=\"10.1016/j.colsurfb.2020.111080\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChandra J, Kuhn DM, Mukherjee PK, Hoyer LL, McCormick T, Ghannoum MA (2001) Biofilm formation by the fungal pathogen Candida albicans: development, architecture, and drug resistance. J Bacteriol 183(18):5385\u0026ndash;5394. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jb.183.18.5385-5394.2001\u003c/span\u003e\u003cspan address=\"10.1128/jb.183.18.5385-5394.2001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNobile CJ, Johnson AD (2015) Candida albicans Biofilms and Human Disease. Annu Rev Microbiol 69:71\u0026ndash;92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev-micro-091014-104330\u003c/span\u003e\u003cspan address=\"10.1146/annurev-micro-091014-104330\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeneviratne CJ, Jin L, Samaranayake LP (2008) Biofilm lifestyle of Candida: a mini review. Oral Dis 14(7):582\u0026ndash;590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1601-0825.2007.01424.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1601-0825.2007.01424.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePurevdorj B, Costerton JW, Stoodley P (2002) Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 68(9):4457\u0026ndash;4464. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aem.68.9.4457-4464.2002\u003c/span\u003e\u003cspan address=\"10.1128/aem.68.9.4457-4464.2002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandez CE, Aspiras MB, Dodds MW, Gonzalez-Cabezas C, Rickard AH (2017) The effect of inoculum source and fluid shear force on the development of in vitro oral multispecies biofilms. J Appl Microbiol 122(3):796\u0026ndash;808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/jam.13376\u003c/span\u003e\u003cspan address=\"10.1111/jam.13376\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEne IV, Adya AK, Wehmeier S, Brand AC, MacCallum DM, Gow NA et al (2012) Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol 14(9):1319\u0026ndash;1335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1462-5822.2012.01813.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1462-5822.2012.01813.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePellon A, Begum N, Sadeghi Nasab SD, Harzandi A, Shoaie S, Moyes DL (2022) Role of Cellular Metabolism during Candida-Host Interactions. Pathogens;11(2):184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pathogens11020184\u003c/span\u003e\u003cspan address=\"10.3390/pathogens11020184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodaki A, Bohovych IM, Enjalbert B, Young T, Odds FC, Gow NA et al (2009) Glucose promotes stress resistance in the fungal pathogen Candida albicans. Mol Biol Cell 20(22):4845\u0026ndash;4855. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1091/mbc.e09-01-0002\u003c/span\u003e\u003cspan address=\"10.1091/mbc.e09-01-0002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrown AJ, Brown GD, Netea MG, Gow NA (2014) Metabolism impacts upon Candida immunogenicity and pathogenicity at multiple levels. Trends Microbiol 22(11):614\u0026ndash;622. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tim.2014.07.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2014.07.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrahl N, Shepardson KM, Chung D, Cramer RA (2012) Hypoxia and fungal pathogenesis: to air or not to air? Eukaryot Cell 11(5):560\u0026ndash;570. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/ec.00031-12\u003c/span\u003e\u003cspan address=\"10.1128/ec.00031-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuhn DM, Balkis M, Chandra J, Mukherjee PK, Ghannoum MA (2003) Uses and limitations of the XTT assay in studies of Candida growth and metabolism. J Clin Microbiol 41(1):506\u0026ndash;508. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jcm.41.1.506-508.2003\u003c/span\u003e\u003cspan address=\"10.1128/jcm.41.1.506-508.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNett JE, Cain MT, Crawford K, Andes DR (2011) Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay. J Clin Microbiol 49(4):1426\u0026ndash;1433. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jcm.02273-10\u003c/span\u003e\u003cspan address=\"10.1128/jcm.02273-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKean R, Delaney C, Rajendran R, Sherry L, Metcalfe R, Thomas R et al (2018) Gaining Insights from Candida Biofilm Heterogeneity: One Size Does Not Fit All. J Fungi (Basel) 4(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/jof4010012\u003c/span\u003e\u003cspan address=\"10.3390/jof4010012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelo AS, Bizerra FC, Freym\u0026uuml;ller E, Arthington-Skaggs BA, Colombo AL (2011) Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex. Med Mycol 49(3):253\u0026ndash;262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/13693786.2010.530032\u003c/span\u003e\u003cspan address=\"10.3109/13693786.2010.530032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J (2011) Adherence and biofilm formation of non-Candida albicans Candida species. Trends Microbiol 19(5):241\u0026ndash;247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tim.2011.02.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2011.02.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":false,"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":"Nakaseomyces glabratus, Candida glabrata, Fungal biofilm, Environmental modulation, Carbon metabolism, In vitro biofilm model","lastPublishedDoi":"10.21203/rs.3.rs-9421212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9421212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiofilm formation is a major virulence trait in \u003cem\u003eNakaseomyces glabratus\u003c/em\u003e, contributing to antifungal tolerance and persistent infections. Environmental and nutritional parameters influence fungal biofilm development, but standardized experimental frameworks for evaluating \u003cem\u003eN. glabratus\u003c/em\u003e biofilm modulation remain limited. This study investigated how defined environmental conditions, including incubation time, agitation, and carbon source supplementation modulate biofilm development under controlled in vitro conditions. Biofilms were formed for 24 or 72 h under static (0 rpm) and dynamic (120 rpm) conditions in Sabouraud dextrose broth supplemented with glucose or galactose as carbon sources. Biofilm formation was assessed using complementary analytical methods, including crystal violet staining for total biomass, XTT reduction assay for metabolic activity, viable cell counts, and scanning electron microscopy for structural characterization. Incubation time and culture medium composition significantly affected biofilm development, whereas agitation exerted a comparatively minor effect. High carbon supplementation acted as a controlled metabolic challenge, enabling the investigation of biofilm adaptability under carbon-excess conditions. Overall, this study establishes a reproducible in vitro framework for investigating environmental and nutritional modulation of \u003cem\u003eN. glabratus\u003c/em\u003e biofilms and provides a basis for future functional and antifungal studies.\u003c/p\u003e","manuscriptTitle":"Influence of Environmental and Nutritional Conditions on Nakaseomyces glabratus Biofilm Development In Vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-27 14:24:51","doi":"10.21203/rs.3.rs-9421212/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T00:36:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T17:52:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113782099844273740178018960529413974296","date":"2026-05-14T14:26:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T13:13:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T14:58:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145068870211831850096313627843631865262","date":"2026-04-22T13:38:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193669434557372131703330846186493044378","date":"2026-04-22T08:20:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154932716442983716872389501870189234173","date":"2026-04-20T10:37:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-19T02:50:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-19T00:52:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-18T08:04:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Microbiology","date":"2026-04-15T03:23:58+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":"a0616f5a-f6e0-4f4c-bfc3-d04e85d57d52","owner":[],"postedDate":"April 27th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T00:36:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-14T17:52:20+00:00","index":41,"fulltext":""},{"type":"reviewerAgreed","content":"113782099844273740178018960529413974296","date":"2026-05-14T14:26:27+00:00","index":40,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T00:38:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-27 14:24:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9421212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9421212","identity":"rs-9421212","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}