The arrangement of dual-species biofilms of Candida albicans and Issatchenkia orientalis can be modified by the medium: effect of Voriconazole.

Both Candida albicans and Issatchenkia orientalis have been isolated from different types of infections over the years. They have the ability to form communities of microorganisms known as biofilms. It has been demonstrated that the medium employed in studies may affect the biofilm development. The aim of this study was to investigate the arrangement of dual-species biofilms of C. albicans and I. orientalis cultivated on either RPMI-1640 or Sabouraud Dextrose Broth (SDB), as well as the inhibitory effect of Voriconazole (VRC). For the experiments performed, ATCC strains were used, and yeast-mixed suspensions were inoculated in 96-well plates with either RPMI-1640 or SDB, in the presence or absence of VRC. The results were observed by counting the number of CFU obtained from scraping off the biofilms produced and plating the content on CHROMagar Candida medium. It was observed that for all conditions tested the medium chosen affected the arrangement of dual-species biofilms: when RPMI-1640 was used, there was a prevalence of C. albicans, while the opposite was noted when SDB was used. It could be suggested that the medium and environment could regulate interactions between both yeast species, including the response to different antifungal drugs.

The sunflower jacalin Helja: biological and structural insights of its antifungal activity against Candida albicans.

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans, capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall of the mnn4∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies.

ASLdC3: A Derivative of Acidic Sophorolipid Disrupts Mitochondrial Function, Induces ROS Generation, and Inhibits Biofilm Formation in Candida albicans.

Fungal infections account for more than 140 million cases of severe and life-threatening conditions each year, causing approximately 1.7 million deaths annually. Candida albicans and related species are the most common human fungal pathogens, causing both superficial (mucosal and cutaneous) and life-threatening invasive infections (candidemia) with a 40-75% mortality rate. Among many virulence factors of Candida albicans, morphological transition from yeast to hyphae, secretion of hydrolytic enzymes, and formation of biofilms are considered to be crucial for pathogenicity. However, the arsenals for the treatment against these pathogens are restricted to only a few classes of approved drugs, the efficacy of which is being compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. In this study, we have described the development of a molecule, exhibiting excellent antifungal activity (MIC 8 µg/mL), by tailoring acidic sophorolipids with aryl alcohols via enzyme catalysis. This novel derivative, ASLdC3, is a surface-active compound that lowers the surface tension of the air-water interface up to 2-fold before reaching the critical micelle concentration of 25 µg/mL. ASLdC3 exhibits excellent antibiofilm properties against Candida albicans and other nonalbicans Candida species. The molecule primarily exhibits its antifungal activity by perturbing mitochondrial function through the alteration of the mitochondrial membrane potential (MMP) and generation of reactive oxygen species (ROS). The ROS damages fungal cell membrane function and cell wall integrity, eventually leading to cell death. ASLdC3 was found to be nontoxic in in vitro assay and nonhemolytic. Besides, it does not cause toxicity in the C. elegans model. Our study provides a valuable foundation for the potential of acidic sophorolipid as a nontoxic, biodegradable precursor for the design and synthesis of novel molecules for use as antimicrobial drugs as well as for other clinical applications.

Enhancing immunity against Candida albicans infections through TIGIT knockout.

T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT) is an inhibitory receptor expressed by T and natural killer cells. Here, we used TIGIT knockout (KO) mice to demonstrate that mouse TIGIT directly interacts with Candida albicans. Reduced fungal growth and colonization were observed when TIGIT-KO splenocytes were co-cultured with C. albicans compared to the wild type (WT). In a systemic candidiasis model, TIGIT-KO mice exhibited improved survival and reduced body weight loss compared to WT mice. Organ-specific fungal burden assessment revealed significantly lower fungal loads in the kidneys, spleen, and lungs of TIGIT-KO mice. Finally, we show that the agglutinin-like sequence proteins ALS6, ALS7, and ALS9 of C. albicans are ligands for TIGIT and that the absence of these proteins abolishes the TIGIT effect in vivo. Our results identify the significance of TIGIT in modulating host defense against C. albicans and highlight the potential therapeutic implications for C. albicans infections. IMPORTANCE: Our results identify the significance of T cell immunoreceptor with immunoglobulin and ITIM domain in modulating host defense against Candida albicans and highlight the potential therapeutic implications for C. albicans infections.

Reactive oxygen species sensitive nanomicelles promote the antifungal activity of ketoconazole against Candida albicans in vulvovaginal candidiasis.

Excessive local accumulation of reactive oxygen species (ROS) in vulvovaginal candidiasis (VVC) leads to oxidative stress and aggravates inflammation. This study aimed to optimize and synthesize four ROS-sensitive polyethylene glycol (PEG)-boride polymers (PB, PCB, BPB, and BCPCB). A nanomicelle (BCPCB-K) was constructed using BCPCB-encapsulated ketoconazole (KTZ). Finally, the depolymerization principle and ROS-sensitive drug release of BCPCB-K as well as its anti-Candida albicans (CA) and therapeutic effects on mice with VVC were explored through in vitro and in vivo experiments. BCPCB-K exhibited low toxicity to mammalian cells in vitro and good biocompatibility in vivo. It also improved the dispersion and solubility of the hydrophobic drug KTZ. Furthermore, BCPCB-K simultaneously scavenged ROS and released the drug, thus facilitating the antifungal and VVC-treating effects of KTZ. Overall, the findings of this study broadened the application of ROS-sensitive materials in the drug-loading and antifungal fields and provided a strategy for VVC treatment.

Otilonium Bromide Exhibits Potent Antifungal Effects by Blocking Ergosterol Plasma Membrane Localization and Triggering Cytotoxic Autophagy in Candida Albicans.

Candidiasis, which presents a substantial risk to human well-being, is frequently treated with azoles. However, drug-drug interactions caused by azoles inhibiting the human CYP3A4 enzyme, together with increasing resistance of Candida species to azoles, represent serious issues with this class of drug, making it imperative to develop innovative antifungal drugs to tackle this growing clinical challenge. A drug repurposing approach is used to examine a library of Food and Drug Administration (FDA)-approved drugs, ultimately identifying otilonium bromide (OTB) as an exceptionally encouraging antifungal agent. Mechanistically, OTB impairs vesicle-mediated trafficking by targeting Sec31, thereby impeding the plasma membrane (PM) localization of the ergosterol transporters, such as Sip3. Consequently, OTB obstructs the movement of ergosterol across membranes and triggers cytotoxic autophagy. It is noteworthy that C. albicans encounters challenges in developing resistance to OTB because it is not a substrate for drug transporters. This study opens a new door for antifungal therapy, wherein OTB disrupts ergosterol subcellular distribution and induces cytotoxic autophagy. Additionally, it circumvents the hepatotoxicity associated with azole-mediated liver enzyme inhibition and avoids export-mediated drug resistance in C. albicans.

A Brg1-Rme1 circuit in Candida albicans hyphal gene regulation.

Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1, established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1Δ/Δ mutation is most prominent under static or "biofilm-like" growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1: hypha-associated gene expression levels are substantially reduced in a brg1Δ/Δ mutant and partially restored in a brg1Δ/Δ rme1Δ/Δ double mutant. An rme1Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1, whose activity is most prominent under biofilm-like growth conditions.

Determination of in vitro synergy and antibiofilm activities of antimicrobials and essential oil components.

Using existing adrentimicrobials with essential oil components to prevent antimicrobial resistance is an alternative strategy. This study aimed to evaluate the resistance status, synergistic combinations, and in vitro biofilm formation activities of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), Stenotrophomonas maltophilia and Candida albicans against antimicrobial agents and cinnamaldehyde, carvacrol, eugenol, limonene and eucalyptol. Antimicrobial activities were evaluated by microdilution, cytotoxicity by XTT, synergy by checkerboard and time-kill, and biofilm inhibition by microplate methods. Cinnamaldehyde and carvacrol showed strong antimicrobial activity. Synergistic effects were observed when using all essential oils with antimicrobials. Only two C. albicans isolates showed antagonism with cinnamaldehyde and fluconazole. The constituents showed cytotoxic effects in the L929 cell line (except limonene). A time-kill analysis revealed a bacteriostatic effect on S. maltophilia and MRSA isolates and a fungicidal effect on C. albicans isolates. These results are important for further research to improve antimicrobial efficacy or to develop new agents.

A customizable and defined medium supporting culturing of Candida albicans, Staphylococcus aureus, and human oral epithelial cells.

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus, allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus, C. albicans, and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus, and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus, addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions. IMPORTANCE: Interaction between microbes and the host are in the center of interest both in disease and in health. In order to study the interactions between microbes of different kingdoms and the host, alternative media are required. Synthetic media are useful as they allow addition of specific components. In addition, well-defined media are required if high-resolution analyses such as metabolomics and proteomics are desired. We describe the development of a synthetic medium to study the interactions between C. albicans, S. aureus, and human oral epithelial cells. Our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions.

Purification of Potential Antimicrobial Metabolites from Endophytic Fusarium oxysporum Isolated from Myrtus communis.

The rise of microbial resistance and emerging infections pose significant health threats. Natural products from endophytic fungi offer a promising source of novel compounds with the potential as major drug leads. This research aims to screen Myrtus communis and Moringa oleifera for endophytic fungi and screen their metabolites for antibacterial and antifungal potential. Six endophytic fungal strains were isolated using a potato dextrose agar (PDA) medium. The M. communis isolates were designated MC1, MC2, and MC3, and the M. oleifera isolates were named MO1, MO2, and MO3. Preliminary bioactivity testing revealed that the MC3 isolate exhibited significant growth inhibition against multidrug-resistant bacterial and fungal pathogens, including Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Candida glabrata. The MC3 isolate was identified as Fusarium oxysporum through morphological and microscopic methods. For metabolite production, the fungal strain was cultured in potato dextrose broth (PDB) medium at 28 °C for 14 days in a shaking incubator. The metabolites were purified using various chromatographic techniques, HPLC and GC-MS. The GC-MS analysis of the bioactive compound containing fungal strain (F. oxysporum) revealed multiple compounds at different retention times using the NIST-20 Library. Based on RSI values and probability indices, two compounds were targeted for further purification. Structure elucidation was performed using 1D and 2D nuclear magnetic resonance (NMR) experiments on a Varian 500 NMR machine. The compounds identified were ethyl iso-allocholate (C26H44O5, exact mass 436.32) and 1-monolinoleoyl glycerol trimethylsilyl ether (C27H56O4Si2, exact mass 500.37). The MS (NIST-20) library facilitated the investigation of the in silico antimicrobial activity of these compounds against the elastase virulence protein of P. aeruginosa and protease Sapp1p from C. parapsilosis. Both the compounds were docked with druggable proteins using the Glide induced fit docking (IFD) algorithm. The ethyl iso-allocholate and 1-monolinoleoyl glycerol trimethylsilyl ether compounds showed binding scores - 10.07 kcal mol-1 and - 7.47 kcal mol-1 against elastase, and - 8.16 kcal mol-1 and - 6.89 kcal mol-1 against aspartic protease, respectively. In vitro studies confirmed the inhibitory activity of these compounds against multidrug-resistant P. aeruginosa and E. faecalis. Ethyl iso-allocholate exhibited higher bioactivity against P. aeruginosa with inhibition rates of 41%, 27%, and 35% at concentrations of 1000, 500, and 250 µg mL-1, respectively. These results suggest that bioactive compounds from F. oxysporum have the potential as antimicrobial agents, warranting further research.

Towards a Wearable Feminine Hygiene Platform for Detection of Invasive Fungal Pathogens via Gold Nanoparticle Aggregation.

Candida albicans is an opportunistic fungus that becomes pathogenic and problematic under certain biological conditions. C. albicans may cause painful and uncomfortable symptoms, as well as deaths in immunocompromised patients. Therefore, early detection of C. albicans is essential. However, conventional detection methods are costly, slow, and inaccessible to women in remote or developing areas. To address these concerns, we have developed a wearable and discrete naked-eye detectable colorimetric platform for C. albicans detection. With some modification, this platform is designed to be directly adhered to existing feminine hygiene pads. Our platform is rapid, inexpensive, user-friendly, and disposable and only requires three steps: (i) the addition of vaginal fluid onto sample pads; (ii) the addition of gold nanoparticle gel and running buffer, and (iii) naked eye detection. Our platform is underpinned by selective thiolated aptamer-based recognition of 1,3-ß-D glucan molecules-a hallmark of C. albicans cell walls. In the absence of C. albicans, wearable sample pads turn bright pink. In the presence of C. albicans, the wearable pads turn dark blue due to significant nanoparticle target-induced aggregation. We demonstrate naked-eye colorimetric detection of 4.4 × 106C. albicans cells per ml and nanoparticle stability over a pH range of 3.0-8.0. We believe that this proof-of-concept platform has the potential to have a significant impact on women's health globally.

Oral Microbiota Alterations in Subjects with SARS-CoV-2 Displaying Prevalence of the Opportunistic Fungal Pathogen Candida albicans.

The oral cavity remains an underappreciated site for SARS-CoV-2 infection despite the myriad of oral conditions in COVID-19 patients. Recently, SARS-CoV-2 was shown to replicate in the salivary gland cells causing tissue inflammation. Given the established association between inflammation and microbiome disruption, we comparatively profiled oral microbial differences at a metagenomic level in a cohort of hospitalized COVID-19 patients and matched healthy controls. Specifically, we aimed to evaluate colonization by the opportunistic fungal pathogen Candida albicans, the etiologic agent of oral candidiasis. Comprehensive shotgun metagenomic analysis indicated that, overall, COVID-19 patients exhibited significantly reduced bacterial and viral diversity/richness; we identified 12 differentially abundant bacterial species to be negatively associated with COVID-19, and the functional pathways of certain bacteria to be highly associated with COVID-19 status. Strikingly, C. albicans was recovered from approximately half of the COVID-19 subjects but not from any of the healthy controls. The prevalence of Candida is likely linked to immune hypo-dysregulation caused by COVID-19 favoring Candida proliferation, warranting investigations into the interplay between Candida and SARS-CoV2 and potential therapeutic approaches directed toward oral candidiasis. Collectively, our findings prompt a reassessment of oral opportunistic infection risks during COVID-19 disease and their potential long-term impacts on oral health.

Using Laser Therapy and Topical Ozone as an Effective Intervention to Revolutionize Denture Stomatitis Treatment: A Case Report.

In dentistry, denture stomatitis (DS), a frequent inflammatory illness of the oral mucosa mostly related to denture wearing, is a major concern. DS is a common oral mycotic infection for those who wear partial or total dentures. The most often found species of Candida that may be isolated from both healthy and diseased oral tissues is Candida albicans. DS is associated with uneven denture surfaces, inadequate oral hygiene, or ill-fitting dentures. The diagnosis and management of DS in a 72-year-old male patient is presented in this case study. The patient complained about burning in his palate and having difficulty chewing. Upon clinical examination, erythema and inflammation were seen in the palate region. The history and clinical findings were consistent with DS. The patient was told to completely stop using dentures. Interventions were included in the treatment plan, such as diode laser therapy, topical ozonated oil application, and teaching about denture hygiene. The third day's follow-up visit revealed a progressive healing of the lesions and symptom relief. The lesion fully resolved on the sixth day. This case emphasizes the value of all-encompassing management techniques in treating DS successfully. It also highlights the significance of patient education, good oral hygiene, and focused therapy in producing favorable results.

Toll-like receptor 4 (TLR4) is the major pattern recognition receptor triggering the protective effect of a Candida albicans extracellular vesicle-based vaccine prototype in murine systemic candidiasis.

Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of ß-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

Expansion microscopy reveals characteristic ultrastructural features of pathogenic budding yeast species.

Candida albicans is the most prevalent fungal pathogen associated with candidemia. Similar to other fungi, the complex life cycle of C. albicans has been challenging to study with high-resolution microscopy due to its small size. We employed ultrastructure expansion microscopy (U-ExM) to directly visualise sub-cellular structures at high resolution in the yeast and during its transition to hyphal growth. NHS-ester pan-labelling in combination with immunofluorescence (IF) via snapshots of various mitotic stages provided a comprehensive map of nucleolar and mitochondrial segregation dynamics and enabled the resolution of inner and outer plaque of spindle pole bodies (SPBs). Analyses of microtubules (MTs) and SPBs suggest that C. albicans displays side-by-side SPB arrangement with a short mitotic spindle and longer astral MTs (aMTs) at the pre-anaphase stage. Modifications to the established U-ExM protocol enabled the expansion of six other human fungal pathogens, revealing that the side-by-side SPB configuration is a plausible conserved feature shared by many fungal species. We highlight the power of U-ExM to investigate sub-cellular organisation at high resolution and low cost in poorly studied and medically relevant microbial pathogens.

Regulatory complexity of cellular differentiation in Candida albicans revealed through systematic screening of protein kinase mutants.

A recent study in mBio reports the construction and preliminary screening of a library containing mutants of 99 of the 119 predicted protein kinases in Candida albicans (the majority of the remaining 20 are probably essential) (J. Kramara, M.-J. Kim, T. L. Ollinger, L. C. Ristow, et al., mBio e01249-24, 2024, https://doi.org/10.1128/mbio.01249-24). Using a quantitative competition assay in 10 conditions that represent nutritional, osmotic, cell wall, and pH stresses that are considered to model various aspects of the host environment allowed them to phenotypically cluster kinases, which highlight both the integration and specialization of signaling pathways, suggesting novel functions for many kinases. In addition, they tackle two complex and partially overlapping differentiation events, hyphal morphogenesis and biofilm formation. They find that a remarkable 88% of the viable kinase mutants in C. albicans affect hyphal growth, illustrating how integrated morphogenesis is in the overall biology of this organism, and begin to dissect the regulatory relationships that control this key virulence trait.

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis.

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

Fluoride-resistant Streptococcus mutans within cross-kingdom biofilms support Candida albicans growth under fluoride and attenuate the in vitro anti-caries effect of fluorine.

Fluoride-resistant Streptococcus mutans (S. mutans) might affect the ecological balance of biofilms in the presence of fluoride. We used a S. mutans and Candida albicans (C. albicans) cross-kingdom biofilm model to investigate whether fluoride-resistant S. mutans in biofilms would support C. albicans growth under fluoride stress and attenuate the in vitro anti-caries effect of fluorine. The impact of fluoride-resistant S. mutans on formation of cross-kingdom biofilms by S. mutans and C. albicans in the presence of fluoride was investigated in vitro using the crystal violet staining assay. Biofilm constitution was determined using colony-forming unit (CFU) counts and fluorescent in situ hybridization (FISH). Extracellular polysaccharide (EPS) generation in biofilms was determined by EPS/bacterial dying and water-insoluble polysaccharide detection. Acid production and demineralization were monitored using pH, lactic acid content, and transversal microradiography (TMR). The gene expression of microorganisms in the cross-kingdom biofilm was measured using qRT-PCR. Our results showed that both C. albicans and fluoride-resistant S. mutans grew vigorously, forming robust cross-kingdom biofilms, even in the presence of sodium fluoride (NaF). Moreover, fluoride-resistant S. mutans-containing cross-kingdom biofilms had considerable cariogenic potential for EPS synthesis, acid production, and demineralization ability in the presence of NaF than fluoride-sensitive S. mutans-containing biofilms. Furthermore, the gene expression of microorganisms in the two cross-kingdom biofilms changed dissimilarly in the presence of NaF. In summary, fluoride-resistant S. mutans in cross-kingdom biofilms supported C. albicans growth under fluoride and might attenuate the anti-caries potential of fluorine by maintaining robust cross-kingdom biofilm formation and cariogenic virulence expression in vitro in the presence of NaF.

Antifungal Activity of Morinda citrifolia Methanolic Extract against Candida albicans: An In Vitro Study.

Aim: Natural medicine used as an alternative and/or complementary treatment to counteract diseases is of great importance in public health. Therefore, the purpose of the present study was to assess the in vitro antifungal activity of Morinda citrifolia methanolic extract of peel, pulp, and seed against Candida albicans. Materials and Methods: The present study was experimental in vitro and cross-sectional. Eight replicates were prepared in Sabouraud dextrose agar with five wells each, where 0.12% chlorhexidine, distilled water, and methanolic extract of seed, peel, and pulp of Morinda citrifolia fruit were placed at concentrations of 10,690, 8,270, and 6,430 mg/mL, respectively, to evaluate sensitivity according to Duraffourd's scale. In addition, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by dilution and agar seeding method. Statistical analysis was performed by analysis of variance (ANOVA) and Tukey's post hoc test, considering a significance level of P < 0.05. Results: The inhibition halos of Morinda citrifolia methanolic extract of seed, peel, and pulp against Candida albicans measured on average 15.94, 11.94, and 11.56 mm, respectively. The MIC of seed, peel, and pulp extract were 1366.25, 2067.5, and 1607.5 mg/mL respectively, whereas the MFC for seed, peel, and pulp extract were 2672.50, 2067.5, and 3215 mg/mL, respectively. Moreover, seed extract presented significantly higher antifungal activity than peel and pulp (P < 0.001). Conclusions: Morinda citrifolia methanolic extract of peel, pulp, and seed showed fungistatic and fungicidal effect against Candida albicans, being this very sensitive to seed extract with a MIC of 1366.25 mg/mL and a MFC of 2672.5 mg/mL, which allows recommending the development of effective pharmacological formulations for the control of candidiasis.

The antioxidant and antimicrobial activity of ethanolic extract in roots, stems, and leaves of three commercial Cymbopogon species.

BACKGROUND: Cymbopogon is a member of the family Poaceae and has been explored for its phytochemicals and bioactivities. Although the antimicrobial activities of Cymbopogon spp. extracts have been extensively studied, comprehensive analyses are required to identify promising compounds for the treatment of antimicrobial resistance. Therefore, this study investigated the antioxidant and antimicrobial properties of Cymbopogon spp. ethanolic extracts in every single organ. METHODS: Ethanolic extracts were obtained from three Indonesian commercial species of Cymbopogon spp., namely Cymbopogon citratus (L.) Rendle, Cymbopogon nardus (DC.) Spatf., and Cymbopogon winterianus Jowitt. The leaf, stem, and root extracts were evaluated via metabolite profiling using gas chromatography-mass spectrometry (GC-MS). In silico and in vitro analyses were used to evaluate the antioxidant and antimicrobial properties of the Cymbopogon spp. ethanolic extracts. In addition, bioactivity was measured using cytotoxicity assays. Antioxidant assays were performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to determine toxicity to Huh7it-1 cells using a tetrazolium bromide (MTT) assay. Finally, the antimicrobial activity of these extracts was evaluated against Candida albicans, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli using a well diffusion assay. RESULTS: GC-MS analysis revealed 53 metabolites. Of these, 2,5-bis(1,1-dimethylethyl)- phenol (27.87%), alpha-cadinol (26.76%), and 1,2-dimethoxy-4-(1-propenyl)-benzene (20.56%) were the predominant compounds. C. winterianus and C. nardus leaves exhibited the highest antioxidant activity against DPPH and ABTS, respectively. Contrastingly, the MTT assay showed low cytotoxicity. C. nardus leaf extract exhibited the highest antimicrobial activity against E. coli and S. aureus, whereas C. winterianus stem extract showed the highest activity against B. substilis. Furthermore, computational pathway analysis predicted that antimicrobial activity mechanisms were related to antioxidant activity. CONCLUSIONS: These findings demonstrate that the leaves had strong antioxidant activity, whereas both the leaves and stems showed great antimicrobial activity. Furthermore, all Cymbopogon spp. ethanolic extracts showed low toxicity. These findings provide a foundation for future studies that assess the clinical safety of Cymbopogon spp. as novel drug candidates.