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.

Protective effects of microbial biosurfactants produced by Bacillus halotolerans and Candida parapsilosis on bleomycin-induced pulmonary fibrosis in mice: Impact of antioxidant, anti-inflammatory and anti-fibrotic properties via TGF-ß1/Smad-3 pathway and miRNA-326.

Idiopathic pulmonary fibrosis (IPF) is an irreversible disease which considered the most fatal pulmonary fibrosis. Pulmonary toxicity including IPF is the most severe adverse effect of bleomycin, the chemotherapeutic agent. Based on the fact that, exogenous surfactants could induce alveolar stabilization in many lung diseases, the aim of this study was to explore the effects of low cost biosurfactants, surfactin (SUR) and sophorolipids (SLs), against bleomycin-induced pulmonary fibrosis in mice due to their antioxidant, and anti-inflammatory properties. Surfactin and sophorolipids were produced by microbial conversion of frying oil and potato peel wastes using Bacillus halotolerans and Candida parapsilosis respectively. These biosurfactants were identified by FTIR, 1H NMR, and LC-MS/MS spectra. C57BL/6 mice were administered the produced biosurfactants daily at oral dose of 200 mg kg-1 one day after the first bleomycin dose (35 U/kg). We evaluated four study groups: Control, Bleomycin, Bleomycin+SUR, Bleomycin+SLs. After 30 days, lungs from each mouse were sampled for oxidative stress, ELISA, Western blot, histopathological, immunohistochemical analyses. Our results showed that the produced SUR and SLs reduced pulmonary oxidative stress and inflammatory response in the lungs of bleomycin induced mice as they suppressed SOD, CAT, and GST activities also reduced NF-κß, TNF-α, and CD68 levels. Furthermore, biosurfactants suppressed the expression of TGF-ß1, Smad-3, and p-JNK fibrotic signaling pathway in pulmonary tissues. Histologically, SUR and SLs protected against lung ECM deposition caused by bleomycin administration. Biosurfactants produced from microbial sources can inhibit the induced inflammatory and fibrotic responses in bleomycin-induced pulmonary fibrosis.

Structure-guided engineered urethanase from Candida parapsilosis with pH and ethanol tolerance to efficiently degrade ethyl carbamate in Chinese rice wine.

Urethane hydrolase can degrade the carcinogen ethyl carbamate (EC) in fermented food, but its stability and activity limit its application. In this study, a mutant G246A and a double mutant N194V/G246A with improved cpUH activity and stability of Candida parapsilosis were obtained by site-directed mutagenesis. The catalytic efficiency (Kcat/Km) of mutant G246A and double mutant N194V/G246A are 1.95 times and 1.88 times higher than that of WT, respectively. In addition, compared with WT, the thermal stability and pH stability of mutant G246A and double mutant N194V/G246A were enhanced. The ability of mutant G246A and double mutant N194V/G246A to degrade EC in rice wine was also stronger than that of WT. The mutation increased the stability of the enzyme, as evidenced by decreased root mean square deviation (RMSD) and increased hydrogen bonds between the enzyme and substrate by molecular dynamics simulation and molecular docking analysis. The molecule modification of new cpUH promotes the industrial process of EC degradation.

Taxonomy of Candida parapsilosis complex isolated from neonates and the role of Hsp90 inhibitors to enhanced the antifungal activity of micafungin.

Species from Candida parapsilosis complex are frequently found in neonatal candidemia. The antifungal agents to treat this infection are limited and the occurrence of low in vitro susceptibility to echinocandins such as micafungin has been observed. In this context, the chaperone Hsp90 could be a target to reduce resistance. Thus, the objective of this research was to identify isolates from the C. parapsilosis complex and verify the action of Hsp90 inhibitors associated with micafungin. The fungal identification was based on genetic sequencing and mass spectrometry. Minimal inhibitory concentrations were determined by broth microdilution method according to Clinical Laboratory and Standards Institute. The evaluation of the interaction between micafungin with Hsp90 inhibitors was realized using the checkerboard methodology. According to the polyphasic taxonomy, C. parapsilosis sensu stricto was the most frequently identified, followed by C. orthopsilosis and C. metapsilosis, and one isolate of Lodderomyces elongisporus was identified by genetic sequencing. The Hsp90 inhibitor geladanamycin associated with micafungin showed a synergic effect in 31.25% of the isolates, a better result was observed with radicicol, which shows synergic effect in 56.25% tested yeasts. The results obtained demonstrate that blocking Hsp90 could be effective to reduce antifungal resistance to echinocandins.

Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

BACKGROUND: Fluconazole-resistant Candida parapsilosis is a matter of concern. OBJECTIVES: To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. PATIENTS/METHODS: We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. RESULTS: Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. CONCLUSIONS: We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

Risk factors associated with fluconazole resistance in Candida parapsilosis candidemia: A case-case study.

BACKGROUND: Candida species are among the most important invasive pathogens in intensive care units (ICUs). Non-albicans species including Candida parapsilosis (C. parapsilosis) has increased in recent years. Fluconazole is the leading antifungal agent but resistance is a concern among C. parapsilosis species. OBJECTIVES: The aim of this study was to determine the factors associated with fluconazole resistance in patients with candidemia due to C. parapsilosis in ICUs. METHODS: This case-case study was conducted in a 750-bed, tertiary hospital between 2015 and 2021. Patients with fluconazole-resistant C. parapsilosis candidemia constituted the 'cases of interest' group and patients with fluconazole-susceptible C. parapsilosis candidemia constituted the 'comparison cases' group. Demographic and clinical data of the patients were recorded. Logistic regression analysis was performed using the backward elimination method to determine the independent predictors of fluconazole-resistant C. parapsilosis bloodstream infections. RESULTS: The study included 177 patients. In the cultures of these patients, 76 (43%) fluconazole-resistant, 13 (7.3%) fluconazole-reduced susceptible, and 88 (49.7%) fluconazole-susceptible isolates were found. In the regression analysis the risk factors for fluconazole-resistant C. parapsilosis bloodstream infection, malignancy, immunosuppressive treatment, history of intra-abdominal surgery, hypoalbunemia, previous fluconazole use, and SOFA score were found to be associated in univariate analysis. In multivariate regression analysis, history of intra-abdominal surgery (OR: 2.16; 95% CI: 1.05-4.44), hypoalbuminemia (OR: 2.56; 95% CI: 1.06-6.17) and previous fluconazole use (OR: 3.35; 95% CI: 1.02-11) were found to be independent predictors. CONCLUSIONS: In this study, a significant correlation was found between candidemia due to fluconazole-resistant C. parapsilosis in ICUs and intra-abdominal surgery, hypoalbuminemia, and previous fluconazole use. C. parapsilosis isolates and fluconazole resistance should be continuously monitored, strict infection control measures should be taken and antifungal stewardship programs should be implemented.

Antifungal activity of Fe3O4@SiO2/Schiff-base/Cu(II) magnetic nanoparticles against pathogenic Candida species.

The antifungal efficacy and cytotoxicity of a novel nano-antifungal agent, the Fe3O4@SiO2/Schiff-base complex of Cu(II) magnetic nanoparticles (MNPs), have been assessed for targeting drug-resistant Candida species. Due to the rising issue of fungal infections, especially candidiasis, and resistance to traditional antifungals, there is an urgent need for new therapeutic strategies. Utilizing Schiff-base ligands known for their broad-spectrum antimicrobial activity, the Fe3O4@SiO2/Schiff-base/Cu(II) MNPs have been synthesized. The Fe3O4@SiO2/Schiff-base/Cu(II) MNPs was characterized by Fourier Transform-Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Energy-dispersive X-ray (EDX), Vibrating Sample Magnetometer (VSM), and Thermogravimetric analysis (TGA), demonstrating successful synthesis. The antifungal potential was evaluated against six Candida species (C. dubliniensis, C. krusei, C. tropicalis, C. parapsilosis, C. glabrata, and C. albicans) using the broth microdilution method. The results indicated strong antifungal activity in the range of 8-64 µg/mL with the lowest MIC (8 µg/mL) observed against C. parapsilosis. The result showed the MIC of 32 µg/mL against C. albicans as the most common infection source. The antifungal mechanism is likely due to the disruption of the fungal cell wall and membrane, along with increased reactive oxygen species (ROS) generation leading to cell death. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for cytotoxicity on mouse L929 fibroblastic cells suggested low toxicity and even enhanced cell proliferation at certain concentrations. This study demonstrates the promise of Fe3O4@SiO2/Schiff-base/Cu(II) MNPs as a potent antifungal agent with potential applications in the treatment of life-threatening fungal infections, healthcare-associated infections, and beyond.

COVID-19 associated candidemia: From a shift in fungal epidemiology to a rise in azole drug resistance.

Our understanding of fungal epidemiology and the burden of antifungal drug resistance in COVID-19-associated candidemia (CAC) patients is limited. Therefore, we conducted a retrospective multicenter study in Iran to explore clinical and microbiological profiles of CAC patients. Yeast isolated from blood, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method M27-A3 protocol. A total of 0.6% of the COVID-19 patients acquired CAC (43/6174). Fluconazole was the most widely used antifungal, and 37% of patients were not treated. Contrary to historic candidemia patients, Candida albicans and C. tropicalis were the most common species. In vitro resistance was high and only noted for azoles; 50%, 20%, and 13.6% of patients were infected with azole-non-susceptible (ANS) C. tropicalis, C. parapsilosis, and C. albicans isolates, respectively. ERG11 mutations conferring azole resistance were detected for C. parapsilosis isolates (Y132F), recovered from an azole-naïve patient. Our study revealed an unprecedented rise in ANS Candida isolates, including the first C. parapsilosis isolate carrying Y132F, among CAC patients in Iran, which potentially threatens the efficacy of fluconazole, the most widely used drug in our centers. Considering the high mortality rate and 37% of untreated CAC cases, our study underscores the importance of infection control strategies and antifungal stewardship to minimize the emergence of ANS Candida isolates during COVID-19.

Isolated Cutaneous Granuloma Caused by Candida Parapsilosis: Case Report and Literature Review.

Candidal granuloma is an uncommon type of deep chronic cutaneous candidiasis. Candida albican is the most common causative pathogen for candidal granuloma. We report herein the original case of a 69-year-old Chinese woman presented with a 3-year of painful cutaneous lesion on the back of left hand. Physical examination revealed a 4 × 5 cm large infiltrative reddish plaque with unclear boundaries. The yellow-white crusts were observed on the uneven surface of plaque. Histopathological examination of biopsy tissue revealed that yeast cells and the horizontal section of hyphae in the dermis by hematoxylin eosin staining and periodic acid-Schiff staining. Finally, the pathogen was identified as Candida parapsilosis by mycological examination and molecular identification. The patient was treated with itraconazole oral 200 mg twice daily combined with topical terbinafine hydrochloride cream for 2 months. The lesions were fully resolved and no recurrence was observed. Since the cutaneous infection caused by C. parasilosis were rarely reported, we also reviewed all 11 cases of cutaneous infection caused by C. parapsilosis in the PubMed. Our study highlighted that chronic unilateral infiltrated plaques or ulcers should be aware of the occurrence of fungal granuloma including candidal granuloma especially in immunocompromised patients.

Evaluation of outbreak persistence caused by multidrug-resistant and echinocandin-resistant Candida parapsilosis using multidimensional experimental and epidemiological approaches.

Candida parapsilosis is known to cause severe and persistent outbreaks in clinical settings. Patients infected with multidrug-resistant C. parapsilosis (MDR Cp) isolates were identified in a large Turkish hospital from 2017-2020. We subsequently identified three additional patients infected with MDR Cp isolates in 2022 from the same hospital and two echinocandin-resistant (ECR) isolates from a single patient in another hospital. The increasing number of MDR and ECR isolates contradicts the general principle that the severe fitness cost associated with these phenotypes could prevent their dominance in clinical settings. Here, we employed a multidimensional approach to systematically assess the fitness costs of MDR and ECR C. parapsilosis isolates. Whole-genome sequencing revealed a novel MDR genotype infecting two patients in 2022. Despite severe in vitro defects, the levels and tolerances of the biofilms of our ECR and MDR isolates were generally comparable to those of susceptible wild-type isolates. Surprisingly, the MDR and ECR isolates showed major alterations in their cell wall components, and some of the MDR isolates consistently displayed increased tolerance to the fungicidal activities of primary human neutrophils and were more immunoevasive during exposure to primary human macrophages. Our systemic infection mouse model showed that MDR and ECR C. parapsilosis isolates had comparable fungal burden in most organs relative to susceptible isolates. Overall, we observed a notable increase in the genotypic diversity and frequency of MDR isolates and identified MDR and ECR isolates potentially capable of causing persistent outbreaks in the future.

In vitro interactions of proton pump inhibitors and azoles against pathogenic fungi.

Introduction: Azole resistance has been increasingly reported and become an issue for clinical managements of invasive mycoses. New strategy with combination therapy arises as a valuable and promising alternative option. The aim of the present study is to investigate the in vitro combinational effect of proton pump inhibitors (PPIs) and azoles against pathogenic fungi. Methods: In vitro interactions of PPIs including omeprazole (OME), lansoprazole (LAN), pantoprazole (PAN), and rabeprazole (RAB), and commonly used azoles including itraconazole (ITC), posaconazole (POS), voriconazole (VRC) and fluconazole (FLC), were investigated via broth microdilution chequerboard procedure adapted from the CLSI M27-A3 and M38-A2. A total of 67 clinically isolated strains, namely 27 strains of Aspergillus spp., 16 strains of Candida spp., and 24 strains of dematiaceous fungi, were studied. C. parapsilosis (ATCC 22019) and A. flavus (ATCC 204304) was included to ensure quality control. Results: PPIs individually did not exert any significant antifungal activity. The combination of OME with ITC, POS, or VRC showed synergism against 77.6%, 86.6%, and 4% strains of tested pathogenic fungi, respectively, while synergism of OME/FLC was observed in 50% strains of Candida spp. Synergism between PAN and ITC, POS, or VRC was observed against 47.8%, 77.6% and 1.5% strains of tested fungi, respectively, while synergism of PNA/FLC was observed in 50% strains of Candida spp. Synergism of LAN with ITC, POS, or VRC was observed against 86.6%, 86.6%, and 3% of tested strains, respectively, while synergism of LAN/FLC was observed in 31.3% strains of Candida spp. Synergy of the combination of RAB with ITC, POS, or VRC was observed against 25.4%, 64.2%, and 4.5% of tested strains, respectively, while synergism of RAB/FLC was observed in 12.5% of Candida spp.. Among PPIs, synergism was least observed between RAB and triazoles, while among triazoles, synergism was least observed between VRC and PPIs. Among species, synergy was much more frequently observed in Aspergillus spp. and dematiaceous fungi as compared to Candida spp. Antagonism between PPIs with ITC or VRC was occasionally observed in Aspergillus spp. and dematiaceous fungi. It is notable that PPIs combined with azoles showed synergy against azole resistant A. fumigatus, and resulted in category change of susceptibility of ITC and POS against Candida spp. Discussion: The results suggested that PPIs combined with azoles has the potential to enhance the susceptibilities of azoles against multiple pathogenic fungi and could be a promising strategy to overcome azole resistance issues. However, further investigations are warranted to study the combinational efficacy in more isolates and more species, to investigate the underlying mechanism of interaction and to evaluate the potential for concomitant use of these agents in human.

In Vivo Confocal Microscopy Characterization of Candida parapsilosis Keratitis.

ABSTRACT: The present clinical case concerns two patients with mycotic keratitis because of Candida parapsilosis in which corneal confocal microscopy presented a characteristic feature of this pathogen. Both described patients used a therapeutic contact lens and administered a therapy with steroid eye drops which are well known predisposing factors for the onset of corneal mycoses. This report can be useful for correctly identifying the pathologic condition and quickly directing the therapy.

Recent gene selection and drug resistance underscore clinical adaptation across Candida species.

Understanding how microbial pathogens adapt to treatments, humans and clinical environments is key to infer mechanisms of virulence, transmission and drug resistance. This may help improve therapies and diagnostics for infections with a poor prognosis, such as those caused by fungal pathogens, including Candida. Here we analysed genomic variants across approximately 2,000 isolates from six Candida species (C. glabrata, C. auris, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis) and identified genes under recent selection, suggesting a highly complex clinical adaptation. These involve species-specific and convergently affected adaptive mechanisms, such as adhesion. Using convergence-based genome-wide association studies we identified known drivers of drug resistance alongside potentially novel players. Finally, our analyses reveal an important role of structural variants and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Our results provide insights on how opportunistic pathogens adapt to human-related environments and unearth candidate genes that deserve future attention.

Breaking barriers in Candida spp. detection with Electronic Noses and artificial intelligence.

The timely and accurate diagnosis of candidemia, a severe bloodstream infection caused by Candida spp., remains challenging in clinical practice. Blood culture, the current gold standard technique, suffers from lengthy turnaround times and limited sensitivity. To address these limitations, we propose a novel approach utilizing an Electronic Nose (E-nose) combined with Time Series-based classification techniques to analyze and identify Candida spp. rapidly, using culture species of C. albicans, C.kodamaea ohmeri, C. glabrara, C. haemulonii, C. parapsilosis and C. krusei as control samples. This innovative method not only enhances diagnostic accuracy and reduces decision time for healthcare professionals in selecting appropriate treatments but also offers the potential for expanded usage and cost reduction due to the E-nose's low production costs. Our proof-of-concept experimental results, carried out with culture samples, demonstrate promising outcomes, with the Inception Time classifier achieving an impressive average accuracy of 97.46% during the test phase. This paper presents a groundbreaking advancement in the field, empowering medical practitioners with an efficient and reliable tool for early and precise identification of candidemia, ultimately leading to improved patient outcomes.

Purification and characterization of crude fructooligosaccharides extracted from red onion (Allium cepa var. viviparum) by yeast treatment.

BACKGROUND: Yeast treatment has been used for purification of fructooligosaccharides (FOSs). However, the main drawback of this approach is that yeast can only partially remove sucrose from crude FOSs. The main objective of this research was to screen yeast strains for the capability of selectively consuming unwanted sugars, namely fructose, glucose, and sucrose, in crude FOSs extracted from red onion (Allium cepa var. viviparum) with minimal effect on FOS content. RESULTS: Among 43 yeast species isolated from Miang, ethnic fermented tea leaves, and Assam tea flowers, Candida orthopsilosis FLA44.2 and Priceomyces melissophilus FLA44.8 exhibited the greatest potential to specifically consume these unwanted sugars. In a shake flask, direct cultivation of C. orthopsilosis FLA44.2 was achieved in the original crude FOSs containing an initial FOSs concentration of 88.3 ± 1.2 g/L and 52.9 ± 1.2 g/L of the total contents of fructose, glucose, and sucrose. This was successful with 93.7% purity and 97.8% recovery after 24 h of cultivation. On the other hand, P. melissophilus FLA48 was limited by initial carbohydrate concentration of crude FOSs in terms of growth and sugar utilization. However, it could directly purify two-fold diluted crude FOSs to 95.2% purity with 92.2% recovery after 72 h of cultivation. Purification of crude FOSs in 1-L fermenter gave similar results to the samples purified in a shake flask. Extracellular ß-fructosidase was assumed to play a key role in the effective removal of sucrose. Both Candida orthopsilosis FLA44.2 and P. melissophilus FLA44.8 showed γ-hemolytic activity, while their culture broth had no cytotoxic effect on viability of small intestinal epithelial cells, preliminarily indicating their safety for food processing. The culture broth obtained from yeast treatment was passed through an activated charcoal column for decolorization and deodorization. After being freeze dried, the final purified FOSs appeared as a white granular powder similar to refined sugar and was odorless since the main sulfur-containing volatile compounds, including dimethyl disulfide and dipropyl trisulfide, were almost completely removed. CONCLUSION: The present purification process is considered simple and straight forward, and provides new and beneficial insight into utilization of alternative yeast species for purification of FOSs.

[Investigation of the Effect of Farnesol on Biofilm Formation by Candida albicans and Candida parapsilosis Complex Isolates]. / Candida albicans ve Candida parapsilosis Kompleks Izolatlarinin Biyofilm Üretimi Üzerine Farnesol Etkisinin Arastirilmasi.

The incidence of infections caused by Candida species has significantly increased over the past three decades. Candida albicans is commonly recognized as the primary causative agent in cases of candidiasis; however, non-albicans Candida species, including Candida parapsilosis, are also frequently defined as pathogens. Treatment-resistant infections arise as a result of biofilm formation, which is one of the effective mechanisms in the pathogenesis of Candida infections. However, the mechanisms of action of farnesol, a quorum sensing (QS) system molecule, on biofilm formation by Candida species remain unclear. This study aimed to demonstrate the changes in the biofilm biomass of C.albicans and C.parapsilosis complex isolates in the presence of farnesol and reveal the expression of the EFG1 and BCR1 genes, which are believed to play a role in the production of QS molecules, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis. C.albicans (n= 91) and C.parapsilosis complex (n= 29) isolates obtained from different clinical samples were included in the study. The minimum inhibitory concentration (MIC) values of farnesol were determined using the broth microdilution method according to the M27-A3 protocol of the Clinical and Laboratory Standards Institute (CLSI). The biofilm biomass of the isolates was examined without farnesol and at the MIC-0 and MIC-2 concentrations of farnesol. Changes in the expression of the biofilm-associated EFG1 and BCR1 genes were investigated using qRT-PCR. According to the results of the study, the MIC values of farnesol were detected in the range of 1-2 mM in 82.4% (n= 75) of the C.albicans isolates and in the range of 0.5-1 mM in 72.4% (n= 21) of the C.parapsilosis complex isolates. Of the C.albicans isolates, 27 (29.7%) exhibited a strong biofilm formation and 58 (63.7%) demonstrated a weaker biofilm formation, while these rates were 34.4% (n= 10) and 62.1% (n= 18), respectively, for the C.parapsilosis complex isolates. At the MIC-0 and MIC-2 concentrations, farnesol was observed to reduce biofilm biomass among C.albicans (n= 24, 88.9%) and C.parapsilosis complex (n= 8, 80.0%) isolates that formed strong biofilms and observed to increase biofilm biomass among those that formed weak biofilms [60.3% (n= 35) and 55.6% (n= 10), respectively]. On completion of the qRT-PCR analysis supporting the results of the biofilm experiment, it was determined that the expressions of the EFG1 and BCR1 genes decreased at the MIC-0 and MIC-2 concentrations of farnesol among the strong biofilm-forming C.albicans and C.parapsilosis complex isolates, but there was an increase in gene expressions among the weak biofilm-forming isolates. In addition to the antifungal effect of farnesol on Candida species, this study provided data on the efficacy of the MIC-0 and MIC-2 concentrations of farnesol against Candida biofilm biomass. Although our results suggest that farnesol can be used as an alternative agent to reduce biofilm formation in Candida infections, they need to be supported by further studies. Moreover, this research has significance as it represents the first study to determine the EFG1 and BCR1 gene expressions among C.parapsilosis complex isolates in the presence of farnesol.

Clonal spread of fluconazole-resistant C. parapsilosis in patients admitted to a referral hospital located in Burgos, Spain, during the COVID-19 pandemic.

BACKGROUND: Fluconazole-resistant Candida parapsilosis (FRCP) is a matter of concern in Spain. OBJECTIVES: We here report a FRCP spread across a 777-bed referral hospital located in Burgos, Spain, during the COVID-19 pandemic. PATIENTS/METHODS: In April 2021, an FRCP isolate (MIC = 64 mg/L, E-test®) from a hospitalised patient was detected. Up to June 2022, all C. parapsilosis isolates (n = 35) from hospitalised patients (n = 32) were stored and genotyped using microsatellite markers, and their antifungal susceptibilities were studied (EUCAST); FRCP isolates were molecularly characterised. RESULTS: We detected 26 FRCP isolates collected between 2021 (n = 8) and 2022 (n = 18); isolates were susceptible to amphotericin B, echinocandins and ibrexafungerp. FRCP isolates were grouped into three genotypes: CP-707 and CP-708 involved isolates harbouring the Y132F + R398I ERG11p substitutions (n = 24) and were clonally related; the remaining CP-675 genotype involved isolates harbouring the G458S ERG11p substitution (n = 2). FRCP genotypes were genetically related to the FRCP genotypes found in Madrid and were unrelated to fluconazole-susceptible ones. Patients harbouring FRCP were mainly (n = 22/23) admitted to intensive care units. Most patients had received broad-spectrum antibiotics (n = 22/23), and/or antifungal therapy with azoles (n = 14/23) within the 30 days prior to FRCP isolation. Thirteen patients were colonised, 10 of whom were infected and presented candidaemia (n = 8/10), endovascular infection (n = 1/10) or complicated urinary infection (n = 1/10). Overall nonattributable 30-day mortality was 17% (n = 4/23). CONCLUSIONS: We report an outbreak caused by FRCP affecting patients admitted to the ICU of a referral hospital located in Burgos. Patients harbouring FRCP had a higher fluconazole use than those carrying susceptible isolates.

Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis.

Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned, and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and ß-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.

Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

OBJECTIVES: We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. METHODS: Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. RESULTS: Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1-29 months. Clusters harbouring Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2-8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. CONCLUSIONS: This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.