Development of a Recombinase-Aided Amplification Assay for the Rapid Detection of Candida auris.

Candida auris (C. auris) was first discovered in Japan in 2009 and has since spread worldwide. It exhibits strong transmission ability, high multidrug resistance, blood infectivity, and mortality rates. Traditional diagnostic techniques for C. auris have shortcomings, leading to difficulty in its timely diagnosis and identification. Therefore, timely and accurate diagnostic assays for clinical samples are crucial. We developed a novel, rapid recombinase-aided amplification (RAA) assay targeting the 18S rRNA, ITS1, 5.8S rRNA, ITS2, and 28S rRNA genes for C. auris identification. This assay can rapidly amplify DNA at 39 °C in 20 min. The analytical sensitivity and specificity were evaluated. From 241 clinical samples collected from pediatric inpatients, none were detected as C. auris-positive. We then prepared simulated clinical samples by adding 10-fold serial dilutions of C. auris into the samples to test the RAA assay's efficacy and compared it with that of real-time PCR. The assay demonstrated an analytical sensitivity of 10 copies/µL and an analytical specificity of 100%. The lower detection limit of the RAA assay for simulated clinical samples was 101 CFU/mL, which was better than that of real-time PCR (102-103 CFU/mL), demonstrating that the RAA assay may have a better detection efficacy for clinical samples. In summary, the RAA assay has high sensitivity, specificity, and detection efficacy. This assay is a potential new method for detecting C. auris, with simple reaction condition requirements, thus helping to manage C. auris epidemics.

Involvement of cGAS/STING Signaling in the Pathogenesis of Candida albicans Keratitis: Insights From Genetic and Pharmacological Approaches.

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.

Multicenter Candida auris outbreak caused by azole-susceptible clade IV in Pernambuco, Brazil.

BACKGROUND: Candida auris is an emerging multidrug-resistant yeast, frequently causing outbreaks in health care facilities. The pathogen persistently colonises human skin and inanimate surfaces such as catheters, aiding to its spread. Moreover, colonisation is a risk factor to develop invasive infection. OBJECTIVES: We investigated 61 C. auris strains isolated from non-sterile human body sites (n = 53) and the hospital environment (n = 8), originating from four different centres in a single Brazilian state. MATERIALS AND METHODS: Antifungal susceptibility testing (AFST) against common antifungals was performed, and resistance-associated genes were evaluated. Genetic relatedness was investigated with short tandem repeat (STR) genotyping and validated with whole-genome sequencing (WGS) single nucleotide polymorphism (SNP) analysis. RESULTS: Antifungal susceptibility testing demonstrated that all isolates were susceptible to azoles, echinocandins and amphotericin B. No mutations were detected in ERG11 and FKS1 genes. With STR typing, isolates were allocated to clade IV and appeared closely related. This was confirmed by WGS SNP analysis of 6 isolates, which demonstrated a maximal difference of only 41 SNPs between these strains. Furthermore, the Brazilian isolates formed a distinct autochthonous branch within clade IV, excluding recent introductions from outside the country. A molecular clock analysis of clade IV isolates from various countries suggests that early in the previous century there was a unique event causing environmental spread of a C. auris ancestor throughout the Latin-American continent, followed by human introduction during the last decades. CONCLUSION: We report the emergence of C. auris patient colonisation in multiple centres by fluconazole-susceptible clade IV close-related strains in Pernambuco State, Brazil.

Antifungal Activity of Mefloquine Against Candida albicans Growth and Virulence Factors: Insights Into Mode of Action.

The antimalarial drug Mefloquine has demonstrated antifungal activity against growth and virulence factors of Candida albicans. The current study focused on the identification of Mefloquine's mode of action in C. albicans by performing cell susceptibility assay, biofilm assay, live and dead assay, propidium iodide uptake assay, ergosterol quantification assay, cell cycle study, and gene expression studies by RT-PCR. Mefloquine inhibited the virulence factors in C. albicans, such as germ tube formation and biofilm formation at 0.125 and 1 mg/ml, respectively. Mefloquine-treated cells showed a decrease in the quantity of ergosterol content of cell membrane in a concentration-dependent manner. Mefloquine (0.25 mg/ml) arrested C. albicans cells at the G2/M phase and S phase of the cell cycle thereby preventing the progression of the normal yeast cell cycle. ROS level was measured to find out oxidative stress in C. albicans in the presence of mefloquine. The study revealed that, mefloquine was found to enhance the ROS level and subsequently oxidative stress. Gene expression studies revealed that mefloquine treatment upregulates the expressions of SOD1, SOD2, and CAT1 genes in C. albicans. In vivo, the antifungal efficacy of mefloquine was confirmed in mice for systemic candidiasis and it was found that there was a decrease in the pathogenesis of C. albicans after the treatment of mefloquine in mice. In conclusion, mefloquine can be used as a repurposed drug as an alternative drug against Candidiasis.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

BACKGROUND: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. OBJECTIVES: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. METHODS: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. RESULTS: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. CONCLUSIONS: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

The vacuolar fusion regulated by HOPS complex promotes hyphal initiation and penetration in Candida albicans.

The transition between yeast and hyphae is crucial for regulating the commensalism and pathogenicity in Candida albicans. The mechanisms that affect the invasion of hyphae in solid media, whose deficiency is more related to the pathogenicity of C. albicans, have not been elucidated. Here, we found that the disruption of VAM6 or VPS41 which are components of the homotypic vacuolar fusion and protein sorting (HOPS) complex, or the Rab GTPase YPT72, all responsible for vacuole fusion, led to defects in hyphal growth in both liquid and solid media, but more pronounced on solid agar. The phenotypes of vac8Δ/Δ and GTR1OE-vam6Δ/Δ mutants indicated that these deficiencies are mainly caused by the reduced mechanical forces that drive agar and organs penetration, and confirmed that large vacuoles are required for hyphal mechanical penetration. In summary, our study revealed that large vacuoles generated by vacuolar fusion support hyphal penetration and provided a perspective to refocus attention on the role of solid agar in evaluating C. albicans invasion.

Molecular Epidemiology and Antifungal Susceptibility Profile in Nakaseomyces glabrata Species Complex: A 5-Year Countrywide Study.

BACKGROUND: The current study aimed to identify Iranian Nakaseomyces (Candida) glabrata complex species in the clinical isolates and determine their antifungal susceptibility profile. METHODS: In total, 320 N. glabrata clinical isolates were collected from patients hospitalized in different geographical regions of Iran. The initial screening was performed by morphological characteristics on CHROMagar Candida. Each isolate was identified by targeting the D1/D2 rDNA using a multiplex-PCR method. To validate the mPCR method and determine genetic diversity, the ITS-rDNA region was randomly sequenced in 40 isolates. Additionally, antifungal susceptibility was evaluated against nine antifungal agents following the CLSI M27-A4 guidelines. RESULTS: All clinical isolates from Iran were identified as N. glabrata. The analysis of ITS-rDNA sequence data revealed the presence of eight distinct ITS clades and 10 haplotypes among the 40 isolates of N. glabrata. The predominant clades identified were Clades VII, V, and IV, which respectively accounted for 22.5%, 17.5%, and 17.5% isolates. The widest MIC ranges were observed for voriconazole (0.016-8 µg/mL) and isavuconazole (0.016-2 µg/mL), whereas the narrowest ranges were seen with itraconazole and amphotericin B (0.25-2 µg/mL). CONCLUSION: Haplotype diversity can be a valuable approach for studying the genetic diversity, transmission patterns, and epidemiology of the N. glabrata complex.

Segmentation of hyphae and yeast in fungi-infected tissue slice images and its application in analyzing antifungal blue light therapy.

Candida albicans is a pathogenic fungus that undergoes morphological transitions between hyphal and yeast forms, adapting to diverse environmental stimuli and exhibiting distinct virulence. Existing research works on antifungal blue light (ABL) therapy have either focused solely on hyphae or neglected to differentiate between morphologies, obscuring potential differential effects. To address this gap, we established a novel dataset of 150 C. albicans-infected mouse skin tissue slice images with meticulously annotated hyphae and yeast. Eleven representative convolutional neural networks were trained and evaluated on this dataset using seven metrics to identify the optimal model for segmenting hyphae and yeast in original high pixel size images. Leveraging the segmentation results, we analyzed the differential impact of blue light on the invasion depth and density of both morphologies within the skin tissue. U-Net-BN outperformed other models in segmentation accuracy, achieving the best overall performance. While both hyphae and yeast exhibited significant reductions in invasion depth and density at the highest ABL dose (180 J/cm2), only yeast was significantly inhibited at the lower dose (135 J/cm2). This novel finding emphasizes the importance of developing more effective treatment strategies for both morphologies.

We studied the effects of blue light therapy on hyphal and yeast forms of Candida albicans. Through image segmentation techniques, we discovered that the changes in invasion depth and density differed between these two forms after exposure to blue light.

Validation of a qualitative real-time PCR assay for the detection of Candida auris in hospital inpatient screening.

Candida auris is a multidrug-resistant opportunistic fungal pathogen capable of causing serious infections and healthcare-associated outbreaks. Screening for colonization with C. auris has become routine and is recommended in many hospitals and healthcare facilities as an infection control and prevention strategy. Subsequently, and since there are currently no FDA-approved tests for this purpose, clinical microbiology laboratories have become responsible for developing protocols to detect C. auris using axial and inguinal screening swabs. In a College of American Pathologists-accredited large academic healthcare center setting, we implemented a laboratory-developed nucleic-acid amplification test for the detection of C. auris DNA. Our test validation evaluated the performance of the DiaSorin C. auris primer set used in a real-time qualitative PCR assay on the LIAISON MDX thermocycler with the Simplexa Universal Disc. The assay was highly sensitive and specific, with a limit of detection of 1-2 CFU/reaction, with no observed cross-reactivity with other Candida spp., bacterial skin commensal organisms or commonly encountered viruses. When run in parallel with a culture-based detection method, the PCR assay was 100% sensitive and specific. The assay was precise, with low variability between replicates within and between runs. Lastly, pre-analytical factors, including swab storage time, temperature, and transport media, were assessed and found to have no significant effect on the detection of C. auris at variable concentrations. Taken together, this study expands the available options for nucleic acid detection of C. auris and characterizes pre-analytical factors for implementation in both high- and low-volume laboratory settings. IMPORTANCE: This study overviews the validation and implementation of a molecular screening tool for the detection of Candida auris in a College of American Pathologist-accredited clinical laboratory. This molecular laboratory-developed test is both highly sensitive and specific and has significant health-system cost-savings associated with significantly reduced turn-around-time compared to traditional standard-of-care culture-based work up. This method and workflow is of interest to support clinical microbiology diagnostics and to help aid in hospital inpatient, and infection prevention control screening.

Virulence factors, antifungal susceptibility and molecular profile in Candida species isolated from the hands of health professionals before and after cleaning with 70% ethyl alcohol-based gel.

Fungal infections in neonatal intensive care units (NICU) are mainly related to Candida species, with high mortality rates. They are predominantly of endogenous origin, however, cross-infection transmitted by healthcare professionals' hands has occurred. The aim of this study was to identify Candida species isolated from the hands of healthcare professionals in a NICU before and after hygiene with 70% ethanol-based gel and evaluate virulence factors DNase, phospholipase, proteinase, hemolysin, biofilm biomass production, and metabolic activity. In vitro antifungal susceptibility testing and similarity by random amplified polymorphic DNA (RAPD) were also performed. C. parapsilosis complex was the most frequent species (57.1%); all isolates presented at least one virulence factor; three isolates (Candida parapsilosis complex) were resistant to amphotericin B, two (Candida famata [currently Debaryomyces hansenii] and Candida guilliermondii [currently Meyerozyma guilliermondii]) was resistant to micafungin, and six (Candida parapsilosis complex, Candida guilliermondii [=Meyerozyma guilliermondii], Candida viswanathi, Candida catenulata [currently Diutina catenulata] and Candida lusitaniae [currently Clavispora lusitaniae]) were resistant to fluconazole. Molecular analysis by RAPD revealed two clusters of identical strains that were in the hands of distinct professionals. Candida spp. were isolated even after hygiene with 70% ethanol-based gel, highlighting the importance of stricter basic measures for hospital infection control to prevent nosocomial transmission.

Biofilm-associated metabolism via ERG251 in Candida albicans.

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.

In silico genome wide identification of long non-coding RNAs differentially expressed during Candida auris host pathogenesis.

Candida auris is an invasive fungal pathogen of high concern due to acquired drug tolerance against antifungals used in clinics. The prolonged persistence on biotic and abiotic surfaces can result in onset of hospital outbreaks causing serious health threat. An in depth understanding of pathology of C. auris is highly desirable for development of efficient therapeutics. Non-coding RNAs play crucial role in fungal pathology. However, the information about ncRNAs is scanty to be utilized. Herein our aim is to identify long noncoding RNAs with potent role in pathobiology of C. auris. Thereby, we analyzed the transcriptomics data of C. auris infection in blood for identification of potential lncRNAs with regulatory role in determining invasion, survival or drug tolerance under infection conditions. Interestingly, we found 275 lncRNAs, out of which 253 matched with lncRNAs reported in Candidamine, corroborating for our accurate data analysis pipeline. Nevertheless, we obtained 23 novel lncRNAs not reported earlier. Three lncRNAs were found to be under expressed throughout the course of infection, in the transcriptomics data. 16 of potent lncRNAs were found to be coexpressed with coding genes, emphasizing for their functional role. Noteworthy, these ncRNAs are expressed from intergenic regions of the genes associated with transporters, metabolism, cell wall biogenesis. This study recommends for possible association between lncRNA expression and C. auris pathogenesis.

Exploring the dynamics of mixed-species biofilms involving Candida spp. and bacteria in cystic fibrosis.

Cystic fibrosis (CF) is an inherited disease that results from mutations in the gene responsible for the cystic fibrosis transmembrane conductance regulator (CFTR). The airways become clogged with thick, viscous mucus that traps microbes in respiratory tracts, facilitating colonization, inflammation and infection. CF is recognized as a biofilm-associated disease, it is commonly polymicrobial and can develop in biofilms. This review discusses Candida spp. and both Gram-positive and Gram-negative bacterial biofilms that affect the airways and cause pulmonary infections in the CF context, with a particular focus on mixed-species biofilms. In addition, the review explores the intricate interactions between fungal and bacterial species within these biofilms and elucidates the underlying molecular mechanisms that govern their dynamics. Moreover, the review addresses the multifaceted issue of antimicrobial resistance in the context of CF-associated biofilms. By synthesizing current knowledge and research findings, this review aims to provide insights into the pathogenesis of CF-related infections and identify potential therapeutic approaches to manage and combat these complex biofilm-mediated infections.

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm-1 attributed to carbon­deuterium bending and at 477 cm-1, attributed to the nitrogen­deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

The modern face of esophageal candidiasis in an oncology center: Correlating clinical manifestations, endoscopic grade, and pathological data in 323 contemporary cancer patients.

OBJECTIVES: Clinical presentation and outcomes of esophageal candidiasis (EC) in cancer patients are scarcely studied in the azole era, as is the correlation between clinical, endoscopic, and histopathological EC manifestations. METHODS: We retrospectively reviewed the risk factors, clinical features, and outcomes of pathology-documented EC cases at MD Anderson Cancer Center. We further assessed associations between presence of symptoms, standardized 4-stage endoscopic grade (Kodsi classification), histopathological data, and fluconazole treatment failure. RESULTS: Among 323 cancer patients with EC, 89% had solid tumors, most commonly esophageal cancer (29%). Thirty-three percent of EC patients were asymptomatic. The proportion of symptomatic EC patients significantly increased with endoscopic grade (P = 0.005). Among 202 patients receiving oral fluconazole, 27 (13%) had treatment failure. Underlying esophageal disease was the only independent predictor of fluconazole treatment failure (odds ratio: 3.88, P = 0.005). Endoscopic grade correlated significantly with Candida organism burden (Correlation coefficient [ρ] = 0.21, P < 0.01) and neutrophilic inflammation (ρ = 0.18, P < 0.01). Candida invasion of the squamous mucosal layer was associated with treatment failure (P = 0.049). CONCLUSIONS: EC was predominantly encountered in patients with solid tumors. One-third of EC patients were asymptomatic, challenging traditional symptom-based diagnosis. The development of integrated clinicopathological scoring systems could further guide the therapeutic management of cancer patients with EC.

Candida albicans accelerates atherosclerosis by activating intestinal hypoxia-inducible factor2α signaling.

The gut microbiota is closely linked to atherosclerosis. However, the role of intestinal fungi, essential members of the complex microbial community, in atherosclerosis is poorly understood. Herein, we show that gut fungi dysbiosis is implicated in patients with dyslipidemia, characterized by higher levels of Candida albicans (C. albicans), which are positively correlated with plasma total cholesterol and low-density lipoprotein-cholesterol (LDL-C) levels. Furthermore, C. albicans colonization aggravates atherosclerosis progression in a mouse model of the disease. Through gain- and loss-of-function studies, we show that an intestinal hypoxia-inducible factor 2α (HIF-2α)-ceramide pathway mediates the effect of C. albicans. Mechanistically, formyl-methionine, a metabolite of C. albicans, activates intestinal HIF-2α signaling, which drives increased ceramide synthesis to accelerate atherosclerosis. Administration of the HIF-2α selective antagonist PT2385 alleviates atherosclerosis in mice by reducing ceramide levels. Our findings identify a role for intestinal fungi in atherosclerosis progression and highlight the intestinal HIF-2α-ceramide pathway as a target for atherosclerosis treatment.

A correlative study of the genomic underpinning of virulence traits and drug tolerance of Candida auris.

Candida auris is an opportunistic fungal pathogen with high mortality rates which presents a clear threat to public health. The risk of C. auris infection is high because it can colonize the body, resist antifungal treatment, and evade the immune system. The genetic mechanisms for these traits are not well known. Identifying them could lead to new targets for new treatments. To this end, we present an analysis of the genetics and gene expression patterns of C. auris carbon metabolism, drug resistance, and macrophage interaction. We chose to study two C. auris isolates simultaneously, one drug sensitive (B11220 from Clade II) and one drug resistant (B11221 from Clade III). Comparing the genomes, we confirm the previously reported finding that B11220 was missing a 12.8 kb region on chromosome VI. This region contains a gene cluster encoding proteins related to alternative sugar utilization. We show that B11221, which has the gene cluster, readily assimilates and utilizes D-galactose and L-rhamnose as compared to B11220, which harbors the deletion. B11221 exhibits increased adherence and drug resistance compared to B11220 when grown in these sugars. Transcriptomic analysis of both isolates grown on glucose or galactose showed that the gene cluster was upregulated when grown on D-galactose. These findings reinforce growing evidence of a link between metabolism and drug tolerance. B11221 resists phagocytosis by macrophages and exhibits decreased ß-1,3-glucan exposure, a key determinant that allows Candida to evade the host immune system, as compared to B11220. In a transcriptomic analysis of both isolates co-cultured with macrophages, we find upregulation of genes associated with transport and transcription factors in B11221. Our studies show a positive correlation between membrane composition and immune evasion, alternate sugar utilization, and drug tolerance in C. auris.

Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

Genetic Mutations in FKS1 Gene Associated with Acquired Echinocandin Resistance in Candida parapsilosis Complex.

Candida parapsilosis complex has recently received special attention due to naturally occurring FKS1 polymorphism associated with high minimal inhibitory concentrations for echinocandin and the increase of clonal outbreaks of strains resistant to commonly used antifungals such as fluconazole. Despite the previous fact, little is known about the genetic mechanism associated with echinocandin resistance. Therefore, the present study was designed to investigate the mechanism of acquired echinocandin resistance in C. parapsilosis complex strains. A total of 15 clinical C. parapsilosis complex isolates were sub-cultured for 30 days at a low concentration of micafungin at ½ the lowest MIC value of the tested isolates (0.12 µg/ml). After culturing, all the isolates were checked phenotypically for antifungal resistance and genotypically for echinocandin resistance by checking FKS1 gene hot spot one (HS1) and HS2 mutations. In vitro induction of echinocandin resistance confirmed the rapid development of resistance at low concentration micafungin, with no difference among C. parapsilosis, C. metapsilosis, and C. orthopsilosis in the resistance development. For the first time we identified different FKS1 HS1 and or HS2 mutations responsible for echinocandin resistance such as R658S and L1376F in C. parapsilosis, S656X, R658X, R658T, W1370X, X1371I, V1371X, and R1373X (corresponding to their location in C. parapsilosis) in C. metapsilosis, and L648F and R1366H in C. orthopsilosis. Our results are of significant concern, since the rapid development of resistance may occur clinically after short-term exposure to antifungals as recently described in other fungal species with the potential of untreatable infections.