Glabrata Isolates Research Articles

Molecular Identification of Candida Species Isolated from Onychomycosis with In Vitro Antifungal Susceptibility Profiles

Background: Onychomycosis is one of the most common fungal infections. The most common etiological agents of onychomycosis in Iran are Candida species, especially fingernails. It is more common in women than men, particularly workers in occupations requiring them to submerge their hands or feet in water for prolonged periods. Objectives: The current study's main aim was to determine the abundance of candidal onychomycosis, identify the Candida species using molecular methods, and evaluate the in vitro antifungal susceptibility profiles. Methods: One hundred forty samples were obtained from patients suspected of onychomycosis, and 51 (36.4%) Candida strains were identified by PCR-RFLP. The in vitro susceptibility of four triazole (FLC, ITC, VRC, and POS) antifungal drug testing of 51 Candida species was performed using broth microdilution. Results: Direct microscopic examination by KOH 20% of 140 nail samples showed that 51 (36.4%) samples were positive in terms of fungal elements, with Candida parapsilosis complex being the most frequently isolated of patients, followed by C. albicans complex, C. glabrata, C. krusei, C. tropicalis, C. guilliermondii, C. famata, C. kefyr, and Candida species. All Candida species showed they were susceptible to four triazoles, except that five C. krusei were resistant to fluconazole. Only one C. glabrata isolates and one C. parapsilosis isolate were resistant to fluconazole. Conclusions: The growing trend towards the frequency of fingernail onychomycosis in housewives has been noticeable in the last decades in Iran. Therefore, accurate identification of Candida species and perform in vitro antifungal susceptibility testing can aid physicians in choosing an effective potential drug for treating onychomycosis patients.

Inter-laboratory variability of caspofungin MICs for Nakaseomyces glabrata isolates – an Irish tertiary hospital experience

Background. Nakaseomyces glabrata, formerly Candida glabrata, is an opportunistic yeast and emerging cause of human infections. The use of broth microdilution (BMD) methodologies for caspofungin (CSP) antifungal susceptibility testing (AFST) against N. glabrata is reported to be prone to high inter-laboratory variation. We aimed to compare CSP MICs of N. glabrata isolates from our institution with those obtained by the Reference Laboratory for the same isolates. Methods. All clinically significant N. glabrata isolates from 2019 to 2021 inclusive were reviewed. AFST was performed locally using the VITEK2 system with the AST-YS08 card, while E-tests were performed at the Mycology Reference Laboratory (MRL), and agreement between these two methods was evaluated – categorical and essential. Results. Forty-one isolates were reviewed during the study period – 30 from blood cultures, seven from intra-operative theatre specimens and four from sterile site drain fluids. Despite an essential agreement of 100 % within ±2 log2 dilutions, marked discrepancies were noted in interpretative breakpoints between assays with 17 Minor and 16 Major category errors. Categorical agreement was 19.5 %, with the VITEK2 over-estimating resistance. A Mann–Whitney U-test assessed the relationship of MICs across the AFST modalities, and a statistically significant difference was noted, P<0.01, with a higher mean rank for VITKEK2 outputs. Conclusion. While the VITEK2 system is highly applicable, its performance for CSP AFST is unreliable and potentially results in the mis-classification of susceptible isolates as highlighted in our study. The use of VITEK2 AST-YS08 micafungin as a sentinel echinocandin should be explored and/or the evaluation of CSP-specific E-tests as utilized by the MRL. These methods appear more consistent and less prone to the variation seen with BMD for CSP.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Molecular fingerprinting by multi-locus sequence typing identifies microevolution and nosocomial transmission of Candida glabrata in Kuwait.

Candida glabrata is a frequently isolated non-albicans Candida species and invasive C. glabrata infections in older patients are associated with high mortality rates. Opportunistic Candida infections in critically ill patients may be either endogenous or nosocomial in origin and this distinction is critical for effective intervention strategies. This study performed multi-locus sequence typing (MLST) to study genotypic relatedness among clinical C. glabrata isolates in Kuwait. Candida glabrata isolates (n = 91) cultured from 91 patients were analyzed by MLST. Repeat isolates (n = 16) from 9 patients were also used. Antifungal susceptibility testing for fluconazole, voriconazole, caspofungin and amphotericin B (AMB) was determined by Etest. Genetic relatedness was determined by constructing phylogenetic tree and minimum spanning tree by using BioNumerics software. Resistance to fluconazole, voriconazole and AMB was detected in 7, 2 and 10 C. glabrata isolates, respectively. MLST identified 28 sequence types (STs), including 12 new STs. ST46 (n = 33), ST3 (n = 8), ST7 (n = 6) and ST55 (n = 6) were prevalent in ≥4 hospitals. Repeat isolates obtained from same or different site yielded identical ST. No association of ST46 with source of isolation or resistance to antifungals was apparent. Microevolution and cross-transmission of infection was indicated in two hospitals that yielded majority (57 of 91, 67%) of C. glabrata. Our data suggest that C. glabrata undergoes microevolution in hospital environment and can be nosocomially transmitted to other susceptible patients. Thus, proper infection control practices during routine procedures on C. glabrata-infected patients may prevent transmission of this pathogen to other hospitalized patients.

Antifungal Susceptibility Pattern of Candida glabrata from a Referral Center and Reference Laboratory: 2012-2022.

The prevalence of invasive candidiasis caused by non-Candida albicans has rapidly increased. Candida glabrata (Nakaseomyces glabrata) is an important pathogen associated with substantial mortality. Our study examined the antifungal temporal susceptibility of C. glabrata and cross-resistance/non-wild-type patterns with other azoles and echinocandins. Laboratory data of all adult patients with C. glabrata isolated from clinical specimens at the Mayo Clinic, Rochester, from 2012 to 2022 were collected. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used. We obtained 1046 C. glabrata isolates from 877 patients. Using CLSI and EUCAST breakpoints, 187 (17.9%) isolates and 256 (24.5%) isolates were fluconazole-resistant, respectively. Focusing on C. glabrata bloodstream infections, fluconazole-resistance ranged from 16 to 22%. Among those 187 fluconazole-resistant isolates, 187 (100%) and 184 (98.4%) isolates were also voriconazole and posaconazole non-wild-type, respectively, with 97 (51.9%) isolates deemed non-wild type for itraconazole. The fluconazole susceptibility pattern has not changed over the past decade. The proportion of fluconazole-resistant C. glabrata is relatively high, which could be due to the complexity of patients and fluconazole exposure. Itraconazole appears to be a compelling step-down therapy for fluconazole-resistant C. glabrata, given the high proportion of wild-type isolates. Further research to examine clinical outcomes is warranted.

P25 Healthcare associated candiduria in ICU patients: experience from a surveillance network in India, 2017–22

Abstract Background Urinary tract infections (UTI) are common healthcare associated infections (HAI) in low-and-middle-income countries including India, with limited information on Candida UTI. An ICU-based HAI surveillance network of tertiary care hospitals across India, coordinated by the All-India Institute of Medical Sciences, New Delhi and the Indian Council of Medical Research, modified the US CDC’s National Healthcare Safety Network (NHSN) case definitions to record UTI. Unlike NHSN, India surveillance includes fungal pathogens, such as Candida spp., as eligible pathogens causing HAI-UTI. We describe the epidemiology, microbiology and antimicrobial resistance (AMR) profile of candiduria cases reported to this network to address the knowledge gap on Candida UTI in India. Methods Patients with at least one UTI symptom and a positive urine culture with Candida sp. who had spent at least 2 calendar days in a participating ICU were included as candiduria events. All candiduria events reported by participating sites from 1 November 2017 to 31 May 2022 were extracted from overall UTI reported for all pathogens. The distribution of events by age, gender, ICU type, urinary catheterization, pathogen and drug resistance profile were performed. UTI rate and device utilization ratio (DUR) were calculated. Network sites performed antifungal susceptibility testing (AST) on Candida isolates using MIC-based method as per CLSI guidelines. Results Data was reported by 131 ICUs in 39 hospitals. Overall network UTI events including all pathogens were 3600; UTI rates were 2.6 (adult ICUs) and 1.3 (paediatric ICUs); DUR was 0.8 (adult ICUs) and 0.4 (paediatric ICUs). There were 1111 candiduria events, 95% were classified as catheter-associated UTI (CAUTI), and the median age of patients was 65 years (IQR 32–74 years) and 58% were male. Among 739 Candida isolates speciated, Candida tropicalis (37%), Candida albicans (36%), Candida glabrata (6%) and Candida auris (6%) were the top four species isolated. Among 220 C. tropicalis isolates tested with AST, 6.9% were fluconazole resistant and 2% of 140 were caspofungin resistant; 7% of 177 C. albicans isolates were fluconazole resistant and 6.5% of 92 isolates were caspofungin resistant; and 48% of 28 C. glabrata isolates were fluconazole resistant and 44% of 18 isolates were caspofungin resistant. Conclusions Candiduria events reported from ICUs to the HAI surveillance network-India are common and the majority are classified as CAUTI. Most of the species isolated are susceptible to fluconazole and caspofungin. Additional work is needed to understand the clinical significance of candiduria events reported to the network.

Evaluation of the CHROMagar Candida Plus medium for presumptive identification of yeasts and MALDI-TOF MS identification.

Rapid and accurate yeasts species identification in clinical laboratories is important for appropriate and timely antifungal treatment. We evaluate the performance of the new medium CHROMagar™ Candida Plus for presumptive identification of yeasts species and MALDI-TOF identification. We identify 303 strains belonging to 60 clinically relevant yeasts species by using the new medium. Presumptive identification was correct at the Candida albicans complex, Candida tropicalis and Pichia kudriavzevii (Candida krusei) species. However, although this medium was able to identify all Candida auris and Candida glabrata strains, other species were misidentified as C. auris or C. glabrata. A total of 215 strains were identified by using MALDI-TOF and evaluated two incubation temperatures (30°C and 37°C) and two incubation times (24 h and 72 h). Most strains (94%; 202/215) were correctly identified at the species (n:190) or complex level (n:12) at both temperatures and incubation times. However, we observed that the time of incubation (24 h vs. 72 h) affects the score values when yeasts are incubated at 37°C, but does not affect score values when yeasts are incubated at 30°C. In conclusion, the new medium has a good performance in the presumptive identification of the C. albicans complex, C. tropicalis and P. kudriavzevii (C. krusei). In addition, this medium is useful for the screening of C. auris and C. glabrata isolates, but identification should be confirmed by other more specific techniques, like MALDI-TOF.

In vitro combination of antifungal drugs with tacrolimus (FK506) holds promise against clinical Candida species including Candida auris.

In vitro interactions between tacrolimus, a calcineurin inhibitor, and fluconazole, itraconazole, caspofungin, or anidulafungin were evaluated against Candida auris, C. albicans, C. parapsilosis, and C. glabrata (each five strains). Tacrolimus-itraconazole, tacrolimus-caspofungin, and tacrolimus-fluconazole combinations resulted in synergistic interactions against 95%, 90%, and 60% of Candida isolates, respectively. Contradictory, tacrolimus-anidulafungin resulted in only a 35% synergistic effect. A combination of tacrolimus and itraconazole was most potent with synergy against 100% of C. auris, C. parapsilosis, and C. glabrata isolates. Of note, no antagonistic interaction was found.

Molecular mechanisms of azole resistance in Candida glabrata isolated from oropharyngeal candidiasis in head and neck cancer patients

ObjectiveThe aim of the current work was to assess the molecular mechanisms of fluconazole-resistant Candida glabrata strains isolated from oropharyngeal candidiasis (OPC) in head and neck patients, as well as evaluation of virulence factors. DesignAntifungal susceptibility pattern of sixty six clinical isolates of C. glabrata were evaluated by broth-microdilution method. The expression of ERG11, CDR1, CDR2, PDR1 genes as well as ERG11 gene capable of possible mutations was also detected in 21 fluconazol-resistant C. glabrata isolates. Phospholipase and proteinase activity of these isolates was estimated, too. The correlation between the virulence factors, antifungal susceptibility patterns and cancer type was also analyzed. ResultsSeven synonymous and four non-synonymous mutations were found in 21 fluconazole-resistant C. glabrata isolates; subsequently, four amino acid substitutions including H257P, Q47H, S487Y and I285N were then reported for the first time. High expression of CDR1 and PDR1 in related to other gene findings were tested in these isolates. Additionally, there was no significant difference between stage of cancer and MIC of all antimicrobial drugs. Significant differences between MIC of fluconazole, voriconazole and cancer types were also, found. The proteinase activity (92.4%) was higher than phospholipase activity in the isolates. Further, no significant difference between proteinase (rs: 0.003), phospholipase (rs: −0.107) activity and fluconazole MICs was observed. ConclusionC. glabrata isolated from OPC in head and neck patients represented high capacities for proteolytic enzymes activity and high mRNA level of CDR1 and PDR1 gene and ERG11 mutations play an important role in azole drug resistance.

Oral candidiasis in patients with kidney transplantation in Iran: prevalence and antifungal susceptibility pattern.

Aim: This study aimed to identify Candida species recovered from the oral cavity of patients with kidney transplantation. Materials & methods: Two swabs were taken from the oral cavities of 40patients before and after transplantation, cultured on Sabouraud dextrose agar, and yeasts identified. Antifungal drug susceptibility testing was performed with fluconazole and itraconazole. Results: Candida glabrata was the most frequently isolated species in patients, followed by Candida albicans and Rhodotorula. C. glabrata isolates from patients before transplantation were resistant to fluconazole, whereas C. albicans was fluconazole-resistant both before and after transplantation. Conclusion: The importance of non-albicans Candida species in the oral cavity of patients sheds light on performing antifungal tests for achieving the best outcome to prevent therapeutic failure.

Antifungal drug susceptibility profiles and molecular mechanisms of azole resistance in Candida blood stream isolates

PurposeThe purpose of this study was to determine the activity of fluconazole, voriconazole, posaconazole, amphotericin B, micafungin and caspofungin against Candida blood stream isolates and to investigate the molecular mechanisms of azole resistance in fluconazole resistant isolates. MethodsThe in vitro susceptibilities of Candida isolates to fluconazole, voriconazole, posaconazole, amphotericin B, micafungin and caspofungin were tested by E-test ERG11, CDR1, CDR2 and MDR1 genes expression of fluconazole resistant, fluconazole (S-DD) and fluconazole intermediate resistant isolates were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). ResultsC. albicans (41%) was the most frequently isolated fungal species from blood stream infections followed by C. parapsilosis (22%). All C. albicans isolates except one and all C. tropicalis isolates were found to be susceptible to fluconazole. Overall, 21% of C. glabrata isolates were resistant to fluconazole. None of the Candida isolates were found to be resistant to caspofungin except 2 ​C. glabrata isolates. Overexpression of ERG11 and CDR1 genes were detected in all fluconazole S-DD and fluconazole resistant C. glabrata isolates, while CDR2 overexpression was observed in 91% fluconazole S-DD and 67 of % fluconazole resistant C.glabrata isolates. The overexpression of MDR1, ERG11 and CDR1 genes were found 100%, 80% and 80%, respectively, in fluconazole resistant C. parapsilosis isolates. ConclusionMost of the Candida isolates were susceptible to posaconazole and caspofungin. Our data also highlighted that overexpression of efflux pump genes major cause of azole resistance.

Epidemiology and susceptibility of Nakaseomyces (formerly Candida) glabrata bloodstream isolates from hospitalised adults in South Africa.

During 2016-2017, Nakaseomyces glabrata (formerly Candida glabrata) caused 14% of cases of candidaemia in South Africa. We aimed to describe the clinical characteristics of adults with N. glabrata candidaemia at 20 sentinel hospitals (accounting for 20% (172/917) of cases) and the antifungal susceptibility of the corresponding isolates. A higher proportion of patients with N. glabrata candidaemia were older (median age: 55 years [interquartile range (IQR): 41-65 years] vs. 49 years [IQR: 35-63 years]; p=0.04), female (87/164, 53% vs. 283/671, 42%; p=0.01), admitted to a public-sector hospital (152/172, 88% vs. 470/745, 63%; p<0.001), treated with fluconazole only (most with suboptimal doses) (51/95, 54% vs. 139/361, 39%; p<0.001), and had surgery (47/172, 27% vs. 123/745, 17%; p=0.001) and a shorter hospital stay (median 7 days [IQR: 2-20 days] vs. 13 days [IQR: 4-27 days]; p<0.001) compared to patients with other causes of candidaemia. Eight N. glabrata isolates (6%, 8/131) had minimum inhibitory concentrations in the intermediate or resistant range for≥1 echinocandin and a R1377K amino acid substitution encoded by the hotspot 2 region of the FKS2 gene. Only 11 isolates (8%, 11/131) were resistant to fluconazole. Patients with confirmed N. glabrata candidaemia are recommended to be treated with an echinocandin (or polyene), thus further guideline training is required.

Fluconazole resistance in non-albicans Candida species in the United States, 2012-2021

Background:Candida spp can cause a variety of infections known as candidiasis, ranging from severe invasive infections to superficial mucosal infections of the mouth and vagina. Fluconazole, a triazole antifungal, is commonly prescribed to treat candidiasis but increasing fluconazole resistance is a growing concern for several Candida spp. Although C. albicans has historically been the most common cause of candidiasis, other species are increasingly common and antifungal resistance is more prevalent in these non-albicans species, including C. glabrata, C. parapsilosis, and C. tropicalis, which were the focus of this analysis. Methods: We used the PINC AI healthcare data (PHD) database to examine fluconazole resistance for inpatient isolates between 2012 and 2021 from 187 US acute-care hospitals with at least 1 Candida spp culture with a fluconazole susceptibility result over the entire period. We calculated annual percentage fluconazole resistance for C. glabrata, C. tropicalis, and C. parapsilosis isolates using the clinical laboratory interpretation for resistance. Results: We identified 4,264 C. glabrata, 2,482 C. parapsilosis, and 2,283 C. tropicalis isolates between 2012 and 2021 with susceptibility results. The percentage of C. glabrata isolates resistant to fluconazole doubled between 2020 and 2021 (14.6% vs 29.3%) (Fig. 1a). The percentage of C. parapsilosis isolates resistant to fluconazole steadily increased since 2017 (Fig. 1b), with an 82% increase in 2021 compared with 2020 (3.8% in 2020 vs 6.9% in 2021). Fluconazole resistance among C. tropicalis isolates varied over the years, with a 0.3% decrease in 2021 from 2020 (Fig. 1c). Of hospitals reporting at least 1 result each year 2020–2021, 44% observed an increase in the proportion of C. glabrata isolates resistant to fluconazole in 2021 compared to 2020. Conclusions: Our analysis highlights a concerning increase in fluconazole resistance among C. glabrata and C. parapsilosis isolates in 2021 compared with previous years. Further investigation of the observed increases in fluconazole resistance among these Candida spp could provide further insight on potential drivers of resistance or limitations in reported results from large databases. More analyses are needed to understand rates, sites of Candida infections, and risk factors (eg, antifungal exposure) associated with resistance.Disclosures: None

Catechin potentiates the antifungal effect of miconazole in Candida glabrata

The rising number of invasive fungal infections caused by drug-resistant Candida strains is one of the greatest challenges for the development of novel antifungal strategies. The scarcity of available antifungals has drawn attention to the potential of natural products as antifungals and in combinational therapies. One of these is catechins—polyphenolic compounds—flavanols, found in a variety of plants. In this work, we evaluated the changes in the susceptibility of Candida glabrata strain characterized at the laboratory level and clinical isolates using the combination of catechin and antifungal azoles. Catechin alone had no antifungal activity within the concentration range tested. Its use in combination with miconazole resulted in complete inhibition of growth in the sensitive C. glabrata isolate and a significant growth reduction in the azole resistant C. glabrata clinical isolate. Simultaneous use of catechin and miconazole leads to increased intracellular ROS generation. The enhanced susceptibility of C. glabrata clinical isolates to miconazole by catechin was accompanied with the intracellular accumulation of ROS and changes in the plasma membrane permeability, as measured using fluorescence anisotropy, affecting the function of plasma membrane proteins.

Thymoquinone Antifungal Activity against Candida glabrata Oral Isolates from Patients in Intensive Care Units-An In Vitro Study.

The number of Candida spp. infections and drug resistance are dramatically increasing worldwide, particularly among immunosuppressed patients, and it is urgent to find novel compounds with antifungal activity. In this work, the antifungal and antibiofilm activity of thymoquinone (TQ), a key bioactive constituent of black cumin seed Nigella sativa L., was evaluated against Candida glabrata, a WHO 'high-priority' pathogen. Then, its effect on the expression of C. glabrata EPA6 and EPA7 genes (related to biofilm adhesion and development, respectively) were analyzed. Swab samples were taken from the oral cavity of 90 hospitalized patients in ICU wards, transferred to sterile falcon tubes, and cultured on Sabouraud Dextrose Agar (SDA) and Chromagar Candida for presumptive identification. Next, a 21-plex PCR was carried out for the confirmation of species level. C. glabrata isolates underwent antifungal drug susceptibility testing against fluconazole (FLZ), itraconazole (ITZ), amphotericin B (AMB), and TQ according to the CLSI microdilution method (M27, A3/S4). Biofilm formation was measured by an MTT assay. EPA6 and EPA7 gene expression was assessed by real-time PCR. From the 90 swab samples, 40 isolates were identified as C. glabrata with the 21-plex PCR. Most isolates were resistant to FLZ (n = 29, 72.5%), whereas 12.5% and 5% were ITZ and AMB resistant, respectively. The minimum inhibitory concentration (MIC50) of TQ against C. glabrata was 50 µg/mL. Importantly, TQ significantly inhibited the biofilm formation of C. glabrata isolates, and EPA6 gene expression was reduced significantly at MIC50 concentration of TQ. TQ seems to have some antifungal, antibiofilm (adhesion) effect on C. glabrata isolates, showing that this plant secondary metabolite is a promising agent to overcome Candida infections, especially oral candidiasis.

Discovery of novel thiosemicarbazone derivatives with potent and selective anti-Candida glabrata activity

A series of 21 novel compounds containing a thiosemicarbazone moiety were designed and synthesised based on hit compound 1 from our in-house compound library screening. Most compounds showed potent antifungal activity in vitro against seven common pathogenic fungi. Notably, all compounds showed high potency against Candida glabrata 537 (MIC = ≤0.0156-2 µg/mL). Of note, compounds 5j and 5r displayed excellent antifungal activity against Candida krusei 4946 and Candida auris 922. Additionally, compounds 5j and 5r also showed high potency against 15 C. glabrata isolates with MIC values ranging from 0.0625 µg/mL to 4 µg/mL, with compound 5r being slightly superior to 5j. Moreover, compound 5r has certain effect against biofilm formation of C. glabrata. Furthermore, compound 5r has minimal cytotoxicity against HUVECs with an IC50 value of 15.89 µg/mL and no haemolysis at 64 µg/mL. Taken together, these results suggest that promising lead compound 5r deserves further investigation.

Combination of Farnesol with Common Antifungal Drugs: Inhibitory Effect against Candida Species Isolated from Women with RVVC.

Background and Objectives: Vulvovaginal candidiasis (VVC) is a mucous membrane infection, with an increased rate of antifungal resistance of Candida species. In this study, the in vitro efficacy of farnesol alone or in combination with traditional antifungals was assessed against resistant Candida strains recovered from women with VVC. Materials and Methods: Eighty Candida isolates were identified by multiplex polymerase chain reaction (PCR), and the antifungal susceptibility to amphotericin B (AMB), fluconazole (FLU), itraconazole (ITZ), voriconazole (VOR), clotrimazole (CTZ), and farnesol was tested by the standard microdilution method. The combinations of farnesol with each antifungal were calculated based on the fractional inhibitory concentration index (FICI). Result: Candida glabrata was the predominant species (48.75%) isolated from vaginal discharges, followed by C. albicans (43.75%), C. parapsilosis (3.75%), a mixed infection of C. albicans and C. glabrata (2.5%) and C. albicans and C. parapsilosis (1%). C. albicans and C. glabrata isolates had lower susceptibility to FLU (31.4% and 23.0%, respectively) and CTZ (37.1% and 33.3%, respectively). Importantly, there was "synergism" between farnesol-FLU and farnesol-ITZ against C. albicans and C. parapsilosis (FICI = 0.5 and 0.35, respectively), reverting the original azole-resistant profile. Conclusion: These findings indicate that farnesol can revert the resistance profile of azole by enhancing the activity of FLU and ITZ in resistant Candida isolates, which is a clinically promising result.

Limosilactobacillus fermentum Limits Candida glabrata Growth by Ergosterol Depletion.

Candida glabrata is a human-associated opportunistic fungal pathogen. It shares its niche with Lactobacillus spp. in the gastrointestinal and vaginal tract. In fact, Lactobacillus species are thought to competitively prevent Candida overgrowth. We investigated the molecular aspects of this antifungal effect by analyzing the interaction of C. glabrata strains with Limosilactobacillus fermentum. From a collection of clinical C. glabrata isolates, we identified strains with different sensitivities to L. fermentum in coculture. We analyzed the variation of their expression pattern to isolate the specific response to L. fermentum. C. glabrata-L. fermentum coculture induced genes associated with ergosterol biosynthesis, weak acid stress, and drug/chemical stress. L. fermentum coculture depleted C. glabrata ergosterol. The reduction of ergosterol was dependent on the Lactobacillus species, even in coculture with different Candida species. We found a similar ergosterol-depleting effect with other lactobacillus strains (Lactobacillus crispatus and Lactobacillus rhamosus) on Candida albicans, Candida tropicalis, and Candida krusei. The addition of ergosterol improved C. glabrata growth in the coculture. Blocking ergosterol synthesis with fluconazole increased the susceptibility against L. fermentum, which was again mitigated by the addition of ergosterol. In accordance, a C. glabrata Δerg11 mutant, defective in ergosterol biosynthesis, was highly sensitive to L. fermentum. In conclusion, our analysis indicates an unexpected direct function of ergosterol for C. glabrata proliferation in coculture with L. fermentum. IMPORTANCE The yeast Candida glabrata, an opportunistic fungal pathogen, and the bacterium Limosilactobacillus fermentum both inhabit the human gastrointestinal and vaginal tract. Lactobacillus species, belonging to the healthy human microbiome, are thought to prevent C. glabrata infections. We investigated the antifungal effect of Limosilactobacillus fermentum on C. glabrata strains quantitively in vitro. The interaction between C. glabrata and L. fermentum evokes an upregulation of genes required for the synthesis of ergosterol, a sterol constituent of the fungal plasma membrane. We found a dramatic reduction of ergosterol in C. glabrata when it was exposed to L. fermentum. This effect extended to other Candida species and other Lactobacillus species. Furthermore, fungal growth was efficiently suppressed by a combination of L. fermentum and fluconazole, an antifungal drug which inhibits ergosterol synthesis. Thus, fungal ergosterol is a key metabolite for the suppression of C. glabrata by L. fermentum.

Correlation of extracellular enzymes activity of Candida glabrata clinical isolates with in vivo pathogenicity in Galleria mellonella larvae

Objective: To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae. Methods: Using experimental research methods, 71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People's Hospital from June 2021 to January 2022. Bovine serum protein agar medium, egg yolk agar medium, sheep blood agar medium, Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity, phospholipase activity, hemolysis activity, esterase activity and lipase activity of Candida glabrata. Median lethal concentration (LC50) was calculated by using 1.25×108 CFU/ml,2.50×108 CFU/ml,3.75×108 CFU/ml,5.00×108 CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae. Histopathological and etiological analysis was performed to determine whether the infection model was successfully established. The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC50 concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae. Results: 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture. Aspartyl protease was detected after 4 days of culture, among which 7 strains (9.86%), 19 strains (26.76%) and 45 strains (63.38%) showed low, medium and high aspartyl protease activity. After 7 days of culture, 71 strains did not detect phospholipase, esterase and lipase activities. Candida glabrata on Galleria mellonella larvae of LC50=2.5×108 CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group, and Candida glabrata was identified by the microbial Mass Spectrometry after culture, while no fungi were found in the pathological section and culture of the control group. Spearman test shows that, there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae (r = 0.73, P<0.01), the difference was statistically significant.Pearson test shows that, there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae (r = 0.16, P = 0.34), the difference was not statistically significant. Conclusion: The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity, but no phospholipase, esterase and lipase activity. The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.

Novel Therapeutic Approaches to Invasive Candidiasis: Considerations for the Clinician.

Invasive candidiasis (IC), due to the yeast pathogen Candida, is still a major cause of in-hospital morbidity and mortality. The limited number of antifungal drug classes and the emergence of multi-resistant Candida species, such as Candida auris and some Candida glabrata isolates, is concerning. However, recent advances in antifungal drug development provide promising perspectives for the therapeutic approach of IC. Notably, three novel antifungal agents, currently in Phase II/III clinical trials, are expected to have an important place for the treatment of IC in the future. Rezafungin is a novel echinocandin with prolonged half-life. Ibrexafungerp and fosmanogepix are two first-in-class antifungal drugs with broad spectrum activity against Candida spp., including C. auris and echinocandin-resistant species. These novel antifungal agents also represent interesting alternative options because of their acceptable oral bioavailability (ibrexafungerp and fosmanogepix) or their large interdose interval (once weekly intravenous administration for rezafungin) for prolonged and/or outpatient treatment of complicated IC. This review discusses the potential place of these novel antifungal drugs for the treatment of IC considering their pharmacologic properties and their preclinical and clinical data.