Glabrata Sensu Stricto Research Articles

Investigation of the Defective Growth Pattern and Multidrug Resistance in a Clinical Isolate of Candida glabrata Using Whole-Genome Sequencing and Computational Biology Applications.

ABSTRACTCandida glabrata is increasingly isolated from blood cultures, and multidrug-resistant isolates have important implications for therapy. This study describes a cholesterol-dependent clinical C. glabrata isolate (ML72254) that did not grow without blood (containing cholesterol) on routine mycological media and that showed azole and amphotericin B (AmB) resistance. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and whole-genome sequencing (WGS) were used for species identification. A modified Etest method (Mueller-Hinton agar supplemented with 5% sheep blood) was used for antifungal susceptibility testing. WGS data were processed via the Galaxy platform, and the genomic variations of ML72254 were retrieved. A computational biology workflow utilizing web-based applications (PROVEAN, AlphaFold Colab, and Missense3D) was constructed to predict possible deleterious effects of these missense variations on protein functions. The predictive ability of this workflow was tested with previously reported missense variations in ergosterol synthesis genes of C. glabrata. ML72254 was identified as C. glabrata sensu stricto with MALDI-TOF, and WGS confirmed this identification. The MICs of fluconazole, voriconazole, and amphotericin B were >256, >32, and >32 μg/mL, respectively. A novel frameshift mutation in the ERG1 gene (Pro314fs) and many missense variations were detected in the ergosterol synthesis genes. None of the missense variations in the ML72254 ergosterol synthesis genes were deleterious, and the Pro314fs mutation was identified as the causative molecular change for a cholesterol-dependent and multidrug-resistant phenotype. This study verified that web-based computational biology solutions can be powerful tools for examining the possible impacts of missense mutations in C. glabrata.IMPORTANCE In this study, a cholesterol-dependent C. glabrata clinical isolate that confers azole and AmB resistance was investigated using artificial intelligence (AI) technologies and cloud computing applications. This is the first of the known cholesterol-dependent C. glabrata isolate to be found in Turkey. Cholesterol-dependent C. glabrata isolates are rarely isolated in clinical samples; they can easily be overlooked during routine laboratory procedures. Microbiologists therefore need to be alert when discrepancies occur between microscopic examination and growth on routine media. In addition, because these isolates confer antifungal resistance, patient management requires extra care.

Candida glabrata Antifungal Resistance and Virulence Factors, a Perfect Pathogenic Combination.

In recent years, a progressive increase in the incidence of invasive fungal infections (IFIs) caused by Candida glabrata has been observed. The objective of this literature review was to study the epidemiology, drug resistance, and virulence factors associated with the C. glabrata complex. For this purpose, a systematic review (January 2001–February 2021) was conducted on the PubMed, Scielo, and Cochrane search engines with the following terms: “C. glabrata complex (C. glabrata sensu stricto, C. nivariensis, C. bracarensis)” associated with “pathogenicity” or “epidemiology” or “antibiotics resistance” or “virulence factors” with language restrictions of English and Spanish. One hundred and ninety-nine articles were found during the search. Various mechanisms of drug resistance to azoles, polyenes, and echinocandins were found for the C. glabrata complex, depending on the geographical region. Among the mechanisms found are the overexpression of drug transporters, gene mutations that alter thermotolerance, the generation of hypervirulence due to increased adhesion factors, and modifications in vital enzymes that produce cell wall proteins that prevent the activity of drugs designed for its inhibition. In addition, it was observed that the C. glabrata complex has virulence factors such as the production of proteases, phospholipases, and hemolysins, and the formation of biofilms that allows the complex to evade the host immune response and generate fungal resistance. Because of this, the C. glabrata complex possesses a perfect pathogenetic combination for the invasion of the immunocompromised host.

Analysis of Biofilm-Related Genes and Antifungal Susceptibility Pattern of Vaginal Candida albicans and Non-Candida albicans Species.

Background Vulvovaginal candidiasis caused by Candida species is a prevalent fungal infection among women. It is believed that the pathogenesis of Candida species is linked with the production of biofilm which is considered a virulence factor for this organism. The aim of this study was molecular identification, antifungal susceptibility, biomass quantification of biofilm, and detection of virulence markers of Candida species. Methods We investigated the molecular identification of 70 vaginal isolates of Candida species, antifungal resistance to amphotericin B, fluconazole, itraconazole, and voriconazole according to CLSI M27-A3 and M27-S4, biofilm formation, and frequency analysis of biofilm-related ALS1, ALS3, and HWP1 genes. Results Our findings showed that the most common yeast isolated from vaginal discharge was C. albicans (67%), followed by the non-Candida albicans species (33%). All C. albicans complex isolates were confirmed as C. albicans by HWP-PCR, and all isolates of the C. glabrata complex were revealed to be C. glabrata sensu stricto using the multiplex PCR method. FLC resistance was observed in 23.4% of C. albicans and 7.7% of C. glabrata. The resistance rate to ITC was found in 10.6% of C. albicans. The frequency of ALS1, ALS3, and HWP1 genes among Candida species was 67.1%, 80%, and 81.4%, respectively. Biofilm formation was observed in 54.3% of Candida species, and the highest frequency detected as a virulence factor was for the ALS3 gene (97.3%) in biofilm-forming species. Discussion. Our results showed the importance of molecular epidemiology studies, investigating antifungal susceptibility profiles, and understanding the role of biofilm-related virulence markers in the pathogenesis of Candida strains.

Molecular epidemiology, antifungal susceptibility and virulence factors of Candida glabrata complex strains in Kayseri/Turkey

BackgroundCandida nivariensis and Candida bracarensis are included in Candida glabrata complex, which are usually misidentified as C. glabrata based on phenotypic identification methods. It was aimed to identify C. glabrata complex isolated from various clinical samples in Kayseri/Turkey to the species level and to determine antifungal susceptibilities, virulence factors, and molecular epidemiology. MethodsEighty three C. glabrata complex strains were studied in this study. Strains were phenotypically and molecularly identified. Phylogenetic analysis was done by the neighbor-joining method. Proteinase, phospholipase, esterase enzyme activity, and biofilm formation of strains were determined phenotypically. Antifungal susceptibility of strains were determined according to M60-Ed2 recommendations. ResultsAll the 83 strains identified as C. glabrata complex by phenotypic tests were confirmed as C. glabrata sensu stricto (C. glabrata) by PCR amplification and sequence analysis, but other complex members C. nivariensis and C. bracarensis were not detected. Phylogenetic analysis results revealed 19 different genotypes. No clonal relationship was detected among the strains. Biofilm formation in 75.9% of strains and esterase activity in 7.2% were found positive. Antifungal resistance rates of strains were determined as 9.2% for fluconazole and 45.8% for itraconazole; 43.4% of the strains for voriconazole were determined as non-wild type. ConclusionIt was determined that biofilm and esterase activity might play an active role in the virulence of C. glabrata. In addition, high resistance rates to azoles in C. glabrata strains isolated in our hospital at Kayseri/Turkey emphasized the significance of epidemiological studies.

Virulence Attributes of Clinical Candida glabrata (Sensu Stricto) Isolates in the West Region of Cameroon

Background: Studies on the assessment of the virulence factors of C. glabrata sensu stricto strains are on the rise. This is partly due to the increase in recurrent C. glabrata infections that have contributed to increased mortality rates. Published data on the virulence characteristics of C. glabrata strains in Cameroon are very rare.
 Aims: This study aimed at assessing some virulence characteristics, including the capacity to form biofilms and hydrolytic enzymes (protease, esterase and phospholipase).
 Methods: Fifty-four (54) molecularly (MALDI-TOF) identified non-duplicate C. glabrata sensu stricto clinical isolates initially collected in a previous study, were used in the present study. These isolates were obtained from stool (S), urine (U), oro-pharyngeal (OPS) and cervico-vaginal (CVS) swabs of pregnant women, diabetic patients (both types 1 and 2 diabetes mellitus), HIV/AIDS and other patients who had neither of these diseases. Phospholipase, protease, esterase and biofilm activities were assessed using previously described methods.
 Results: Our results revealed that our isolates were more able to produce phospholipase (37.04%) than they were able to produce protease (1.85%) and esterase (0%). The high producers of phospholipase (Pz < 0.7) originated mostly from oro-pharyngeal swabs (41.17%) of some diabetic patients and pregnant women. Also, all our isolates were formers of biofilm, most (74.42%) of which had lower (< 100%) biofilm formation activity compared to our reference strain. To be able to give a significant conclusion about the virulence characteristics of C. glabrata strains in the west region, we recommend that more studies be carried on a larger number of strains.

In vitro activity of isavuconazole against clinically isolated yeasts from Chile.

Isavuconazole is the last antifungal agent approved by the FDA and available for treatment of fungal infections. In the present study, the in vitro activity of isavuconazole against several yeasts was investigated. Two hundred forty-six isolates were included: 64 Candida albicans, 53 Candida parapsilosis sensu stricto, 48 Cryptococcus neoformans species complex, 27 C. glabrata sensu stricto, 17 C. lusitaniae, 17 C. tropicalis, 5 C. orthopsilosis, 4 C. krusei, 3 C. guilliermondii sensu stricto, 3 C. pelliculosa, 2 C. dubliniensis, 1 C. auris, 1 C. fermentati and 1 Trichosporon asahii. All isolates were recovered from clinical isolates from Chile, being 221 from hemoculture, 22 from cerebrospinal fluid, 1 pleural fluid, and 1 from tissue culture. The minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) of isavuconazole were determined. Isavuconazole demonstrated good in vitro activity against all species tested. MIC90 values and MFC ranges of isavuconazole for Candida albicans were 0.03mg/L and 0.03- > 16mg/L respectively. Non-Candida albicans species isolates were inhibited by ≤ 1mg/L, with MFC ranges from < 0.03- > 16mg/L. Also, isavuconazole was active against the non-Candida yeasts, being inhibited with MIC values ≤ 0.06mg/L. Isavuconazole has exhibited potent in vitro activity against clinical isolates of Candida spp., Cryptococcus neoformans species complex, and other clinical yeast in Chile. Despite the results obtained in the present work, additional clinical studies are necessary to verify the level of efficacy of this azole.

Antifungal drug susceptibility, molecular basis of resistance to echinocandins and molecular epidemiology of fluconazole resistance among clinical Candida glabrata isolates in Kuwait

Candida glabrata readily develops resistance to echinocandins. Identification, antifungal susceptibility testing (AST) and resistance mechanism to echinocandins among C. glabrata was determined in Kuwait. C. glabrata isolates (n = 75) were tested by Vitek2, multiplex PCR and/or PCR-sequencing of rDNA. AST to fluconazole, caspofungin, micafungin and amphotericin B was determined by Etest and to micafungin by broth microdilution (BMD). Mutations in hotspot-1/hotspot-2 of FKS1/FKS2 and ERG11 were detected by PCR-sequencing. All isolates were identified as C. glabrata sensu stricto. Seventy isolates were susceptible and five were resistant to micafungin by Etest and BMD (essential agreement, 93%; categorical agreement, 100%). Three micafungin-resistant isolates were resistant and two were susceptible dose-dependent to caspofungin. Four and one micafungin-resistant isolate contained S663P and ∆659 F mutation, respectively, in hotspot-1 of FKS2. Micafungin-resistant isolates were genotypically distinct strains. Only one of 36 fluconazole-resistant isolate contained nonsynonymous ERG11 mutations. Thirty-four of 36 fluconazole-resistant isolates were genotypically distinct strains. Our data show that micafungin susceptibility reliably identifies echinocandin-resistant isolates and may serve as a surrogate marker for predicting susceptibility/resistance of C. glabrata to caspofungin. All micafungin-resistant isolates also harbored a nonsynonymous/deletion mutation in hotspot-1 of FKS2. Fingerprinting data showed that echinocandin/fluconazole resistance development in C. glabrata is not clonal.

Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study

Candidemia is the most common, serious fungal infection and Candida antifungal resistance is a challenge. We report recent surveillance of candidemia in China. The study encompassed 77 Chinese hospitals over 3 years. Identification of Candida species was by mass spectrometry and DNA sequencing. Antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute broth microdilution method. In total, 4010 isolates were collected from candidemia patients. Although C. albicans was the most common species, non-albicans Candida species accounted for over two-thirds of isolates, predominated C. parapsilosis complex (27.1%), C. tropicalis (18.7%), and C. glabrata complex (12.0%). Most C. albicans and C. parapsilosis complex isolates were susceptible to all antifungal agents (resistance rate <5%). However, there was a decrease in voriconazole susceptibility to C. glabrata sensu stricto over the 3 years and fluconazole resistance rate in C. tropicalis tripled. Amongst less common Candida species, over one-third of C. pelliculosa isolates were coresistant to fluconazole and 5-flucytocine, and >56% of C. haemulonii isolates were multidrug resistance. Non-albicans Candida species are the predominant cause of candidemia in China. Azole resistance is notable amongst C. tropicalis and C. glabrata. Coresistance and multidrug resistance has emerged in less common Candida species.

Rapid identification of the Candida glabrata species complex by high-resolution melting curve analysis.

Background Candida glabrata is a common pathogen that causes invasive candidiasis. Among non‐albicans Candida infections, C glabrata infections are associated with the highest fatality rates. Candida glabrata sensu stricto, Candida nivariensis, and Candida bracarensis have been identified and together form the C glabrata species complex. It is difficult to detect the two rare species by traditional laboratory methods. This study established a method for the rapid identification of members of the C glabrata species complex based on high‐resolution melting curve (HRM) analysis and evaluated its practical application.MethodsThe internal transcribed spacer (ITS) region was used as target gene region to design specific primers. HRM analysis was performed with three subspecies of the C glabrata species complex and negative controls to test its specificity and sensitivity. To evaluate its practical application, the HRM technique was tested with clinical isolates, and the results were compared with the DNA sequencing results.ResultsDifferences were detected among the melting profiles of the members of the C glabrata species complex. The negative controls were not amplified, indicating the high specificity of the method. The minimum detection limits of C glabrata sensu stricto, C nivariensis, and C bracarensis were approximately 1 × 101 copies/µL or less. The results of the HRM analysis of the clinical isolates were consistent with the DNA sequencing results.ConclusionsThe HRM method is sensitive and can be used to rapidly identify the members of the C glabrata species complex. The method can allow early and targeted treatment of patients with invasive candidiasis.

Lack of detection of Candida nivariensis and Candida bracarensis among 440 clinical Candida glabrata sensu lato isolates in Kuwait.

Occurrence of Candida nivariensis and Candida bracarensis, two species phenotypically similar to Candida glabrata sensu stricto, in human clinical samples from different geographical settings remains unknown. This study developed a low-cost multiplex PCR (mPCR) and three species-specific singleplex PCR assays. Reference strains of common Candida species were used during development and the performance of mPCR and singleplex PCR assays was evaluated with 440 clinical C. glabrata sensu lato isolates. The internal transcribed spacer (ITS) region of rDNA was also sequenced from 85 selected isolates and rDNA sequence variations were used for determining genetic relatedness among the isolates by using MEGA X software. Species-specific amplicons for C. glabrata (~360 bp), C. nivariensis (~250 bp) and C. bracarensis (~180 bp) were obtained in mPCR while no amplicon was obtained from other Candida species. The three singleplex PCR assays also yielded expected results with reference strains of Candida species. The mPCR amplified ~360 bp amplicon from all 440 C. glabrata sensu lato isolates thus identifying all clinical isolates in Kuwait as C. glabrata sensu stricto. The results of mPCR were confirmed for all 440 isolates as they yielded an amplicon only in C. glabrata sensu stricto-specific singleplex PCR assay. The rDNA sequence data identified 28 ITS haplotypes among 85 isolates with 18 isolates belonging to unique haplotypes and 67 isolates belonging to 10 cluster haplotypes. In conclusion, we have developed a simple, low-cost mPCR assay for rapid differentiation of C. glabrata sensu stricto from C. nivariensis and C. bracarensis. Our data obtained from a large collection of clinical C. glabrata sensu lato isolates show that C. nivariensis and C. bracarensis are rare pathogens in Kuwait. Considerable genetic diversity among C. glabrata sensu stricto isolates was also indicated by rDNA sequence analyses.

Prevalence and Antifungal Susceptibility of the Emerging Fungal Species, Candida nivariensis, Isolated in a Teaching Hospital in Poland.

The data on susceptibility to antifungals of new species within Candida glabrata complex are limited. Our study was to enrich a global knowledge of yeast epidemiology and drug resistance. The study was focused on the identification of species within clinical isolates of the C. glabrata complex and on the determination of their resistance to antifungals. Four hundred forty-five clinical C. glabrata sensu lato strains were isolated from different clinical samples at routine mycological exams at the Infant Jesus Teaching Hospital in Warsaw. The identification of the most of tested isolates to species complex level was performed using the ID 32 C system. The identification of C. nivariensis and C. bracarensis species within the C. glabrata complex was performed by DNA sequencing. The MICs of amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, anidulafungin, and micafungin were determined by E-test. Twenty-four isolates did not have an ITS-1 region, characteristic of C. glabrata sensu stricto and their D1/D2 regions of the 26S rRNA were 99% homologous to C. nivariensis 26S rRNA. No strains of C. bracarensis were recovered. C. nivariensis strains were very susceptible to amphotericin B, anidulafungin, micafungin, and caspofungin. Ninety-two percent of C. nivariensis were resistant to itraconazole. The halves of the strains was resistant to posaconazole. Eighty-three percent of C. nivariensis were susceptible to voriconazole. None of the tested strains were susceptible to fluconazole. In the present study, none of the C. nivariensis strains were simultaneously resistant to azoles and echinocandins. C. nivariensis should be recognized as an emerging pathogen, resistant to azoles.

Candida glabrata complex from patients with healthcare-associated infections in Mansoura University Hospitals, Egypt: distribution, antifungal susceptibility and effect of fluconazole and polymyxin B combination.

Candida glabrata complex is composed of three cryptic species, Candida glabrata sensu stricto, C. bracarensis and C. nivariensis. Its reduced susceptibility to fluconazole is responsible for treatment failure of its infections. Combination therapy is recommended to treat these resistant strains. This study assessed the distribution of C. glabrata complex collected from patients in Mansoura University Hospitals and their antifungal susceptibility, with evaluation of fluconazole and polymyxin B combination against them. C. glabrata complex was collected from patients with healthcare-associated infections. The isolates were identified biochemically then the species were detected using multiplex PCR. The susceptibility of isolates to antifungals and polymyxin B was tested by the microdilution assay. The effect of fluconazole and polymyxin B combination was assessed by checkerboard microdilution and time kill assays. This study included 45 isolates of Candida glabrata complex. The common isolate was Candida glabrata sensu stricto (38 isolates). There were 4 isolates of C. bracarensis and three isolates of C. nivariensis. All isolates were susceptible to amphotericin B, and 17.8% were susceptible to itraconazole. Regarding fluconazole, 48.9% of isolates were susceptible dose-dependent and 51.1% were resistant. Synergistic effect of the combination was observed in 68.9% of isolates by the checkerboard method and in 66.7% of isolates using the time kill assay. Candida glabrata sensu stricto is the main member of the complex. Combination of fluconazole and polymyxin B can be considered a treatment option for fluconazole resistant C. glabrata complex infections. In vivo studies are needed to validate these results.

In Vitro Determination of Hydrolytic Enzymes and Echinocandin Susceptibility in Mexican Clinical Isolates of Candida glabrata Sensu Stricto

Background: Candida glabrata is an opportunistic yeast that has emerged as a cause of human fungal disease, and is a complex of three closely related species. Until now, there is not enough information about its virulence attributes. Moreover, its resistance to echinocandins has been documented, which is a cause of clinical concern. Objectives: The objective of this study was to evaluate the in vitro production of aspartyl proteinase, phospholipase, esterase, hemolysin, DNase, and coagulase in a subset of 107 Mexican clinical isolates of C. glabrata sensu stricto, as well as to determinate its echinocandin susceptibility. Methods: The enzymatic determinations were carried out in plate assays using specific substrates, excepting coagulase, which was determined by the classic tube test. Antifungal susceptibility testing was determined in accordance with the CLSI broth microdilution method. Results: Aspartyl proteinase, hemolysin, and phospholipase were detected in 100%, 95%, and 79% of isolates, respectively. Blood isolates were associated with a very strong activity of aspartyl proteinase and hemolysin, and those recovered from vaginal swabs were associated with very strong production of aspartyl proteinase, phospholipase, and hemolysin. All isolates were susceptible to echinocandins, except one bloodstream isolate, which was resistant to echinocandins. Conclusions: A very strong activity of aspartyl proteinase and hemolysin was particularly associated with both, bloodstream, and vaginal swab isolates. Echinocandin resistance was rare.

Candida albicans and non-albicans Isolates from Bloodstream Have Different Capacities to Induce Neutrophil Extracellular Traps.

Neutrophils activated with pathogens or their products induce formation of extracellular traps (NETs), but if this constitutes a general response against all pathogenic species in a single genus or intrageneric differences exist remains unknown, yet this is of great importance for the establishment of effective treatments. To determine this, we analyzed neutrophil extracellular traps formation after the stimulation with bloodstream isolates from different Candida species (Candida albicans, C. tropicalis, C. parapsilosis, and C. glabrata), and found that each species has a different capacity to induce DNA extrusion, which is independent of their morphology (yeast or hyphae). We observed that phospholipase producer’s strains and their secretion products were able to induce NETs, a property not observed with phospholipase deficient strains, with exception of some Candida glabrata sensu stricto isolates, which showed no NETs induction although they did show phospholipase production. To further analyze this, we extended our study to include Candida glabrata cryptic species (C. bracarensis and C. nivariensis) and no extracellular traps formation was observed. Here, we contribute to the understanding of how neutrophils initiate NETs, and we found that certain strains may have a differential capacity to trigger these structures, which may explain the high mortality of some isolates.

Usefulness of a multiplex PCR for the rapid identification of Candida glabrata species complex in Mexican clinical isolates.

ABSTRACT Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.

New species within Candida parapsilosis and Candida glabrata

Introduction: Candida parapsilosis and Candida glabrata are another yeasts that form complexes of crypospecies. Although these species have been described more than a decade ago, knowledge about them is still limited. The reason for this is the large phenotypic similarity that unables them from being differentiated by classical diagnostic methods. The aim of the study was to identify species of clinical strains within C. glabrata and C. parapsilosis complexes. Material and methods: Standard PCR-RFLP of the secondary alcohol dehydrogenase gene (SADH) with BanI restriction enzyme served to determine species affiliation within the C. parapsilosis complex. The internal transcribed spacer was used to confirm the identification of C. glabrata sensu stricto. The D1/D2 domain of the 26S rDNA gene was sequenced in order to identify C. nivariensis and C. bracarensis strains. Results: As a result of the molecular analysis, 24 Candida nivariensis isolates and 4 C. metapsilosis strains and 9 C. orthopsilosis strains were detected. Conclusions: Prevalence of new cryptic species was relatively low.

Prevalence of human pathogens of the clade Nakaseomyces in a culture collection\u2014the first report on Candida bracarensis in Poland

Human pathogens belonging to the Nakaseomyces clade include Candida glabrata sensu stricto, Candida nivariensis and Candida bracarensis. Their highly similar phenotypic characteristics often lead to misidentification by conventional laboratory methods. Therefore, limited information on the true epidemiology of the Candida glabrata species complex is available. Due to life-threatening infections caused by these species, it is crucial to supplement this knowledge. The aim of the study was to estimate the prevalence of C. bracarensis and C. nivariensis in a culture collection of C. glabrata complex isolates. The study covered 353 isolates identified by biochemical methods as C. glabrata, collected from paediatric and adult patients hospitalised at four medical centres in Southern Poland. The multiplex PCR was used to identify the strains. Further species confirmation was performed via sequencing and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis. One isolate was recognised as C. bracarensis (0.28%). To our knowledge, it is the first isolate in Poland. C. glabrata sensu stricto species has been confirmed for all the remaining isolates. No C. nivariensis was found. Our study has shown that the prevalence of C. nivariensis and C. bracarensis strains is infrequent. However, it should be emphasised that the incidence of these strains may differ locally and depend on environmental factors and the population.

Prevalence and antifungal susceptibility profiles of Candida glabrata, Candida parapsilosis and their close-related species in oral candidiasis

ObjectiveTo evaluate the importance of Candida glabrata, Candida parapsilosis and their close-related species, Candida bracarensis, Candida nivariensis, Candida metapsilosis and Candida orthopsilosis in patients with oral candidiasis and, to determine the in vitro activities of antifungal drugs currently used for the treatment. MethodsOne hundred fourteen isolates of C. glabrata and 97 of C. parapsilosis, previously identified by conventional mycological methods, were analysed by molecular techniques. In vitro antifungal susceptibility to fluconazole, itraconazole, miconazole, and nystatin was evaluated by CLSI M44-A2 disk diffusion test, and by CLSI M27-A3 microdilution for fluconazole. ResultsAll C. glabrata isolates were identified as C. glabrata sensu stricto, 93 out of 97 C. parapsilosis isolates as C. parapsilosis sensu stricto, three as C. orthopsilosis and one as C. metapsilosis. Candida glabrata was mainly isolated in mixed cultures but C. parapsilosis complex was more frequent in pure culture. Candida metapsilosis and C. orthopsilosis were isolated as pure culture and both species were susceptible to all antifungal agents tested. Most C. glabrata isolates were susceptible to miconazole and nystatin, but resistant to fluconazole and itraconazole. Azole cross resistance was also observed. Candida parapsilosis isolates were susceptible to fluconazole although azole cross resistance to miconazole and itraconazole was observed. ConclusionThis study highlights the importance of accurate identification and antifungal susceptibility testing of oral Candida isolates in order to have an in-depth understanding of the role of C. glabrata and C. parapsilosis in oral candidiasis.

Identification of Candida Glabrata Complex Species: use of Vitek MS ® RUO &amp; Bruker Clinprotools ®

Distinction of species within the Candida glabrata complex (i.e., C. glabrata sensu stricto, Candida nivariensis and Candida bracarensis) is relevant for epidemiological purposes and antifungal management. Two commercial matrix-assisted laser desorption ionization-time of flight mass spectrometry systems were comprehensively evaluated for the identification of fungi within this complex. None of the species (C. nivariensis and C. bracarensis) were identified correctly by Vitek mass spectrometry (MS®) v2.0 In Vitro Diagnosis system and Bruker Biotyper MS® v3.1, but all were correct by the Vitek MS® Research Use Only system. The Bruker ClinProTools software showed 100% recognition capability and cross validation for the discrimination of C. nivariensis and C. bracarensis. Using Vitek MS Research Use Only and Bruker ClinProTools can overcome limitations of the Vitek MS In Vitro Diagnosis and Bruker Biotyper databases in the identification of C. glabrata complex.

Association between Candida biofilm-forming bloodstream isolates and the clinical evolution in patients with candidemia: An observational nine-year single center study in Mexico

BackgroundCandidemia is one of the most common nosocomial infections globally and it is associated with considerable excess mortality and costs. Abreast, biofilm-forming strains are associated with even higher mortality rates and poor prognosis for the patient. AimsTo evaluate a possible association between the biofilm-forming capability of Candida bloodstream isolates and the clinical evolution in patients with candidemia. MethodsAn observational, retrospective study was conducted at a tertiary care university hospital during 9 years (2006–2015). The biofilm quantitation of the Candida bloodstream isolates was determined by crystal violet staining and XTT reduction assay. ResultsA total of 218 cases of candidemia had been diagnosed and 89 isolates were obtained. The mortality rate was 36% and the main risk factors were antibiotic exposure and the use of catheters. Candida tropicalis (52.8%) was the most frequent species, followed by Candida albicans (30.4%), Candida parapsilosis sensu stricto (10.1%), Candida orthopsilosis (3.4%), Candida krusei (2.2%) and Candida glabrata sensu stricto (1.1%). All the strains were biofilm producers, which is an important contribution to the patient's mortality. C. tropicalis showed the highest production of biomass biofilm, whereas C. glabrata exhibited the highest metabolic activity. ConclusionsThis study contributes to expand the knowledge about the local epidemiology of candidemia and highlights the impact of Candida biofilm on patient's outcome.