Clumping Morphology Influences Virulence Uncoupled from Echinocandin Resistance in Candida glabrata.

Here, we report two paired sets of an index wild-type Candida glabrata bloodstream isolate and subsequent echinocandin-resistant FKS mutant. One paired set exhibited a higher proportion of clumping cells and was more virulent in the invertebrate host Galleria mellonella than the other paired set. No virulence difference between the paired index and FKS strains was observed. These findings imply a potential link of clumping morphology with virulence in C. glabrata that is uncoupled from FKS-mediated echinocandin resistance. IMPORTANCE Candida glabrata is a leading cause of invasive candidiasis. In contrast to other species, it has a high propensity for developing resistance to echinocandins, which are the first-line treatment. Unlike the dimorphic Candida albicans which can grow invasive filamentous hyphae, C. glabrata lacks this ability. Here, we report a link between virulence and clumping cell morphology in two different sets of clinical C. glabrata strains obtained from patients failing echinocandin therapy. One set of paired strains (echinocandin-susceptible and subsequent resistant mutant) had a high proportion of clumping cells in the population and were significantly more virulent than another set which had fewer clumping cells. Additionally, we corroborate that echinocandin resistance does not impart a significant fitness cost. Our findings suggest that clumping morphology may be an important but previously underestimated virulence factor for C. glabrata and also aid our understand for the high prevalence of resistance observed in this species.

Dynamic regulation of membrane integrity to enhance l-malate stress tolerance in Candida glabrata.

Microbial cell factories provide a sustainable and economical way to produce chemicals from renewable feedstocks. However, the accumulation of targeted chemicals can reduce the robustness of the industrial strains and affect the production performance. Here, the physiological functions of Mediator tail subunit CgMed16 at l-malate stress were investigated. Deletion of CgMed16 decreased the survival, biomass, and half-maximal inhibitory concentration (IC50 ) by 40.4%, 34.0%, and 30.6%, respectively, at 25 g/L l-malate stress. Transcriptome analysis showed that this growth defect was attributable to changes in the expression of genes involved in lipid metabolism. In addition, tolerance transcription factors CgUSV1 and CgYAP3 were found to interact with CgMed16 to regulate sterol biosynthesis and glycerophospholipid metabolism, respectively, ultimately endowing strains with excellent membrane integrity to resist l-malate stress. Furthermore, a dynamic tolerance system (DTS) was constructed based on CgUSV1, CgYAP3, and an l-malate-driven promoter Pcgr-10 to improve the robustness and productive capacity of Candida glabrata. As a result, the biomass, survival, and membrane integrity of C. glabrata 012 (with DTS) increased by 22.6%, 31.3%, and 53.8%, respectively, compared with those of strain 011 (without DTS). Therefore, at shake-flask scale, strain 012 accumulated 35.5 g/L l-malate, and the titer and productivity of l-malate increased by 32.5% and 32.1%, respectively, compared with those of strain 011. This study provides a novel strategy for the rational design and construction of DTS for dynamically enhancing the robustness of industrial strains.

Transient Mitochondria Dysfunction Confers Fungal Cross-Resistance against Phagocytic Killing and Fluconazole.

Loss or inactivation of antivirulence genes is an adaptive strategy in pathogen evolution. Candida glabrata is an important opportunistic pathogen related to baker's yeast, with the ability to both quickly increase its intrinsic high level of azole resistance and persist within phagocytes. During C. glabrata's evolution as a pathogen, the mitochondrial DNA polymerase CgMip1 has been under positive selection. We show that CgMIP1 deletion not only triggers loss of mitochondrial function and a petite phenotype, but increases C. glabrata's azole and endoplasmic reticulum (ER) stress resistance and, importantly, its survival in phagocytes. The same phenotype is induced by fluconazole and by exposure to macrophages, conferring a cross-resistance between antifungals and immune cells, and can be found in clinical isolates despite a slow growth of petite strains. This suggests that petite constitutes a bet-hedging strategy of C. glabrata and, potentially, a relevant cause of azole resistance. Mitochondrial function may therefore be considered a potential antivirulence factor. IMPORTANCE Candida glabrata is an opportunistic pathogen whose incidence has been increasing in the last 40 years. It has risen to become the most prominent non-Candida albicans Candida (NCAC) species to cause candidemia, constituting about one-third of isolates in the United States, and steadily increasing in European countries and in Australia. Despite its clinical importance, C. glabrata's pathogenicity strategies remain poorly understood. Our research shows that loss of mitochondrial function and the resulting petite phenotype is advantageous for C. glabrata to cope with infection-related stressors, such as antifungals and host immune defenses. The (cross-)resistance against both these factors may have major implications in the clinical outcome of infections with this major fungal pathogen.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation.

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.

An aspartyl protease-mediated cleavage regulates structure and function of a flavodoxin-like protein and aids oxidative stress survival.

A family of eleven glycosylphosphatidylinositol-anchored aspartyl proteases, commonly referred to as CgYapsins, regulate a myriad of cellular processes in the pathogenic yeast Candida glabrata, but their protein targets are largely unknown. Here, using the immunoprecipitation-mass spectrometry approach, we identify the flavodoxin-like protein (Fld-LP), CgPst2, to be an interactor of one of the aspartyl protease CgYps1. We also report the presence of four Fld-LPs in C. glabrata, which are required for survival in kidneys in the murine model of systemic candidiasis. We further demonstrated that of four Fld-LPs, CgPst2 was solely required for menadione detoxification. CgPst2 was found to form homo-oligomers, and contribute to cellular NADH:quinone oxidoreductase activity. CgYps1 cleaved CgPst2 at the C-terminus, and this cleavage was pivotal to oligomerization, activity and function of CgPst2. The arginine-174 residue in CgPst2 was essential for CgYps1-mediated cleavage, with alanine substitution of the arginine-174 residue also leading to elevated activity and oligomerization of CgPst2. Finally, we demonstrate that menadione treatment led to increased CgPst2 and CgYps1 protein levels, diminished CgYps1-CgPst2 interaction, and enhanced CgPst2 cleavage and activity, thereby implicating CgYps1 in activating CgPst2. Altogether, our findings of proteolytic cleavage as a key regulatory determinant of CgPst2, which belongs to the family of highly conserved, electron-carrier flavodoxin-fold-containing proteins, constituting cellular oxidative stress defense system in diverse organisms, unveil a hidden regulatory layer of environmental stress response mechanisms.

Experimental Evolution of Candida by Serial Passaging in Host Cells.

Experimental evolution is an experiment class of its own; instead of requiring an a priori hypothesis, the genetic adaptation of microbes to defined environments tells us about the underlying pathways and mechanisms. Such experiments are often deceptively simple in their design, based on a single abiotic stressor and what is in essence a long-term continuous culture. However, they generally provide a starting point to thorough follow-up analyses (which are specific for the organism at hand and not part of this method chapter). In this chapter, we describe a method to use a biotic stressor which is frequently encountered by pathogenic fungi-macrophage-like cells-in a serial passaging regime. Experimental evolution under such conditions can reveal new virulence attributes and mechanisms by selecting for adaptive mutations against the host cell-induced stress.It is important to note that every evolution experiment is different, and these techniques should be taken as a general guideline to be adapted to different organisms and questions. Then, it is a powerful tool with many potential applications in pathobiology research.

Multifunctional properties and safety evaluation of lactic acid bacteria and yeasts associated with fermented cereal doughs.

Spontaneous cereal fermentations involve diverse lactic acid bacteria (LAB) and yeasts which may include multifunctional and safe or unsafe strains. This study assessed acidification ability, safety, antifungal activity and free amino acids release ability of LAB and yeasts previously isolated from spontaneously fermented cereal doughs in Benin. Fourteen LAB and thirteen yeast strains were studied in liquid media and/or in a model cereal dough prepared in laboratory conditions. Antifungal activity was assessed against Candida glabrata in liquid medium. Amino acids were determined by pre-column derivatization and separation with reversed-phase HPLC. Antimicrobial susceptibility was analysed by minimum inhibitory concentration determination. The acidification ability was higher for LAB compared to yeast strains. All LAB strains retarded the growth of C. glabrata Cg1 with the highest inhibition recorded for Weissella confusa Wc1 and Wc2. The highest free amino acid content was found in the doughs fermented with Pichia kudriavzevii Pk2 and Pk3. All the LAB strains were susceptible to ampicillin, chloramphenicol, erythromycin, but displayed phenotypic resistance to kanamycin, streptomycin and tetracycline. Positive PCR amplicon of resistance genes were detected in the following cases: 2 LAB strains were positive for kanamycin (aph(3)III), 5 strains were positive for streptomycin (aadA and/or strA and/or strB) and 3 strains were positive for tetracycline (tet (L) and/or tet (M)). For yeasts, most of the P. kudriavzevii strains were resistant to amphotericin B, fluconazole and itraconazole opposite to K. marxianus and Saccharomyces cerevisiae strains which were susceptible. The results obtained are valuable for selecting safe and multifunctional strains for cereal fermentation in West Africa.

Abundance interaction in Candida albicans and Candida glabrata mixed biofilms under diverse conditions.

Candida albicans and Candida glabrata are frequently coisolated from the oral cavity in immunosuppressive or immunocompromised individuals. Their relationship is usually defined as competition as C. glabrata can inhibit growth of C. albicans in cohabitation. In this study, eight C. albicans isolates as well as two C. glabrata strains were used to investigate the effects of culture medium (Roswell Park Memorial Institute [RPMI]-1640, YPD, YND), incubation time (24 h, 48 h, 72 h, 96 h), initial inoculum (C. glabrata: C. albicans = 2:1, 1:1, 1:2), and medium state (static and dynamic states) on viable cell enumeration and relative abundance in both Candida SB and MB. The results showed that in most cases, C. glabrata and C. albicans SB and MB flourished in RPMI-1640 at 24 h under dynamic state compared with other conditions. Except YPD medium, there were high proportions of preponderance of C. albicans over C. glabrata in MB compared with SB. High initial inoculum promoted corresponding Candida number in both SB and MB and its abundance in MB relative to SB. This study revealed an impact of several environmental conditions on the formation of C. albicans and C. glabrata SB and MB and their abundance in MB in comparison with SB, deepening our understanding of both Candida interaction and their resistance mechanism in MB. LAY SUMMARY: This study described the effects of diverse experimental conditions on the numbers of Candida albicans and Candida glabrata single biofilms and mixed biofilms and their abundance.

Physicochemical and Antifungal Properties of Clotrimazole in Combination with High-Molecular Weight Chitosan as a Multifunctional Excipient.

Chitosans represent a group of multifunctional drug excipients. Here, we aimed to estimate the impact of high-molecular weight chitosan on the physicochemical properties of clotrimazole-chitosan solid mixtures (CL-CH), prepared by grinding and kneading methods. We characterised these formulas by infrared spectroscopy, differential scanning calorimetry, and powder X-ray diffractometry, and performed in vitro clotrimazole dissolution tests. Additionally, we examined the antifungal activity of clotrimazole-chitosan mixtures against clinical Candida isolates under neutral and acid conditions. The synergistic effect of clotrimazole and chitosan S combinations was observed in tests carried out at pH 4 on Candida glabrata strains. The inhibition of C. glabrata growth reached at least 90%, regardless of the drug/excipient weight ratio, and even at half of the minimal inhibitory concentrations of clotrimazole. Our results demonstrate that clotrimazole and high-molecular weight chitosan could be an effective combination in a topical antifungal formulation, as chitosan acts synergistically with clotrimazole against non-albicans candida strains.

A homologous overexpression system to study roles of drug transporters in Candida glabrata.

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.

Ethno-medicinal study of Artemisia ordosica Krasch. (traditional Chinese/Mongolian medicine) extracts for the treatment of allergic rhinitis and nasosinusitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia ordosica Krasch. (AOK) has been used for rheumatic arthritis, cold headache, sore throat, etc. in traditional Chinese/Mongolian medicine and is used for nasosinusitis by local Mongolian "barefoot" doctors. Up to now, their mechanisms are still unclear. AIM: To evaluate the in vivo anti-inflammatory and allergic rhinitis (AR) alleviating effect as well as in vitro antimicrobial activities of AOK extracts to verify its ethno-medicinal claims. MATERIALS AND METHODS: Crude extracts (methanol/95%-ethanol/ethyl acetate) of AOK root/stem/leaf and fractions (petroleum ether/ethyl acetate/n-butanol/aqueous) of AOK root extract were prepared. Xylene-induced ear swelling model in mouse and ovalbumin (OVA)-induced AR model in guinea pig were established. Ear swelling degrees of mice were measured. The numbers of rubbing movement and sneezes of guinea pigs were counted to evaluate the symptoms of AR. The serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1 were measured by ELISA assay. The histological changes of nasal mucosa were investigated by light microscope after H&E staining. Antimicrobial activities of AOK extracts were also tested. LC-MS/MS analysis was performed to characterize the constituents of active extract and molecular docking was conducted to predict the biological mechanism. RESULTS: In ear-swelling model, extract (100.00 mg/kg) from the ethyl acetate layer of 95% ethanol (100.00 mg/kg) showed better swelling inhibition in mice than positive control (dexamethasone, 191.91 mg/kg). In AR model, extract from the ethyl acetate layer of 95% ethanol significantly alleviated the AR symptoms in guinea pigs, decreased the serum levels of histamine, INF-γ, IL-2/4/10, and VCAM-1, and reduced the infiltration of eosinophil in nasal mucosa. For Staphylococcus aureus, the ethyl acetate extract of AOK stem showed the highest inhibition (MIC=1.25 mg/mL), for Escherichia coli, n-butanol layer of 95% ethanol extract of AOK root showed the highest inhibition (MIC=15.00 mg/mL), for Candida glabrata, 95%-ethyl acetate extract of AOK leaf showed the best inhibition (MIC=0.064 mg/mL), while ethyl acetate and n-butanol layers showed similar inhibition on MRSA (MIC=7.50 mg/mL). LC-MS/MS characterization showed that dicaffeoylquinic acids account for more than 30% of ethyl acetate layer of AOK extract. Dicaffeoylquinic acids bind with histamine-1 receptor with high affinities and interesting modes. CONCLUSIONS: Extracts from AOK had interesting anti-inflammatory activity in mice, alleviating effect against OVA-induced AR in guinea pigs, and antimicrobial activities in vitro, which support the ethno-medicinal use of it. The main constituents in ethyl acetate layer of AOK root extract are dicaffeoylquinic acids and could bind with histamine-1 receptor well. These findings highlighted the importance of natural product chemistry study of AOK.

Deletion of CDR1 reveals redox regulation of pleiotropic drug resistance in Candida glabrata.

Microbial cells sense the presence of xenobiotics and, in response, upregulate genes involved in pleiotropic drug resistance (PDR). In yeast, PDR activation to a major extent relies on the transcription factor Pdr1. In addition, many xenobiotics induce oxidative stress, which may upregulate PDR independently of Pdr1 activity. Mitochondria are important sources of reactive oxygen species under stressful conditions. To evaluate the relevance of this redox pathway, we studied the activation of PDR in the yeast Candida glabrata, which we treated with a mitochondrially targeted antioxidant plastoquinonyl-decyl-triphenylphosphonium and dodecyltriphenylphosphonium (C12TPP) as a control. We found that both compounds induced activation of PDR genes and decreased the intracellular concentration of the PDR transporter substrate Nile red. Interestingly, the deletion of PDR transporter gene CDR1 inhibited the decrease in Nile red accumulation induced by antioxidant plastoquinonyl-decyl-triphenylphosphonium but not that by C12TPP. Moreover, antioxidant alpha-tocopherol inhibited C12TPP-mediated activation of PDR in Δcdr1 but not in the wild-type strain. Furthermore, pre-incubation of yeast cells with low concentrations of hydrogen peroxide induced a decrease in the intracellular concentration of Nile red in Δcdr1 and Δpdr1 as well as in control cells. Deletion of PDR1 inhibited the C12TPP-induced activation of CDR1 but not that of FLR1, which is a redox-regulated PDR transporter gene. It appears that disruption of the PDR1/CDR1 regulatory circuit makes auxiliary PDR regulation mechanisms crucial. Our data suggest that redox regulation of PDR is dispensable in wild-type cells because of redundancy in the activation pathways, but is manifested upon deletion of CDR1.

Iontophoresis enhances voriconazole antifungal potency and corneal penetration.

Strategies to enhance corneal penetration of voriconazole (VOR) could improve the treatment of fungal keratitis. Here, we evaluated the use of iontophoresis for ocular VOR delivery from either: (i) a cyclodextrin inclusion complex (CD VOR), (ii) a liposome (LP VOR), and (iii) a chitosan-coated liposome (LP VOR CS). LP VOR CS presented mean diameter of 139.2 ±â€¯1.3 nm and zeta potential equal to + 3.3 ±â€¯1.5 mV compared to 134.6 ±â€¯1.7 and -8.2 ±â€¯3.0 mV of LP VOR, which, together with mucin mucoadhesion study, confirmed chitosan-coating. Both drug and liposomal formulations were stable under the influence of an applied electric current. Interestingly, in vitro studies in Candida glabrata culture indicated a decrease in VOR MIC values following iontophoresis (from 0.28 to 0.14 µg/mL). Iontophoresis enhanced drug penetration into the cornea. After 10 min of a 2 mA/cm2 applied current, corneal retained amounts were 45.4 ±â€¯11.2, 30.4 ±â€¯2.1 and 30.6 ±â€¯2.9 µg/cm2 for, respectively, CD VOR, LP VOR, and LP VOR CS. In conclusion, iontophoresis increases drug potency and enhances drug penetration into the cornea, showing potential to be used as "an emergency burst delivery approach".

Release of different amphotericin B formulations from PMMA bone cements and their activity against Candida biofilm.

Amphotericin B is used for local delivery from polymethylmethacrylate to treat fungal prosthetic joint infections. The optimal amphotericin B formulation and the influence of different poragens in the bone cements are unknown. To investigate the necessary amount of amphotericin B in the bone cement to prevent Candida biofilm several amphotericin B formulations were studied: non-liposomal and liposomal with or without poragen gentamicin. For the non-liposomal formulation, standard bile salt, the sodium deoxycholate, was used and additionally N-methyl-D-glucamine/palmitate was applied. The activity of the released amphotericin B was tested against C. albicans, C. glabrata, C. parapsilosis and C. krusei biofilms with application of the isothermal calorimeter and standard microbiological methods. Compressive strength was measured before and after antifungal elution from the cements. There is less aggregated N-methyl-D-glucamine/palmitate amphotericin B released but its antifungal activity is equivalent with the deoxycholate amphotericin B. The minimum quantity of antifungal preventing the Candida biofilm formation is 12.5 mg in gram of polymer powder for both non-liposomal formulations. The addition of gentamicin reduced the release of sodium deoxycholate amphotericin B. Gentamicin can be added to N-methyl-D-glucamine/palmitate amphotericin B in order to boost the antifungal release. When using liposomal amphotericin B more drug is released. All amphotericin B formulations were active against Candida biofilms. Although compressive strength slightly decreased, the obtained values were above the level of strength recommended for the implant fixation. The finding of this work might be beneficial for the treatment of the prosthetic joint infections caused by Candida spp.

Epidemiology and prognostic factors of nosocomial candidemia in Northeast Brazil: A six-year retrospective study.

Candidemia has been considered a persistent public health problem with great impact on hospital costs and high mortality. We aimed to evaluate the epidemiology and prognostic factors of candidemia in a tertiary hospital in Northeast Brazil from January 2011 to December 2016. Demographic and clinical data of patients were retrospectively obtained from medical records and antifungal susceptibility profiling was performed using the broth microdilution method. A total of 68 episodes of candidemia were evaluated. We found an average incidence of 2.23 episodes /1000 admissions and a 30-day mortality rate of 55.9%. The most prevalent species were Candida albicans (35.3%), Candida tropicalis (27.4%), Candida parapsilosis (21.6%) and Candida glabrata (11.8%). Higher mortality rates were observed in cases of candidemia due to C. albicans (61.1%) and C. glabrata (100%), especially when compared to C. parapsilosis (27.3%). Univariate analysis revealed some variables which significantly increased the probability of death: older age (P = 0.022; odds ratio [OR] = 1.041), severe sepsis (P < 0.001; OR = 8.571), septic shock (P = 0.035; OR = 3.792), hypotension (P = 0.003; OR = 9.120), neutrophilia (P = 0.046; OR = 3.080), thrombocytopenia (P = 0.002; OR = 6.800), mechanical ventilation (P = 0.009; OR = 8.167) and greater number of surgeries (P = 0.037; OR = 1.920). Multivariate analysis showed that older age (P = 0.040; OR = 1.055), severe sepsis (P = 0.009; OR = 9.872) and hypotension (P = 0.031; OR = 21.042) were independently associated with worse prognosis. There was no resistance to amphotericin B, micafungin or itraconazole and a low rate of resistance to fluconazole (5.1%). However, 20.5% of the Candida isolates were susceptible dose-dependent (SDD) to fluconazole and 7.7% to itraconazole. In conclusion, our results could assist in the adoption of strategies to stratify patients at higher risk for developing candidemia and worse prognosis, in addition to improve antifungal management.

Moonlighting proteins are variably exposed at the cell surfaces of Candida glabrata, Candida parapsilosis and Candida tropicalis under certain growth conditions.

BACKGROUND: Adaptability to different environmental conditions is an essential characteristic of pathogenic microorganisms as it facilitates their invasion of host organisms. The most external component of pathogenic yeast-like fungi from the Candida genus is the multilayered cell wall. This structure is composed mainly of complex polysaccharides and proteins that can undergo dynamic changes to adapt to the environmental conditions of colonized niches. RESULTS: We utilized cell surface shaving with trypsin and a shotgun proteomic approach to reveal the surface-exposed proteins of three important non-albicans Candida species-C. glabrata, C. parapsilosis and C. tropicalis. These proteinaceous components were identified after the growth of the fungal cells in various culture media, including artificial saliva, artificial urine and vagina-simulative medium under aerobic conditions and anaerobically in rich YPD medium. Several known proteins involved in cell wall maintenance and fungal pathogenesis were identified at the cell surface as were a number of atypical cell wall components-pyruvate decarboxylase (Pdc11), enolase (Eno1) and glyceraldehyde-3-phosphate dehydrogenase (Tdh3) which are so-called 'moonlighting' proteins. Notably, many of these proteins showed significant upregulation at the cell surface in growth media mimicking the conditions of infection compared to defined synthetic medium. CONCLUSIONS: Moonlighting proteins are expressed under diverse conditions at the cell walls of the C. glabrata, C. parapsilosis and C. tropicalis fungal pathogens. This indicates a possible universal surface-associated role of these factors in the physiology of these fungi and in the pathology of the infections they cause.

Anidulafungin Susceptibility Testing of Candida glabrata Isolates from Blood Cultures by the MALDI Biotyper Antibiotic (Antifungal) Susceptibility Test Rapid Assay.

Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100 C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

EUCAST Reference Testing of Rezafungin Susceptibility and Impact of Choice of Plastic Plates.

Rezafungin is a new long-acting echinocandin currently in phase 3 development. Epidemiological cutoff values are necessary for breakpoint setting but have not been established due to unexplained interlaboratory MIC variations observed in a prior multicenter study. Here we investigated if the choice of microtiter plates affected the variability when anidulafungin was included as a comparator. Testing by the EUCAST E.Def 7.3.1 reference method using tissue and cell culture-treated polystyrene plates (TC plates) and untreated polystyrene plates (UT plates) from four manufacturers was performed. Six control strains (Candida albicans, n = 3; C. krusei, n = 2; C. parapsilosis, n = 1) were tested (520 MICs). Subsequently, 5 or 6 wild-type isolates and 4 or 5 fks mutants of C. albicans, C. glabrata, C. krusei, C. parapsilosis (wild type only), and C. tropicalis were tested (930 MICs). For each strain-plate combination, ≥98% of the repetitive MICs were within 3 dilutions. The rezafungin modal MICs for the collated C. albicans control strain distributions were 0.016 mg/liter across TC plates but 0.03 mg/liter across UT plates, whereas they were 0.004 mg/liter and 0.016 mg/liter, respectively, for anidulafungin. The difference was most pronounced with Falcon plates and was not observed for C. krusei and C. parapsilosis Eleven rezafungin MICs for mutants overlapped with the MICs for wild-type isolates (TC plates, n = 4; UT plates, n = 7). For anidulafungin, five overlaps (all UT plates) were observed. Most overlaps (rezafungin, n = 5; anidulafungin, n = 3) were caused by fks mutants of C. tropicalis (Fks1, F650F/L) and C. glabrata (Fks2. D666Y; rezafungin, n = 2; anidulafungin, n = 1). Interlaboratory variation was low. The use of TC plates resulted in lower MICs, particularly for C. albicans and Falcon plates, ad this was more often the case for anidulafungin than for rezafungin. Adoption of TC plates for EUCAST antifungal susceptibility testing would improve interlaboratory reproducibility and the separation of non-wild-type and wild-type strains.

The regulation of iron homeostasis in the fungal human pathogen Candida glabrata.

Iron is an essential element to most microorganisms, yet an excess of iron is toxic. Hence, living cells have to maintain a tight balance between iron uptake and iron consumption and storage. The control of intracellular iron concentrations is particularly challenging for pathogens because mammalian organisms have evolved sophisticated high-affinity systems to sequester iron from microbes and because iron availability fluctuates among the different host niches. In this review, we present the current understanding of iron homeostasis and its regulation in the fungal pathogen Candida glabrata. This yeast is an emerging pathogen which has become the second leading cause of candidemia, a life-threatening invasive mycosis. C. glabrata is relatively poorly studied compared to the closely related model yeast Saccharomyces cerevisiae or to the pathogenic yeast Candida albicans. Still, several research groups have started to identify the actors of C. glabrata iron homeostasis and its transcriptional and post-transcriptional regulation. These studies have revealed interesting particularities of C. glabrata and have shed new light on the evolution of fungal iron homeostasis.

CgHog1-Mediated CgRds2 Phosphorylation Alters Glycerophospholipid Composition To Coordinate Osmotic Stress in Candida glabrata.

Under stress conditions, Hog1 is required for cell survival through transiently phosphorylating downstream targets and reprogramming gene expression. Here, we report that Candida glabrata Hog1 (CgHog1) interacts with and phosphorylates CgRds2, a zinc cluster transcription factor, in response to osmotic stress. Additionally, we found that deletion of CgRDS2 led to decreases in cell growth and cell survival by 23.4% and 39.6%, respectively, at 1.5 M NaCl, compared with levels of the wild-type strain. This is attributed to significant downregulation of the expression levels of glycerophospholipid metabolism genes. As a result, the content of total glycerophospholipid decreased by 30.3%. Membrane integrity also decreased 47.6% in the Cgrds2Δ strain at 1.5 M NaCl. In contrast, overexpression of CgRDS2 increased the cell growth and cell survival by 10.2% and 6.3%, respectively, owing to a significant increase in the total glycerophospholipid content and increased membrane integrity by 27.2% and 12.1%, respectively, at 1.5 M NaCl, compared with levels for the wild-type strain. However, a strain in which the CgRDS2 gene encodes the replacement of Ser64 and Thr97 residues with alanines (Cgrds22A ), harboring a CgRds2 protein that was not phosphorylated by CgHog1, failed to promote glycerophospholipid metabolism and membrane integrity at 1.5 M NaCl. Thus, the above results demonstrate that CgHog1-mediated CgRds2 phosphorylation enhanced glycerophospholipid composition and membrane integrity to resist osmotic stress in C. glabrataIMPORTANCE This study explored the role of CgHog1-mediated CgRds2 phosphorylation in response to osmotic stress in Candida glabrataCgHog1 interacts with and phosphorylates CgRds2, a zinc cluster transcription factor, under osmotic stress. Phosphorylated CgRds2 plays an important role in increasing glycerophospholipid composition and membrane integrity, thereby enhancing cell growth and survival.