The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCENakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.

Our understanding of the relationship between oral Candida and systemic conditions has significantly increased recently, which this study aims to extend further by investigating the risks of oral candidiasis. A total of 314 patients were involved in this study and underwent an oral swab test at the Department of Oral Medicine, Hokkaido University Hospital, between January and December 2021. Data were collected on age, sex, white and red blood cell counts, Hb, total protein, vitamin B12, as well as serum albumin, iron, copper, and zinc levels. The clinical fungus samples were swabbed to identify those with Candida species using a MALDI Biotyper, then applied analysis of covariance and multivariant logistic regression analysis. It was possible to assess the oral swab test results without considering the difference between sex (p = 0.946). The oral swab test results were associated with aging (odds ratio: 1.03) and serum albumin levels (odds ratio: 0.32). In summary, the results of our study suggest a relationship between aging and oral candidiasis and offer in-depth insights into how to prevent or treat oral candidiasis onset.

UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

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.

Candida infective endocarditis is a rare but serious entity that often requires aggressive treatment. However, treatment can be challenging in patients infected with drug-resistant fungi and/or with substantial comorbidity. Moreover, recommendations in treatment guidelines for these patients are based on limited clinical data due to their rarity. Here we report a case of Nakaseomyces glabrata (Candida glabrata) prosthetic valve endocarditis in a patient with congenital heart disease. This case illustrates a therapeutic dilemma for Nakaseomyces glabrata prosthetic valve endocarditis and the need for novel antifungal drugs and further clinical studies.