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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.

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 rme1 Δ/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 rme1 Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1 Δ/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 brg1 Δ/Δ mutant and partially restored in a brg1 Δ/Δ rme1 Δ/Δ double mutant. An rme1 Δ/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.IMPORTANCE Candida 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., Competing Interests: The authors declare no conflict of interest.

Objective: Adhesion to dentin is a first step for a successful microbial root canal colonization. Cell hydrophobicity seems to have some influence in the Candida species adhesion to surfaces. To measure cell surface hydrophobicity and to investigate the adherence ability to human dentin among Candida albicans strains isolated from root canal and lingual dorsum via an in vitro study., Methodology: adhesion was quantified in function of dentin area covered by blastospores and/or hyphae presence detected by epifluorescence microscope. Cell surface hydrophobicity was estimated by assessing the percentage migration of cells from an aqueous phase to a hydrocarbon phase. Contact angles were measured by the sessile drop technique on the dentin surface using a contact angle measurements apparatus. We also examined the correlation between adhesion ability and hydrophobicity., Results: although there was some intra-species variation in cell surface hydrophobicity, most isolates were characterized by moderate hydrophobicity. There was no significant difference in this parameter when the isolation niche was considered. Both root canal and lingual dorsum yeasts were able to adhere to dentin. No association was found between the strains' site of isolation and adhesion. Moreover, cell surface hydrophobicity and adhesion ability were not correlated., Conclusion: although hydrophobicity can influence Candida albicans virulence in many ways, this study suggests that this parameter by itself was not a good predictor of adhesion to dentin.

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log 10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log 10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms., (© 2024. The Author(s).)

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.

An important host defence mechanism against pathogens is intracellular killing, which is achieved through phagocytosis, a cellular process for engulfing and neutralizing extracellular particles. Phagocytosis results in the formation of matured phagolysosomes, which are specialized compartments that provide a hostile environment and are considered the end point of the degradative pathway. However, all fungal pathogens studied to date have developed strategies to manipulate phagosomal function directly and also indirectly by redirecting phagosomes from the degradative pathway to a non-degradative pathway with the expulsion and even transfer of pathogens between cells. Here, using the major human fungal pathogens Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Histoplasma capsulatum as examples, we discuss the processes involved in host phagosome-fungal pathogen interactions, with a focus on fungal evasion strategies. We also discuss recent approaches to targeting intraphagosomal pathogens, including the redirection of phagosomes towards degradative pathways for fungal pathogen eradication., (© 2024. Springer Nature Limited.)

Skin infections are a major threat to human health. Early diagnosis of bacterial infections is of great significance for implementing protective measures on the skin. Therefore, in this study, we designed an electrospun membrane (PPBT) for visual monitoring of colonized bacteria and responsive antibacterial ability. Specifically, the acidity of the microenvironment caused by bacterial metabolism was applied to drive the color change of bromothymol blue (BTB) on the PPBT membrane from green to yellow, thereby facilitating the early warning of infection and timely treatment. Within 4 h, different concentrations of Staphylococcus aureus (∼10 5 CFU mL -1 ), Escherichia coli (∼10 5 CFU mL -1 ), Pseudomonas aeruginosa (∼10 5 CFU mL -1 ) and Candida albicans (∼10 4 CFU mL -1 ) were visually monitored. Moreover, as the local acidity was enhanced via microbial metabolism, ZIF-8 nanoparticles loaded with TCS (TCS@ZIF-8) on the PPBT membrane could release TCS in an acid-responsive manner. At the same time, ROS were generated under 405 nm irradiation to achieve synergistic antibacterial ability. Experiments confirmed that the PPBT membrane has ideal and controllable antibacterial features based on acid responsive release and a synergistic photocatalytic antibacterial mechanism after monitoring. Therefore, the PPBT membrane developed in this work provides a feasible solution for bacterial monitoring and inactivation devices. More importantly, it can be beneficial for meeting the needs of clinical diagnosis and timely treatment of bacterial infection.

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., Competing Interests: The authors declare no conflict of interest.

At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation ( PRP19 ) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans ., Competing Interests: L.E.C. is a co-founder and shareholder in Bright Angel Therapeutics, a platform company for the development of novel antifungal therapeutics. L.E.C. is a science advisor for Kapoose Creek, a company that harnesses the therapeutic potential of fungi. All other authors have no conflicts of interest to declare.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans . An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCE Candida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions., Competing Interests: The authors declare no conflict of interest.

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50% by VEA purified from accessions #5 and #36. VEA antibiofilm variability between accessions was observed, likely due to co-purified small molecules rather than differences in VEA protein sequences. In conclusion, VEA seed extracts from the accessions with the highest antibiofilm activity could represent a valid approach for the development of an effective antibiofilm agent., (© 2024. The Author(s).)

Vulvovaginal candidiasis (VVC) is characterized as a very common fungal infection that significantly affects women's health worldwide. Essential oils (EOs) are currently being evaluated as an alternative therapy. The development of efficient techniques such as micro- or nanoencapsulation for protecting and controlling release is essential to overcome the limitations of EO applications. Therefore, the aim of this study was to develop and characterize oregano EO-loaded keratin microparticles (OEO-KMPs) as a potential treatment for VVC. OEO-KMPs were produced using high-intensity ultrasonic cycles and characterized in terms of morphological and physicochemical parameters. In vitro evaluation included assessing the toxicity of the OEO-KMPs and their effect against Candida albicans using microdilution and agar diffusion, while the activity against biofilm was quantified using colony forming units (CFU). The efficacy of the OEO-KMPs in an in vivo VVC mouse model was also studied. Female BALB/c mice were intravaginally infected with C. albicans , 24 h postinfection animals were treated intravaginally with 15 μL of OEO-KMPs and 24 h later vaginal fluid was analyzed for C. albicans and Lactobacillus growth (CFU mL -1 ). The results showed the stability of the OEO-KMPs over time, with high encapsulation efficiency and controlled release. This nanoparticle size facilitated penetration and completely inhibited the planktonic growth of C. albicans . In addition, an in vitro application of 2.5% of the OEO-KMPs eradicated mature C. albicans biofilms while preserving Lactobacillus species. In in vivo , a single intravaginal application of OEO-KMPs induced a reduction in C. albicans growth, while maintaining Lactobacillus species. In conclusion, this therapeutic approach with OEO-KMPs is promising as a potential alternative or complementary therapy for VVC while preserving vaginal microflora.

Candida albicans is the main fungal species involved in oral candidiasis, and its increasing resistance to pharmacological treatment encourages the search for improved antifungal agents. Lavandula dentata L. essential oil (LD-EO) has been recognized for its antimicrobial activity, but little is known about its role against oral C. albicans. This study evaluated the antifungal and antibiofilm activities, mechanisms of action, and toxicity of LD-EO from Brazil against oral strains of C. albicans. Antifungal activity was assessed based on Minimum Inhibitory Concentration (MIC), Minimum Fungicidal Concentration (MFC), association study with miconazole (Checkerboard method), and sorbitol and ergosterol assays. Inhibition of biofilm formation and disruption of preformed biofilm were considered when studying the effects of the product. Additionally, the toxicity of LD-EO was evaluated by a hemolysis assay on human erythrocytes. Phytochemical analysis by gas chromatography-mass spectrometry identified eucalyptol (33.1%), camphor (18.3%), and fenchone (15.6%) as major constituents. The test substance showed mainly fungicidal activity (MIC100 = 8 μg/mL; MFC = 16 μg/mL), including against two miconazole-resistant isolates of C. albicans. The effects of LD-EO were synergistic with those of miconazole and appeared not to involve damage to the fungal cell wall or plasma membrane. Its effectiveness in inhibiting biofilm formation was higher than the effect of disrupting preformed biofilm. Finally, the product exhibited low hemolytic activity at MIC. Based on the favorable and novel results described here, LD-EO could constitute a promising therapeutic alternative for oral candidiasis, including miconazole-resistant cases.

Introduction: Candida albicans and Streptococcus mutans co-exist in biofilms in the oral cavity. In this study, the impact of S. mutans on the growth of C. albicans within a mixed-species biofilm was examined., Materials and Methods: Single species C. albicans biofilms and mixed species biofilms containing C. albicans and S. mutans at 1:3 and 1:10 ratios were constructed in 6-well microtiter plates. After 24 hours of incubation, the density of resuspended biofilm cells was determined as CFU/ml and used to compare the growth of C. albicans in single species and mixed species biofilms., Results: The CFU/ml of C. albicans in mixed-species biofilms was found to be higher than that in single-species biofilms., Conclusion: S. mutans promotes the growth of C. albicans in a co-inhabited biofilm.

Objectives: Guided bone regeneration (GBR) is a core procedure used to regenerate bone defects. The aim of the study was to investigate the adherence of Candida albicans on six commercially available polytetrafluoroethylene (PTFE) membranes used in GBR procedures and the subsequent clinical consequences., Materials and Methods: Six commercially available PTFE membranes were tested. Two of the membranes had a textured surface and the other four a plane, nontextured one. C. albicans (ATCC 24433) was cultured for 24 h, and its cell surface hydrophobicity was assessed using a modified method. C. albicans adhesion to membrane discs was studied by scanning electron microscopy (SEM) and real-time polymerase chain reaction (PCR)., Results: C. albicans was found to be hydrophobic (77.25%). SEM analysis showed that C. albicans adherence to all membranes examined was characterized by patchy, scattered, and small clustered patterns except for one nontextured membrane with a most rough surface in which a thick biofilm was observed. Real-time PCR quantification revealed significantly greater adhesion of C. albicans cells to PTFE membranes than the control membrane (p ≤ .001) with the membranes having a textured surface exhibiting the highest count of 2680 × 10 4 cells/ml compared to the count of 707 × 10 4  cells/mL on those with a nontextured one (p ≤ .001). One membrane with nontextured surface, but with most rough surface was found to exhibit the highest count of 3010 × 10 4 cells/ml (p ≤ .05)., Conclusion: The results of this study indicate that C. albicans adhesion on membranes' surfaces depends on the degree of surface roughness and/or on the presence of a texture. Textured PTFE membranes and/or membranes high roughness showed significantly more adhered C. albicans cells. These findings can impact the surgeon's choice of GBR membrane and postoperative maintenance., (© 2024 The Author(s). Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Root caries is a subtype of dental caries that predominantly impacts older adults. The occurrence and progression of root caries are associated with the homeostasis of dental plaque biofilm, and microbial synergistic and antagonistic interactions in the biofilm play a significant role in maintaining the oral microecological balance. The objective of the current study was to investigate the role of Veillonella parvula in the microbial interactions and the pathogenesis of root caries. The analysis of clinical samples from patients with/without root caries revealed that Veillonella and V. parvula were abundant in the saliva of patients with root caries. More importantly, a significantly increased colonization of V. parvula was observed in root carious lesions. Further in vitro biofilm and animal study showed that V. parvula colonization increased the abundance and virulence of Streptococcus mutans and Candida albicans, leading to the formation of a polymicrobial biofilm with enhanced anti-stress capacity and cariogenicity, consequently exacerbating the severity of carious lesions. Our results indicate the critical role of V. parvula infection in the occurrence of root caries, providing a new insight for the etiological investigation and prevention of root caries., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Aim: To evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA)., Material and Methods: Titanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM)., Results: AE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups., Conclusions: The nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa , Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Candida albicans is often implicated in nosocomial infections with fatal consequences. Its virulence is contributed to hydrolytic enzymes and biofilm formation. Previous research focused on studying these virulence factors individually. Therefore, this study aimed to investigate the impact of biofilm formation on the hydrolytic activity using an adapted low-cost method. Eleven strains of C. albicans were used. The biofilms were formed on pre-treated silicone discs using 24-well plates and then deposited on the appropriate agar to test each enzyme, while the planktonic cells were conventionally seeded. Biofilms were analysed using Raman spectroscopy, fluorescent and scanning electron microscopy. The adapted method provided an evaluation of hydrolytic enzymes activity in C. albicans biofilm and showed that sessile cells had a higher phospholipase and proteinase activities compared with planktonic cells. These findings were supported by spectroscopic and microscopic analyses, which provided valuable insights into the virulence mechanisms of C. albicans during biofilm formation.

Epithelial cells orchestrate immune responses against fungal pathogens. This review highlights advances in integrating epithelial cells in immune responses against inhaled molds and dimorphic fungi, and against Candida species that colonize mucosal surfaces. In the lung, epithelial cells respond to interleukin-1 (IL-1) and interferon signaling to regulate effector cell influx and fungal killing. In the alimentary and vulvovaginal tracts, epithelial cells modulate fungal commensalism, invasive growth, and local immune tone, in part by responding to damage caused by candidalysin, a C. albicans peptide toxin, and through IL-17-dependent release of antimicrobial peptides that contribute to Candida colonization resistance. Understanding fungal-epithelial interactions in mammalian models of disease is critical to predict vulnerabilities and to identify opportunities for immune-based strategies to treat fungal infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Candida albicans is one of the agents of invasive candidiasis, a life-threatening disease strongly associated with hospitalization, particularly among patients in intensive care units with central venous catheters. This study aimed to evaluate the synergistic activity of the antifungal peptide ToAP2 combined with fluconazole against C. albicans biofilms grown on various materials. We tested combinations of different concentrations of the peptide ToAP2 with fluconazole on C. albicans biofilms. These biofilms were generated on 96-well plates, intravenous catheters, and infusion tubes in RPMI medium at two maturation stages. Scanning electron microscopy and atomic force microscopy were employed to assess the biofilm structure. We also evaluated the expression of genes previously proven to be involved in C. albicans biofilm formation in planktonic and biofilm cells after treatment with the peptide ToAP2 using qPCR. ToAP2 demonstrated a synergistic effect with fluconazole at concentrations up to 25 µM during both the early and mature stages of biofilm formation in 96-well plates and on medical devices. Combinations of 50, 25, and 12.5 µM of ToAP2 with 52 µM of fluconazole significantly reduced the biofilm viability compared to individual treatments and untreated controls. These results were supported by substantial structural changes in the biofilms observed through both scanning and atomic force microscopy. The gene expression analysis of C. albicans cells treated with 25 µM of ToAP2 revealed a decrease in the expression of genes associated with membrane synthesis, along with an increase in the expression of genes involved in efflux pumps, adhesins, and filamentation. Our results highlight the efficacy of the combined ToAP2 and fluconazole treatment against C. albicans biofilms. This combination not only shows therapeutic potential but also suggests its utility in developing preventive biofilm tools for intravenous catheters.

To evaluate the effect of antiseptic soap on single and dual-species biofilms of Candida albicans and Streptococcus mutans on denture base and reline resins. Samples of the resins were distributed into groups (n = 9) according to the prevention or disinfection protocols. In the prevention protocol, samples were immersed in the solutions (Lifebuoy, 0.5% sodium hypochlorite solution and PBS) for 7, 14 and 28 days before the single and dual-species biofilms formation. Overnight denture disinfection was simulated. In the disinfection protocol, samples were immersed in the same solutions during 8 hours after the single and dual-species biofilms formation. Antimicrobial activity was analyzed by counting colony-forming units (CFU/mL) and evaluating cell metabolism. Cell viability and protein components of the biofilm matrix were evaluated using confocal laser scanning microscopy (CLSM). Data were submitted to ANOVA, followed by Tukey's post-test (α = 0.05) or Dunnett's T3 multiple comparisons test. In the prevention protocol, Lifebuoy solution effectively reduced the number of CFU/mL of both species. In addition, the solution decreased the cell metabolism of the microorganisms. Regarding disinfection protocol, the Lifebuoy solution was able of reduce approximately of 2-3 logs for all the biofilms on the denture base and reline resin. Cellular metabolism was also reduced. The images obtained with CLSM corroborate these results. Lifebuoy solution was effective in reducing single and dual-species biofilms on denture base and reline resins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tasso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Multiple host and microbial factors dictate whether Candida albicans can colonize the mammalian gastrointestinal tract. In this issue of Cell Host & Microbe, Savage et al. demonstrate that restoration of intestinal epithelial hypoxia is sufficient to restore Candida albicans colonization resistance, even when other Candida inhibitory effectors remain depleted., Competing Interests: Declaration of interests A.Y.K. is a co-founder of Aumenta Biosciences. A.Y.K. received research funding from Novartis., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vulvovaginal candidiasis (VVC) is a prevalent condition affecting a significant portion of women worldwide. Licochalcone A (LA), a natural compound with diverse biological activities, holds promise as a protective agent against Candida albicans (C. albicans) infection. This study aims to investigate the potential of LA to safeguard vaginal epithelial cells (VECs) from C. albicans infection and elucidate the underlying molecular mechanisms. To simulate VVC in vitro, VK2-E6E7 cells were infected with C. albicans. Candida albicans biofilm formation, C. albicans adhesion to VK2-E6E7 cells, and C. albicans-induced cell damage and inflammatory responses were assessed by XTT reduction assay, fluorescence assay, LDH assay, and ELISA. CCK-8 assay was performed to evaluate the cytotoxic effects of LA on VK2-E6E7 cells. Western blotting assay was performed to detect protein expression. LA dose-dependently hindered C. albicans biofilm formation and adhesion to VK2-E6E7 cells. Furthermore, LA mitigated cell damage, inhibited the Bax/Bcl-2 ratio, and attenuated the secretion of pro-inflammatory cytokines in C. albicans-induced VK2-E6E7 cells. The investigation into LA's impact on the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway revealed that LA downregulated TLR4 expression and inhibited NF-κB activation in C. albicans-infected VK2-E6E7 cells. Furthermore, TLR4 overexpression partially abated LA-mediated protection, further highlighting the role of the TLR4/NF-κB pathway. LA holds the potential to safeguard VECs against C. albicans infection, potentially offering therapeutic avenues for VVC management., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

The human fungal pathogen Candida albicans can cause lethal systemic infections due to its ability to resist stress from the host and to undergo invasive hyphal growth. Previous studies showed that plasma membrane MCC/eisosome domains were important for virulence by promoting the ability of Sur7 to mediate normal cell wall morphogenesis and stress resistance. The sur7Δ mutant displayed abnormal clusters of PI 4,5 P 2 , suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI 4,5 P 2 levels by deleting combinations of the three PI 4,5 P 2 5' phosphatase genes ( INP51 , INP52 , and INP54 ) and found that some combinations, such as inp51Δ inp52Δ , gave phenotypes similar the sur7Δ mutant. In contrast, deleting one copy of MSS4 , the gene that encodes the 5' kinase needed to create PI 4,5 P 2 , reduced the abnormal PI 4,5 P 2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI 4,5 P 2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI 4,5 P 2 and highlight the critical role of PI 4,5 P 2 in the regulation of C. albicans virulence properties.

Candida albicans is known for its pathogenicity, although it lives within the human body as a commensal member. The commensal nature of C. albicans is well controlled and regulated by the host's immune system as they live in the harmonized microenvironment. However, the development of certain unusual microhabitat conditions (change in pH, co-inhabiting microorganisms' population ratio, debilitated host-immune system) pokes this commensal fungus to transform into a pathogen in such a way that it starts to propagate very rapidly and tries to breach the epithelial barrier to enter the host's systemic circulations. In addition, Candida is infamous as a major nosocomial (hospital-acquired infection) agent because it enters the human body through venous catheters or medical prostheses. The hysterical mode of C. albicans growth builds its microcolony or biofilm, which is pathogenic for the host. Biofilms propose additional resistance mechanisms from host immunity or extracellular chemicals to aid their survival. Differential gene expressions and regulations within the biofilms cause altered morphology and metabolism. The genes associated with adhesiveness, hyphal/pseudo-hyphal growth, persister cell transformation, and biofilm formation by C. albicans are controlled by myriads of cell-signaling regulators. These genes' transcription is controlled by different molecular determinants like transcription factors and regulators. Therefore, this review has focused discussion on host-immune-sensing molecular determinants of Candida during biofilm formation, regulatory descriptors (secondary messengers, regulatory RNAs, transcription factors) of Candida involved in biofilm formation that could enable small-molecule drug discovery against these molecular determinants, and lead to disrupt the well-structured Candida biofilms effectively., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Candida albicans is an immunogen for anti- Saccharomyces cerevisiae antibodies (ASCA), a serological marker of Crohn's disease. ASCA has also been reported in other autoimmune diseases, including coeliac disease (CeD). A strong antibody response against Hwp1, a protein associated with invasive hyphal form of C. albicans which presents peptide sequence homologies with gliadin, has also been described in CeD. This observation supports the hypothesis that C. albicans hyphal transition in C. albicans may trigger CeD onset through a mechanism of molecular/antigenic mimicry. In this study, we assessed whether the anti- C. albicans oligomannose and anti-Hwp1 protein responses may be linked despite their different pathophysiological significance. The measurement of ASCA levels in a cohort of patients involved in our previous Hwp1 study showed a significant correlation between the two biomarkers. This new observation further reinforces the link between C. albicans and CeD.

Trained Immunity is defined as a biological process normally induced by exogenous or endogenous insults that triggers epigenetic and metabolic reprogramming events associated with long-term adaptation of innate immune cells. This trained phenotype confers enhanced responsiveness to subsequent triggers, resulting in an innate immune "memory" effect. Trained Immunity, in the past decade, has revealed important benefits for host defense and homeostasis, but can also induce potentially harmful outcomes associated with chronic inflammatory disorders or autoimmune diseases. Interestingly, evidence suggest that the "trainers" prompting trained immunity are frequently glycans structures. In fact, the exposure of different types of glycans at the surface of pathogens is a key driver of the training phenotype, leading to the reprogramming of innate immune cells through the recognition of those glycan-triggers by a variety of glycan-binding proteins (GBPs) expressed by the immune cells. β-glucan or mannose-enriched structures in Candida albicans are some of the examples that highlight the potential of glycans in trained immunity, both in homeostasis and in disease. In this review, we will discuss the relevance of glycans exposed by pathogens in establishing key immunological hubs with glycan-recognizing receptors expressed in immune cells, highlighting how this glycan-GBP network can impact trained immunity. Finally, we discuss the power of glycans and GBPs as potential targets in trained immunity, envisioning potential therapeutic applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Contact lenses can be contaminated with various microorganisms, including pathogenic yeasts of the genus Candida, which are known for their ability to adhere to abiotic surfaces, including plastic materials used for various medical purposes. Microbial contamination of the lenses can lead to infection of the wearer's eyes. The purpose of this study was to simulate the contamination of contact lenses with C. albicans and C. parapsilosis, analyze the interaction of the microorganisms with the lens material, and optimize the protocol for PCR-based analysis of the microbial agents responsible for lens contamination., Methods: Hilafilcon lenses were exposed to C. albicans and C. parapsilosis cultures, washed, and examined for their ability to further spread the contamination. Scanning electron microscopy was used to analyze the attachment of yeast cells to the lenses. Infrared spectroscopy was used to examine the potential changes in the lens material due to Candida contamination. The protocol for DNA isolation from contaminated lenses was established to enable PCR analysis of microbes attached to the lenses., Results: Hilafilcon lenses contaminated with Candida were able to spread the contamination even after washing with saline or with a commercial cleaning solution. In the present experimental settings, the yeasts did not grow into the lenses but began to form biofilms on the surface. However, the ability of the lenses to retain water was altered. The PCR-based protocol could be used to help identify the type of contamination of contact lenses., Conclusion: Once contaminated with Candida albicans or Candida parapsilosis, Hilafilcon contact lenses are difficult to clean. Yeasts began to form biofilms on lens surfaces., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: To develop a protocol for forming subsurface caries lesions on bovine enamel by dual-species biofilms of Streptococcus mutans and Candida albicans in vitro., Design: Biofilms were grown on bovine enamel specimens in artificial saliva (AS) for seven days. After 24 h of formation, the AS was supplemented or not with fluoride (F) using sodium fluoride (0.005 or 0.008 ppm F), and the biofilms were exposed or not to a 20 % sucrose solution (reproducing a cariogenic challenge) once/day. On the seventh day, the biofilms were harvested and had their extracellular polysaccharides (EPS) and inorganic components analyzed. The specimens were subjected to computed X-ray microtomography analysis to determine their mineral concentration. Data were compared using two-way analyses of variance, followed by Fisher's LSD or Student-Newman-Keuls tests (p < 0.05)., Results: Biofilms exposed to the cariogenic challenge had significantly higher EPS concentrations than those not exposed, regardless of the presence of F. For biofilms grown with 0.008 ppm F, those exposed to the cariogenic challenge had lower F levels than those not exposed. For biofilms exposed to the cariogenic challenge, those grown with 0.008 ppm F had lower lesion depths and integrated mineral loss, and higher outer layers than those grown without F., Conclusions: The dual biofilm model assessed was able to create subsurface caries lesions in bovine enamel in vitro, which was influenced by the presence of F in the culture medium and exposure to sucrose., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SUMMARY Candida albicans remains a major fungal pathogen colonizing humans and opportunistically invading tissue when conditions are predisposing. Part of the success of C. albicans was attributed to its capacity to form hyphae that facilitate tissue invasion. However, in 1987, a second developmental program was discovered, the "white-opaque transition," a high-frequency reversible switching system that impacted most aspects of the physiology, cell architecture, virulence, and gene expression of C. albicans . For the 15 years following the discovery of white-opaque switching, its role in the biology of C. albicans remained elusive. Then in 2002, it was discovered that in order to mate, C. albicans had to switch from white to opaque, a unique step in a yeast mating program. In 2006, three laboratories simultaneously identified a putative master switch gene, which led to a major quest to elucidate the underlying mechanisms that regulate white-opaque switching. Here, the evolving discoveries related to this complicated phenotypic transition are reviewed in a quasi-chronological order not only to provide a historical perspective but also to highlight several unique characteristics of white-opaque switching, which are fascinating and may be important to the life history and virulence of this persistent pathogen. Many of these characteristics have not been fully investigated, in many cases, leaving intriguing questions unresolved. Some of these include the function of unique channeled pimples on the opaque cell wall, the capacity to form opaque cells in the absence of the master switch gene WOR1 , the formation of separate "pathogenic" and "sexual" biofilms, and the possibility that a significant portion of natural strains colonizing the lower gastrointestinal tract may be in the opaque phase. This review addresses many of these characteristics with the intent of engendering interest in resolving questions that remain unanswered., Competing Interests: The author declares no conflict of interest.

Candida albicans is a commensal of the human microbiota that can form biofilms on implanted medical devices. These biofilms are tolerant to antifungals and to the host immune system. To identify novel genes modulating C. albicans biofilm formation, we performed a large-scale screen with 2,454 C. albicans doxycycline-dependent overexpression strains and identified 16 genes whose overexpression significantly hampered biofilm formation. Among those, overexpression of the ZCF15 and ZCF26 paralogs that encode transcription factors and have orthologs only in biofilm-forming species of the Candida clade, caused impaired biofilm formation both in vitro and in vivo. Interestingly, overexpression of ZCF15 impeded biofilm formation without any defect in hyphal growth. Transcript profiling, transcription factor binding, and phenotypic microarray analyses conducted upon overexpression of ZCF15 and ZCF26 demonstrated their role in reprogramming cellular metabolism by regulating central metabolism including glyoxylate and tricarboxylic acid cycle genes. Taken together, this study has identified a new set of biofilm regulators, including ZCF15 and ZCF26, that appear to control biofilm development through their specific role in metabolic remodeling., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

IFN regulatory factor 7 (IRF7) exerts anti-infective effects by promoting the production of IFNs in various bacterial and viral infections, but its role in highly morbid and fatal Candida albicans infections is unknown. We unexpectedly found that Irf7 gene expression levels were significantly upregulated in tissues or cells after C. albicans infection in humans and mice and that IRF7 actually exacerbates C. albicans infection in mice independent of its classical function in inducing IFNs production. Compared to controls, Irf7-/- mice showed stronger phagocytosis of fungus, upregulation of C-type lectin receptor CD209 expression, and enhanced P53-AMPK-mTOR-mediated autophagic signaling in macrophages after C. albicans infection. The administration of the CD209-neutralizing Ab significantly hindered the phagocytosis of Irf7-/- mouse macrophages, whereas the inhibition of p53 or autophagy impaired the killing function of these macrophages. Thus, IRF7 exacerbates C. albicans infection by compromising the phagocytosis and killing capacity of macrophages via regulating CD209 expression and p53-AMPK-mTOR-mediated autophagy, respectively. This finding reveals a novel function of IRF7 independent of its canonical IFNs production and its unexpected role in enhancing fungal infections, thus providing more specific and effective targets for antifungal therapy., (Copyright © 2024 by The American Association of Immunologists, Inc.)

The interaction of foreign implants with their surrounding environment is significantly influenced by the adsorption of proteins on the biomaterial surfaces, playing a role in microbial adhesion. Therefore, understanding protein adsorption on solid surfaces and its effect on microbial adhesion is essential to assess the associated risk of infection. The aim of this study is to evaluate the effect of conditioning by fibronectin (Fn) or bovine serum albumin (BSA) protein layers of silica (SiO 2 ) surfaces on the adhesion and detachment of two pathogenic microorganisms: Pseudomonas aeruginosa PAO1-Tn 7-gfp and Candida albicans CIP 48.72. Experiments are conducted under both static and hydrodynamic conditions using a shear stress flow chamber. Through the use of very low wall shear stresses, the study brings the link between the static and dynamic conditions of microbial adhesion. The results reveal that the microbial adhesion critically depends on: (i) the presence of a protein layer conditioning the SiO 2 surface, (ii) the type of protein and (iii) the protein conformation and organization in the conditioning layer. In addition, a very distinct adhesion behaviour of P. aeruginosa is observed towards the two tested proteins, Fn and BSA. This effect is reinforced by the amount of proteins adsorbed on the surface and their organization in the layer. The results are discussed in the light of atomic force microscopy analysis of the organization and conformation of proteins in the layers after adsorption on the SiO 2 surface, as well as the specificity in bacterial behaviour when interacting with these protein layers. The study also demonstrates the very distinctive behaviours of the prokaryote P. aeruginosa PAO1-Tn 7-gfp compared to the eukaryote C. albicans CIP 48.72. This underscores the importance of considering species-specific interactions between the protein conditioning layer and different pathogenic microorganisms, which appear crucial in designing tailored anti-adhesive surfaces.

Candida albicans ( C. albicans ) and Lactiplantibacillus plantarum subsp. plantarum ( L. plantarum ) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans , including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS . Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans , possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans , indicating the potential interruption of pathogenic activities by L. plantarum . Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans . The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti- Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans ., Competing Interests: The authors declare no conflict of interest.

Purpose: Fungal keratitis (FK) is an invasive corneal infection associated with significant risk to vision. Although the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway has been recognized for its role in defending against viral infections, its involvement in FK still remains largely unclear. This study sought to elucidate the contribution of the cGAS/STING signaling pathway to the pathogenesis of FK., Methods: The expression of cGAS/STING signaling components was assessed in a murine model of Candida albicans keratitis through RNA sequencing, western blot analysis, immunofluorescence staining, and real-time PCR. Both genetic (utilizing Sting1gt/gt mice) and pharmacological (using C176) interventions were employed to inhibit STING activity, allowing for the evaluation of resultant pathogenic alterations in FK using slit-lamp examination, clinical scoring, hematoxylin and eosin (H&E) staining, fungal culture, and RNA sequencing. Subconjunctival administration of the NOD-like receptor protein 3 (NLRP3) inflammasome inhibitor MCC950 was performed to evaluate FK manifestations following STING activity blockade. Furthermore, the impact of the STING agonist diABZI on FK progression was investigated., Results: Compared to uninfected corneas, those infected with C. albicans exhibited increased expression of cGAS/STING signaling components, as well as its elevated activity. Inhibiting cGAS/STING signaling exacerbated the advancement of FK, as evidenced by elevated clinical scores, augmented fungal load, and heightened inflammatory response, including NLRP3 inflammasome activation and pyroptosis. Pharmacological inhibition of the NLRP3 inflammasome effectively mitigated the exacerbated FK by suppressing STING activity. Conversely, pre-activation of STING exacerbated FK progression compared to the PBS control, characterized by increased fungal burden and reinforced inflammatory infiltration., Conclusions: This study demonstrates the essential role of the cGAS/STING signaling pathway in FK pathogenesis and highlights the necessity of its proper activation for the host against FK.

Aim: To evaluate the influence of different preparation tapers on the reduction in planktonic bacteria and biofilms of Enterococcus faecalis and Candida albicans in the apical third (4 mm) of the mesial roots of mandibular molars, correlating decontamination with canal shape., Methodology: After microtomography analysis for morphological standardization of the canals, 48 mandibular molar roots, each containing two canals (96 canals), were contaminated with E. faecalis and C. albicans and divided into four groups (n = 11) for canal instrumentation using ProDesign Logic 2 files with different tapers G (.03): # 25.03; G (.04): # 25.04; G (.05): # 25.05; and G (.06): # 25.06 and irrigation with 2.5% sodium hypochlorite. Four roots were examined under a scanning electron microscope (SEM) to qualitatively assess biofilm formation. Eight roots were used as the negative control group (samples were not contaminated). Bacteriological samples were taken exclusively from the apical third of the roots before and after chemical-mechanical preparation and bacterial counts were determined (CFU/mL). The final micro-CT scan was used to quantify the volume variation and unprepared canal area in the apical third. Statistical analysis was performed using the Kruskal-Wallis, Student-Newman-Keuls and Wilcoxon tests for analysis of microbiological data. anova and the Tukey or Games-Howell test were used for analysis of micro-CT data and Spearman's test for correlations (α = 5%)., Results: All groups showed a significant reduction in bacteria (p < .05), with no statistically significant difference between groups. There was no significant difference in per cent volume increase between groups. The unprepared area (Δ%) was affected by the file used (p = .026) and was significantly lower for G (.06) compared to G (.03). There was no statistically significant correlation among bacterial reduction, volume and unprepared area (p > .05)., Conclusion: The different preparation tapers influenced root canal shaping in the apical third but did not improve decontamination in this region., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

The oral cavity, a warm and moist environment, is prone to the proliferation of microorganisms like Candida albicans ( C. albicans ), which forms robust biofilms on biotic and abiotic surfaces, leading to challenging infections. These biofilms are resistant to conventional treatments due to their resilience against antimicrobials and immune responses. The dynamic nature of the oral cavity, including the salivary flow and varying surface properties, complicates the delivery of therapeutic agents. To address these challenges, we introduce dendritic microparticles engineered for enhanced adhesion to dental surfaces and effective delivery of antifungal agents and antibiofilm enzymes. These microparticles are fabricated using a water-in-oil-in-water emulsion process involving a blend of poly(lactic- co -glycolic acid) (PLGA) random copolymer (RCP) and PLGA- b -poly(ethylene glycol) (PLGA- b -PEG) block copolymer (BCP), resulting in particles with surface dendrites that exhibit strong adhesion to oral surfaces. Our study demonstrates the potential of these adhesive microparticles for oral applications. The adhesion tests on various oral surfaces, including dental resin, hydroxyapatite, tooth enamel, and mucosal tissues, reveal superior adhesion of these microparticles compared to conventional spherical ones. Furthermore, the release kinetics of nystatin from these microparticles show a sustained release pattern that can kill C. albicans . The biodegradation of these microparticles on tooth surfaces and their efficacy in preventing fungal biofilms have also been demonstrated. Our findings highlight the effectiveness of adhesive microparticles in delivering therapeutic agents within the oral cavity, offering a promising approach to combat biofilm-associated infections.

Candida albicans (C. albicans) can behave as a commensal yeast colonizing the vaginal mucosa, and in this condition is tolerated by the epithelium. When the epithelial tolerance breaks down, due to C. albicans overgrowth and hyphae formation, the generated inflammatory response and cell damage lead to vulvovaginal candidiasis (VVC) symptoms. Here, we focused on the induction of mitochondrial reactive oxygen species (mtROS) in vaginal epithelial cells after C. albicans infection and the involvement of fungal burden, morphogenesis and candidalysin (CL) production in such induction. Bioluminescent (BLI) C. albicans, C. albicans PCA-2 and C. albicans 529L strains were employed in an in vitro infection model including reconstituted vaginal epithelium cells (RVE), produced starting from A-431 cell line. The production of mtROS was kinetically measured by using MitoSOX™ Red probe. The potency of C. albicans to induced cell damage to RVE and C. albicans proliferation have also been evaluated. C. albicans induces a rapid mtROS release from vaginal epithelial cells, in parallel with an increase of the fungal load and hyphal formation. Under the same experimental conditions, the 529L C. albicans strain, known to be defective in CL production, induced a minor mtROS release showing the key role of CL in causing epithelial mithocondrial activation. C. albicans PCA-2, unable to form hyphae, induced comparable but slower mtROS production as compared to BLI C. albicans yeasts. By reducing mtROS through a ROS scavenger, an increased fungal burden was observed during RVE infection but not in fungal cultures grown on abiotic surface. Collectively, we conclude that CL, more than fungal load and hyphae formation, seems to play a key role in the rapid activation of mtROS by epithelial cells and in the induction of cell-damage and that mtROS are key elements in the vaginal epithelial cells response to C. albicans., Competing Interests: The Authors have declared that no competing interests exist., (Copyright: © 2024 Spaggiari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Xiong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The prevalence of Candida albicans infection has increased during the past few years, which contributes to the need for new, effective treatments due to the increasing concerns regarding antifungal drug toxicity and multidrug resistance. Butyl isothiocyanate (butylITC) is a glucosinolate derivative, and has shown a significant antifungal effect contrary to Candida albicans. Additionally, how butylITC affects the virulence traits of C. albicans and molecular mode of actions are not well known. Present study shows that at 17.36 mM concentration butylITC inhibit planktonic growth. butylITC initially slowed the hyphal transition at 0.542 mM concentration. butylITC hampered biofilm development, and inhibits biofilm formation at 17.36 mM concentration which was analysed using metabolic assay (XTT assay) and Scanning Electron Microscopy (SEM). In addition, it was noted that butylITC inhibits ergosterol biosynthesis. The permeability of cell membranes was enhanced by butylITC treatment. Moreover, butylITC arrests cells at S-phase and induces intracellular Reactive Oxygen Species (ROS) accumulation in C. albicans. The results suggest that butylITC may have a dual mode of action, inhibit virulence factors and modulate cellular processes like inhibit ergosterol biosynthesis, cell cycle arrest, induces ROS production which leads to cell death in C. albicans., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLC F F - /H + antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLC F transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans , increased the relative proportion of oral commensal S. gordonii , and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLC F transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity., Importance: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans , outcompete beneficial species, such as Streptococcus gordonii , in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries., Competing Interests: The authors declare no conflict of interest.

Background: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level., Materials and Methods: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method., Results: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001)., Conclusions: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm., (© 2024. The Author(s).)

Objective: Microbial dysbiosis and microbiome-induced inflammation may play a role in the etiopathogenesis of oral squamous cell carcinoma (OSCC). Candida albicans (C. albicans) is the most prevalent opportunistic pathogenic fungus in the oral cavity, and Candida infection is considered as one of its high-risk factors. Although oral microbiota-host interactions are closely associated with the development of OSCC, the interrelationship between fungi and OSCC is poorly understood compared to that between bacteria and viruses., Results: We accumulated knowledge of the evidence, pathogenic factors, and possible multiple mechanisms by which C. albicans promotes malignant transformation of OSCC, focusing on the induction of epithelial damage, production of carcinogens, and regulation of the tumor microenvironment. In addition, we highlight the latest treatment strategies for Candida infection., Conclusion: This review provides a new perspective on the interrelationship between C. albicans and OSCC and contributes to the establishment of a systematic and reliable clinical treatment system for OSCC patients with C. albicans infection., (© 2023 Wiley Periodicals LLC.)

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by P. aeruginosa and the fungal pathogen Candida albicans. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-h-old dual-species biofilms but showed only negligible activity against 24-h-old biofilms. This study lays the first foundation for potential therapeutic approaches to treat co-infections caused by bacteria and fungi using phage-antibiotic combinations., (© 2024. The Author(s).)

Objective: This study evaluated the surface roughness, wettability and adhesion of multispecies biofilms (Candida albicans, Staphylococcus aureus and Streptococcus mutans) on 3D-printed resins for complete denture bases and teeth compared to conventional resins (heat-polymerized acrylic resin; artificial pre-fabricated teeth)., Methodology: Circular specimens (n=39; 6.0 mm Ø × 2.0 mm) of each group were subjected to roughness (n=30), wettability (n=30) and biofilm adhesion (n=9) tests. Three roughness measurements were taken by laser confocal microscopy and a mean value was calculated. Wettability was evaluated by the contact angle of sessile drop method, considering the mean of the three evaluations per specimen. In parallel, microorganism adhesion to resin surfaces was evaluated using a multispecies biofilm model. Microbial load was evaluated by determining the number of Colony Forming Units (CFU/mL) and by scanning electron microscopy (SEM). Data were subjected to the Wald test in a generalized linear model with multiple comparisons and Bonferroni adjustment, as well as two-way ANOVA (α=5%)., Results: The roughness of the conventional base resin (0.01±0.04) was lower than that of the conventional tooth (0.14±0.04) (p=0.023) and 3D-printed base (0.18±0.08) (p<0.001). For wettability, conventional resin (84.20±5.57) showed a higher contact angle than the 3D-printed resin (60.58±6.18) (p<0.001). Higher microbial loads of S. mutans (p=0.023) and S. aureus (p=0.010) were observed on the surface of the conventional resin (S. mutans: 5.48±1.55; S. aureus: 7.01±0.57) compared to the 3D-printed resin (S. mutans: 4.11±1.96; S. aureus: 6.42±0.78). The adhesion of C. albicans was not affected by surface characteristics. The conventional base resin showed less roughness than the conventional dental resin and the printed base resin., Conclusion: The 3D-printed resins for base and tooth showed less hydrophobicity and less adhesion of S. mutans and S. aureus than conventional resins.

Fungal keratitis (FK) is a highly blinding infectious corneal disease caused by pathogenic fungi. Candida albicans (C. albicans) is one of the main pathogens of fungal keratitis. Extracellular vesicles (EVs), lipid bilayer compartments released by almost all living cells, including fungi, have garnered attention for their role in pathogenic microbial infection and host immune responses in recent years. Studies have reported that pretreating the host with fungal EVs can reduce the inflammatory response of the host when attacked by fungi and reduce the lethality of fungal infection. However, there are no studies that have evaluated whether C. albicans EVs can modulate the inflammatory response associated with C. albicans keratitis. Our study revealed that C. albicans EVs could activate the polymorphonuclear cells (PMNs) and promote their secretion of proinflammatory cytokines and nitric oxide (NO), enhance their phagocytic and fungicidal abilities against C. albicans. C. albicans EVs also induced a proinflammatory response in RAW264.7 cells, which was characterized by increased production of inflammatory cytokines and elevated expression of the chemokine CCL2. Similarly, stimulation of C. albicans EVs to RAW264.7 cells also enhanced the phagocytosis and killing ability of cells against C. albicans. Besides, in our in vivo experiments, after receiving subconjunctival injection of C. albicans EVs, C57BL/6 mice were infected with C. albicans. The results demonstrated that pre-exposure to C. albicans EVs could effectively diminish the severity of keratitis, reduce fungal load and improve prognosis. Overall, we conclude that C. albicans EVs can modulate the function of immune cells and play a protective role in C. albicans keratitis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: The present systematic review and meta-analysis investigated the available evidence about the adherence of Candida Albicans to the digitally-fabricated acrylic resins (both milled and 3D-printed) compared to the conventional heat-polymerized acrylic resins., Methods: This study followed the guidelines of the Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA). A comprehensive search of online databases/search tools (Web of Science, Scopus, PubMed, Ovid, and Google Scholar) was conducted for all relevant studies published up until May 29, 2023. Only in-vitro studies comparing the adherence of Candida albicans to the digital and conventional acrylic resins were included. The quantitative analyses were performed using RevMan v5.3 software., Results: Fourteen studies were included, 11 of which were meta-analyzed based on Colony Forming Unit (CFU) and Optical Density (OD) outcome measures. The pooled data revealed significantly lower candida colonization on the milled digitally-fabricated compared to the heat-polymerized conventionally-fabricated acrylic resin materials (MD = - 0.36; 95%CI = - 0.69, - 0.03; P = 0.03 and MD = - 0.04; 95%CI = - 0.06, - 0.01; P = 0.0008; as measured by CFU and OD respectively). However, no differences were found in the adhesion of Candida albicans between the 3D-printed digitally-fabricated compared to the heat-polymerized conventionally-fabricated acrylic resin materials (CFU: P = 0.11, and OD: P = 0.20)., Conclusion: The available evidence suggests that candida is less likely to adhere to the milled digitally-fabricated acrylic resins compared to the conventional ones., (© 2024. The Author(s).)

Background: The interaction between the host and Candida albicans is dynamic and intricate. We performed proteomic analysis to explore monocyte -C. albicans hyphae interaction. Materials & methods: Primary human monocytes were stimulated by heat-killed C. albicans hyphae and their proteins were profiled by tandem liquid chromatography with mass spectrometry (LC-MS/MS). Results: Based on the protein database of different species for analysis, we found that stimulation of monocytes by hyphae was accompanied by upregulation of histones and activation of extracellular traps (ETs) formation pathway. Meanwhile, monocyte ETs (MoETs) were evoked by synthesis or alteration of C. albicans cell wall proteins expression during the morphological switch to hyphal. Conclusion: MoETs formation is linked to cell wall proteins of C. albicans hyphae.

Candida albicans and Porphyromonas gingivalis are prevalent in the subgingival area where the frequency of fungal colonization increases with periodontal disease. Candida's transition to a pathogenic state and its interaction with P. gingivalis exacerbate periodontal disease severity. However, current treatments for these infections differ, and combined therapy remains unexplored. This work is based on an antimicrobial peptide that is therapeutic and induces a color change in a nanoparticle reporter. Methods: We built and characterized two enzyme-activatable prodrugs to treat and detect C. albicans and P. gingivalis via the controlled release of the antimicrobial peptide. The zwitterionic prodrug quenches the antimicrobial peptide's activity until activation by a protease inherent to the pathogens (SAP9 for C. albicans and RgpB for P. gingivalis ). The toxicity of the intact prodrugs was evaluated against fungal, bacterial, and mammalian cells. Therapeutic efficacy was assessed through microscopy, disk diffusion, and viability assays, comparing the prodrug to the antimicrobial peptide alone. Finally, we developed a colorimetric detection system based on the aggregation of plasmonic nanoparticles. Results: The intact prodrugs showed negligible toxicity to cells absent a protease trigger. The therapeutic impact of the prodrugs was comparable to that of the antimicrobial peptide alone, with a minimum inhibitory concentration of 3.1 - 16 µg/mL. The enzymatic detection system returned a detection limit of 10 nM with gold nanoparticles and 3 nM with silver nanoparticles. Conclusion: This approach offers a convenient and selective protease sensing and protease-induced treatment mechanism based on bioinspired antimicrobial peptides., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)