Krusei Isolates Research Articles

Biology and genetic diversity of Candida krusei isolates from fermented vegetables and clinical samples in China

ABSTRACT Candida krusei, also known as Pichia kudriavzevii, is an emerging non-albicans Candida (NAC) species causing both superficial and deep-seated infections in humans. This fungal pathogen is inherently resistant to the first-line antifungal drug, fluconazole, and is widely distributed in natural environments such as soil, foods, vegetables, and fruits. In this study, we collected 86 C. krusei strains from clinical settings and traditional fermented vegetables from different areas of China. Compared to C. krusei strains from fermented vegetables, clinical isolates exhibited a higher ability to undergo filamentation and biofilm development, which could facilitate its host colonization and infections. Isolates from fermented vegetables showed higher resistance to several antifungal drugs including fluconazole, voriconazole, itraconazole, amphotericin B, and caspofungin, than clinical strains, while they were more susceptible to posaconazole than clinical strains. Although C. krusei has been thought to be a diploid organism, we found that one-fourth of clinical strains and the majority of isolates from fermented vegetables (87.5%) are triploid. Whole-genome sequencing and population genetic analyses demonstrated that isolates from clinical settings and fermented food are genetically associated, and distributed across a wide range of genetic clusters. Additionally, we found that six nucleotide substitutions at the promoter region of the ABC11 gene, encoding a multidrug efflux pump, could play a critical role in antifungal resistance in this species. Given the ubiquitous distribution of C. krusei strains in fermented vegetables and their genetic association with clinical strains, a One Health approach will be necessary to control the prevalence of this pathogen.

Antifungal susceptibility pattern of Candida species isolated from pregnant women.

Candidaspecies, opportunistic yeast, are the second most common cause of female vulvovaginal candidiasis. This study aimed to evaluate the antifungal susceptibility profile of the isolatedCandidaspecies in pregnant women in Hajjah governorate, Yemen. A hospital-based cross-sectional study was conducted among 396 pregnant women attending Authority AL-Gumhorri Hospital Hajjah between February and July 2023. Vaginal swabs were collected, andCandidaspecies were isolated and identified based on the standard laboratory method. Furthermore, the antifungal drug susceptibility ofCandidaspecies was determined by the Kirby-Bauer technique. The prevalence of vaginal Candida infection among pregnant women was 61.4%. Candida albicanswas the most predominant species (59.26%), followed by Candida krusei(13.58%), Candida Tropicalis (11.12%), Candida Grabata (9.87%), and Candida dubliniensis (6.17%). The highest rate ofCandidainfections was among women aged 24-30 years (71.9%) who finished primary school (77.8%), with the third trimester (80%), multigravida (66.1%), and recurrent infection (67.7%) showing significant differences (P< 0.05). The Candida albicans isolates were resistant to clotrimazole and itraconazole at 34.7% and 23.6%, respectively.In addition, the resistance of Candida krusei, Candida tropicalis, Candida glabrata, and Candida dublinensis isolates to fluconazole, voriconazole, voriconazole, and nystatin was 57.6%, 63%, 43.8%, and 60%, respectively. Additionally, approximately 46.2% of isolated Candida albicans exhibited one kind of antifungal drug resistance, whereas 38.7% of isolated non-albicans exhibited resistance to three different antifungal agents. According to the above findings, Candida infection is highly prevalent in Yemen and quite widespread. Interventions in health education are advised to increase women's knowledge of vaginitis and its prevention. The antifungal susceptibility test may also be helpful in determining the best medication for each patient.

Candida spp. isolated from recreational coastal waters of Rio de Janeiro – Brazil: Focus on antifungal resistance and virulence attributes

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1–5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87–3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

Comparative pharmacodynamics and dose optimization of liposomal amphotericin B against Candida species in an in vitro pharmacokinetic/pharmacodynamic model.

As comparative pharmacokinetic/pharmacodynamic (PK/PD) studies of liposomal amphotericin B (L-AMB) against Candida spp. are lacking, we explored L-AMB pharmacodynamics against different Candida species in an in vitro PK/PD dilution model. Eight Candida glabrata, Candida parapsilosis, and Candida krusei isolates (EUCAST/CLSI AMB MIC 0.125-1 mg/L) were studied in the in vitro PK/PD model simulating L-AMB Cmax = 0.25-64 mg/L and t1/2 = 9 h. The model was validated with one susceptible and one resistant Candida albicans isolate. The Cmax/MIC-log10CFU/mL reduction from the initial inoculum was analyzed with the Emax model, and Monte Carlo analysis was performed for the standard (3 mg/kg with Cmax = 21.87 ± 12.47 mg/L) and higher (5 mg/kg with Cmax = 83 ± 35.2 mg/L) L-AMB dose. A ≥1.5 log10CFU/mL reduction was found at L-AMB Cmax = 8 mg/L against C. albicans, C. parapsilosis, and C. krusei isolates (MIC 0.25-0.5 mg/L) whereas L-AMB Cmax ≥ 32 mg/L was required for C. glabrata isolates. The in vitro PK/PD relationship followed a sigmoidal pattern (R2 ≥ 0.85) with a mean Cmax/MIC required for stasis of 2.1 for C. albicans (close to the in vivo stasis), 24/17 (EUCAST/CLSI) for C. glabrata, 8 for C. parapsilosis, and 10 for C. krusei. The probability of target attainment was ≥99% for C. albicans wild-type (WT) isolates with 3 mg/kg and for wild-type isolates of the other species with 5 mg/kg. L-AMB was four- to eightfold less active against the included non-C. albicans species than C. albicans. A standard 3-mg/kg dose is pharmacodynamically sufficient for C. albicans whereas our data suggest that 5 mg/kg may be recommendable for the included non-C. albicans species.

The fungicidal effectiveness of 2-Chloro-3-hydrazinylquinoxaline, a newly developed quinoxaline derivative, against Candida species.

Candida represents a prevalent fungal infection, notable for its substantial implications on morbidity and mortality rates. In the landscape of prospective treatments, quinoxaline derivatives emerge as a category of compact compounds exhibiting notable potential in addressing infections. These derivatives showcase promising antimicrobial efficacy coupled with favorable pharmacokinetic and safety characteristics. The central aim of this investigation was to examine the antifungal characteristics of 2-Chloro-3-hydrazinylquinoxaline against diverse strains of Candida and Aspergillus in vitro. Additionally, we endeavored to assess the in vivo efficacy of 2-Chloro-3-hydrazinylquinoxaline using a murine model for oral candidiasis induced by C. albicans cells ATCC 10231. 2-Chloro-3-hydrazinylquinoxaline demonstrated noteworthy effectiveness when tested against various reference strains of Candida species. It exhibited heightened efficacy, particularly against Candida krusei isolates. However, its performance against Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida auris isolates exhibited variability. Notably, 2-Chloro-3-hydrazinylquinoxaline manifests variable efficacy against Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus and Aspergillus flavus and no effect against Aspergillus brasiliensis. In a murine model, 2-Chloro-3-hydrazinylquinoxaline exhibited significant efficacy in combating the C. albicans cells ATCC 10231 strain, underscoring its potential as a viable treatment option. 2-Chloro-3-hydrazinylquinoxaline has demonstrated substantial potential in effectively addressing various Candida and Aspergillus species, showcasing dual attributes of antifungal and anti-inflammatory properties. However, to attain a more comprehensive understanding of its therapeutic capabilities, further investigations, incorporating additional tests and experiments, are imperative.

Pulmonary surfactant impacts in vitro activity of selected antifungal drugs against Candida krusei and Candida albicans

PurposeThis study investigates how surfactants affect the in-vitro anti-infective efficacy of micafungin, caspofungin, anidulafungin, and amphotericin B in treating pulmonary mycoses.MethodsMIC values for antifungal agents were determined against Candida krusei (now Pichia kudriavzevii) ATCC 6258, Candida albicans ATCC 90028, and 18 clinical isolates using the broth microdilution method in RPMI medium, following EUCAST recommendations. MIC assays included testing with and without Curosurf® surfactant at 1 mg/mL for C. krusei ATCC 6258 and all C. krusei isolates. Subsequent Time-kill studies in Sabouraud broth involved testing both C. albicans ATCC 90028 and C. krusei ATCC 6258 strains at concentrations equal their respective MIC values, with and without surfactant, using all four antifungals. CFU/mL were assessed at multiple time points up to 24 h. TKCs with different surfactant concentrations for C. krusei ATCC 6258 and mini-TKCs at various concentrations relative to the MIC of C. krusei isolates and the reference strain were conducted with micafungin, anidulafungin, and caspofungin.ResultsMIC results showed that 1 µg/mL surfactant reduced killing of micafungin and anidulafungin against C. krusei, while caspofungin was unaffected. Amphotericin B's MIC decreased by half. TKCs demonstrated significant effects of surfactant on micafungin and anidulafungin against C. krusei, with complete abolition of anidulafungin's activity against C. albicans.ConclusionThis in-vitro study highlights the concentration-dependent inhibitory effect of surfactant on antifungal activity against C. krusei and, to some extent, C. albicans, necessitating further clinical validation for invasive lung mycoses treatment.

Molecular typing and antifungal susceptibility profile of Candida krusei bloodstream isolates from Türkiye.

Candida krusei also known as Pichia kudriavzevii is a potentially multidrug-resistant yeast because it is intrinsically resistant to fluconazole and develops acquired resistance to echinocandins and polyenes. Here, we aim to provide a better understanding of the epidemiology and transmission modes of C. krusei infections by comparing invasive bloodstream (n=35) and non-invasive vaginal (n=20) C. krusei isolates. The genetic relatedness of the isolates was assessed using a newly described short tandem repeat (STR) analysis and their sensitivity to eight antifungal compounds was evaluated by antifungal susceptibility testing using the CLSI microbroth dilution method. All C. krusei isolates revealed unique STR genotypes, indicating the absence of clonal transmission in the study group. Furthermore, no drug-resistant or non-wild-type isolates were identified. Our findings demonstrated high resolution of STR genotyping for the detection and simultaneous genetic analysis of multiple C. krusei strains in clinical samples and excellent in vitro activity of common antifungal agents against invasive strains.

Candida species isolated in female patients of reproductive age with vaginal candidiasis in Gualeguaychú, Entre Ríos, Argentina

Objective: To identify the Candida species isolated in female patients of reproductive age with vaginal candidiasis. To determine the antifungal (ATF) sensitivity profile of the isolated Candida species. Materials and methods: Cross-sectional descriptive study, in which 124 Candida spp strains from vaginal discharge samples were isolated in Sabouraud medium supplemented with glucose. CHRO Magar Candida was used for species identification and complementary tests such as germ tube, chlamydoconidia development and investigation of trehalose assimilation were performed. In vitro sensitivity was investigated by diffusion method with ATF discs fluconazole (FLU), itraconazole (ITRA) and amphotericin B (AMB). Results: C. albicans was observed in 85.5% of the isolates followed by C. glabrata with 7.3%, C. krusei 4%, C. tropicalis 2.4% and other Candida species. For FLU, 0.9% of C. albicans, 11.1% of C. glabrata and 100% of C. krusei isolates showed resistance. For ITRA, 17% of C. albicans isolates, 55.6% of C. glabrata and 100% of C. krusei were resistant. There were no isolates resistant to AMB. Conclusion: Vaginal candidiasis continues to present Candida albicans as the main etiological agent, which is widely sensitive to ATFs. C. glabrata and C. krusei species show increased resistance to azoles. The results obtained ratify the growing need for Candida species identification tests and determination of in vitro sensitivity to ATFs in order to guide the treatment of vaginal candidiasis towards therapeutic success.

Antifungal Activity and Type of Interaction of Melissa officinalis Essential Oil with Antimycotics against Biofilms of Multidrug-Resistant Candida Isolates from Vulvovaginal Mucosa.

(1) Background: Vulvovaginal candidosis (VVC) is a major therapy issue due to its high resistance rate and virulence factors such as the ability to form biofilms. The possibility of combining commonly used antifungals with natural products might greatly improve therapeutic success. (2) Methods: A total of 49 vulvovaginal isolates, causative agents of recurrent VVC, were tested for their susceptibility to fluconazole, nystatin, and Melissa officinalis essential oil (MOEO). This examination included testing the antibiofilm potential of antifungals and MOEO and the determination of their types of interaction with mature biofilms. (3) Results: Antimicrobial testing showed that 94.4% of the Candida albicans isolates and all the Candida krusei isolates were resistant to fluconazole, while all strains showed resistance to nystatin. The same strains were susceptible to MOEO in 0.156-2.5 mg/mL concentrations. Additionally, the results revealed very limited action of fluconazole, while nystatin and MOEO reduced the amount of biofilm formed by as much as 17.7% and 4.6%, respectively. Testing of the combined effect showed strain-specific synergistic action. Furthermore, the lower concentrations exhibited antagonistic effects even in cases where synergism was detected. (4) Conclusions: This study showed that MOEO had a very good antibiofilm effect. However, combining MOEO with antimycotics demonstrated that the type of action depended on the choice of antifungal drugs as well as the applied concentration.

Antifungal Resistance Profile, Biofilm Formation, and Virulence Factor Production in Candida krusei Isolates From HIV-Infected Patients in Cameroon.

Background Fungal infections mainly caused by Candida krusei are increasing rapidly and represent a serious public health problem in human immunodeficiency virus (HIV)-infected patients. This study aimed to investigate the antifungal susceptibility profile and virulence factors in C. krusei isolated from HIV-infected patients. Methodology Isolates were identified by biochemical and molecular methods. The antifungal resistance profile was established based on the antifungal susceptibility test performed using the Sensititre YeastOne™ (Thermo Fisher Scientific, Waltham, MA) microdilution technique. The production of phospholipase and proteinase was detected by standard methods. Biofilm formation was performed by the microtiter plate method. Results A total of 73 isolates of C. krusei were recovered from stool, oral swabs, vaginal swabs, and urine samples. The highest number of C. krusei isolates (49, 67.05%)was recovered from stool samples. A total of 32.56% of the C. krusei isolates were multidrug-resistant (MDR). The patients living with HIV and not receiving antiretroviral treatment displayed the highest number of C. krusei isolates (29, 39.76%), whereas the patients living with HIV on antiretroviral therapy exhibited the lowest number of C. krusei isolates (2, 2.72%). All isolateswere categorized as strong biofilm producers. Among the production of hydrolytic enzymes, 25 (58.13%) and 24 (55.81%) of C. krusei isolates were classified as strong phospholipase and proteinase producers, respectively. Conclusion The C. krusei isolates obtained in this study were MDR and strongly expressed biofilm formation and both phospholipase and proteinase hydrolytic enzymes. The results show how pathogenic C. krusei is in the HIV-infected population and will contribute toward the management of C. krusei-relatedinfections, which may help improve the life quality of people living with HIV.

Whole genome sequencing data of Candida krusei, the pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia.

Candida krusei is a Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against Candida spp., and both inherent and acquired resistance to fluconazole have been reported. C. krusei species is also reported as the only Candida sp. which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964.

Genetic Diversity of Candida spp. Isolates Colonizing Twins and Their Family Members.

A wide range of options for studying Candida species are available through genetic methods. Twins, particularly monozygotic ones and their families may be fitting subjects for studying those microorganisms. The question is: How specific can yeast flora be in an individual? The study aimed to analyze the strain relatedness among commensal yeasts isolated from various parts of the bodies of healthy people and to compare correlations between the genotypes of the isolates. Yeasts were isolated from 63 twins and their family members (n = 25) from the oral cavity, anus, interdigital space and navel. After species identification, Candida albicans (n = 139), C. parapsilosis (n = 39), C. guilliermondii (n = 25), C. dubliniensis (n = 11) and C. krusei (n = 9) isolates were analyzed using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) optimization method. The similarities between the strains were calculated based on the Dice (Sab) coefficient and are displayed graphically as dendrograms. Using cluster analysis, the following relatedness was distinguished: 13 genotypes and three unique (Un) patterns among C. albicans; 10 genotypes and four Un patterns among C. parapsilosis; three genotypes and one Un pattern among C. guilliermondii and C. dubliniensis; and three genotypes among C. krusei isolates. The presence of identical, similar or both genotypes among the strains isolated from family members shows the transmission of yeasts between ontocenoses in the same person and between individuals. The similarity between the genotypes of C. albicans, C. guilliermondii, C. dubliniensis and C. krusei was more remarkable than between the genotypes of C. parapsilosis in the strains isolated from ontocenoses of the same individual and their family members. The degrees of genetic similarity between Candida spp. strains isolated from monozygotic twins and those obtained from their relatives did not differ.

P082 Synergistic activity, anti-adherence and anti-fungal abilities of fluconazole and voriconazole combined with thymol and carvacrol against C andida species

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThe current study aimed to assess the anti-adherence and antifungal activities of thymol and carvacrol against Candida albicans, C. glabrata, and C. krusei isolates obtained from patients with oral candidiasis concerning growth inhibition and fungal death as compared to the synthetic antifungals such as fluconazole and voriconazole.MethodsThe susceptibility assay for the test compounds was performed using the disk diffusion method against all Candida isolates. Also, anti-adherence activity was examined using a rapid and highly reproducible 96 well microtiter-based method.ResultsBoth natural phenols and antifungal drugs revealed various efficacies against studied Candida species. The susceptibility to fluconazole and voriconazole were 100% for C. albicans, 50% and 90% for C. glabrata, and 0% and 100% for C. krusei isolates, respectively. The mean diameter of the inhibition zone was greater for thymol than carvacrol in C. albicans (19.89-0.80 mm vs 17.05-0.61 mm), C. glabrata (18.87-0.71 mm vs 15.77-0.57 mm), and C. krusei (15.11-0.91 mm vs 13.91-1.04 mm) isolates tested.Thymol showed more effective inhibition on adherence of all Candida species than other treatments. The mean relative adherence ratios for C. albicans, C. glabrata, and C. krusei were 0.50, 0.60, and 0.64, respectively.ConclusionsThis study demonstrated significant inhibitory properties of thymol and carvacrol on the adherence and growth of azole susceptible- and -resistant Candida isolates. Also, thymol was more effective for preventing the adherence of yeast cells to polystyrene in comparison to carvacrol.

S2.4d Combination antifungal effects of eugenol with voriconazole against Candida tropicalis and Candida krusei strains isolated from the genital tract of mares

S2.4 Veterinary mycology research, September 21, 2022, 3:00 PM - 4:30 PM ObjectivesThe objective of the current study was to investigate the possibility that eugenol synergizes the antifungal effects of voriconazole on genital isolates of Candida krusei and Candida tropicalis from mares.MethodsThe antifungal activity of eugenol and voriconazole was evaluated using the broth microdilution assay (CLSI- M27-A3). The synergism of eugenol and voriconazole against genital Candida isolates was evaluated by the microdilution checkerboard method.ResultsMinimum inhibitory concentration (MIC) values for eugenol and voriconazole ranged from 400 to 800 μg/ml and 1 to 8 μg/ml, respectively, for C. tropicalis isolates, and from 200 to 400 μg/ml for eugenol and 2 to 16 μg/ml for voriconazole against C. krusei isolates. Eugenol decreased the arithmetic mean of MIC for voriconazole against C. tropicalis and C. krusei isolates from 2.66 to 0.46 μg/ml and 7.77 to 0.41 μg/ml respectively. The fractional inhibitory concentration index (FICI) values for the eugenol voriconazole combination ranged from 0.25 to 0.88 and 0.19 to 0.63 for C. tropicalis and C. krusei isolates respectively. A synergistic effect of eugenol in combination with voriconazole was observed for 83.3% of C. tropicalis and 77.7% of C. krusei isolates. The antagonistic activity was not seen in any of the isolates tested.ConclusionsEugenol showed fungistatic and fungicidal effects against genital Candida isolates and, in combination, synergized the antifungal effects of voriconazole. The eugenol-voriconazole combination can lay the foundation for a therapeutic approach against isolates in which azole resistance has increased over time.

P008 Molecular mechanisms associated with fluconazole resistance and genetic diversity in clinical Candida krusei isolates from North India

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives Candida krusei accounts for 2.8% of invasive candidiasis worldwide. Fluconazole resistance and its underlying mechanism in clinical isolates of C. krusei (n = 137) collected from eight hospitals in India were investigated. Also, genetic diversity of C. krusei strains among different hospitals was studied through short tandem repeat (STR) genotyping.Material and MethodAll the isolates were identified by MALDI-TOF MS. Antifungal susceptibility test was done by using broth microdilution method (CLSI-M27). To evaluate the genetic relatedness among the strains, STR typing was done by using 9 STR markers. To understand the fluconazole-resistant mechanisms in C. krusei, known fluconazole resistance mechanisms such as alterations in target enzyme ERG11 and drug transporters ABC1, and ABC2 were investigated in 35 C. krusei isolates [18 fluconazole-susceptible (FLU-S), and 17 fluconazole-susceptible dose-dependent (FLU-SDD)]. Furthermore, transcriptomics of one FLU-SDD (MIC 32 mg/L) and one FLU-S (MIC 4 mg/l) isolate was performed.ResultsMajority (77%) of C. krusei isolates were from bloodstream infections. Notably, 70% of candidemia cases occurred in neonatal intensive care units (NICUs). Remarkably, 81% (n = 110) were detected as fluconazole-SDD (MIC 16-32 mg/l), and the remaining 19% were FLU-S (MIC ≤ 8 mg/l). Marked genetic diversity with 51 diverse STR types was noticed among the 106 isolates. Interestingly, two ongoing candidemia outbreaks were observed in two geographically separated hospitals both representing NICU isolates. In addition, a large cluster containing isolates from six different hospitals was observed. ERG11 mutation analysis revealed that it did not harbor any mutation contributing to the flu-resistance. Overexpression of the ABC1 gene in 11 FLU-SDD isolates out of 17 as compared to FLU-S isolates was noted. However, no alteration was observed in the expression of ERG11 and ABC2 in both groups.Transcriptomics analysis revealed a significant number of differentially regulated genes were distributed in various gene-ontology terms including transport (10 genes), mitogen-activated protein kinase (MAPK) signaling (8 genes, MSG5, PTP3, STE50, BNR1, OPY2, STE5, SKN7, and RLM1), ergosterol biosynthesis (3 genes, ERG24, ERG25, and ERG26) and transcription factors (7 genes). In addition to the up-regulation of ergosterol pathway genes, overexpression of key transcriptional regulator of ergosterol biosynthesis genes UPC2 was observed in FLU-SDD isolates as compared with susceptible. Additionally, FLU-SDD isolate showed 2-fold increased expression of PDR12, plasma membrane ATP-binding cassette (ABC) transporter. Next, ICL1 (Isocitrate Lyase), a major glyoxylate-synthesizing enzyme was found to be 5-fold down-regulated in FLU SDD isolate compared to susceptible. The loss of ICL1 alters the expression of the FKS1, ERG11, and CDR2 genes in C. albicans. Taken together, the increased expression of PDR12 and altered MAPK singling network may partially account for the FLU resistance in C. krusei FLU-SDD isolate.Conclusion Candida krusei isolates among different hospitals showed large genetic diversity (54 different genotypes). Also, the presence of C. krusei clonal strains in six different hospitals suggests possible introduction from a widespread environmental source and human-to-human transmission. In comparison to other Candida species, the resistant mechanism in C. krusei seems to be more complex. Therefore, an in-depth study of other resistance mechanism pathways in C. krusei is further warranted.

Distribution, trends, and antifungal susceptibility of Candida species causing candidemia in Japan, 2010-2019: A retrospective observational study based on national surveillance data.

The increasing incidence of candidemia and the emergence of drug-resistant Candida species are major concerns worldwide. Therefore, long-term surveillance studies are required. Here, we provide one of the largest longitudinal overviews of the trends in the prevalence of Candida species using national data of 57 001 candidemia isolates obtained from > 2000 hospitals for the 2010–2019 period in the Japan Nosocomial Infections Surveillance database. The proportion of Candida species, except Candida krusei and Candida guilliermondii, was almost the same during the study period. The proportion of C. guilliermondii surpassed that of C. krusei in 2014. The incidence of candidemia due to C. albicans (P < 0.0001), C. parapsilosis (P = 0.0002), and C. tropicalis (P < 0.0001) have decreased significantly over this period. Azole susceptibility of C. tropicalis was low, with 17.8% of isolates resistant to fluconazole and 13.5% resistant to voriconazole. The micafungin susceptibility of C. glabrata was low, with 8.0% of isolates showing resistance. The resistance rate of C. krusei toward amphotericin B fluctuated considerably (between 3.2% and 35.7%) over this period. The incidence rate of candidemia caused by C. parapsilosis and C. guilliermondii in hospitals responsible for bone marrow transplantation was significantly higher than that in other hospitals. Overall, our study suggests that in Japan, the species distribution of Candida was almost the same in this period and similar to that reported in North America and Europe. A relatively high resistance to azoles and micafungin was observed in C. glabrata, C. tropicalis, and C. krusei isolates, which require continued surveillance.

Combining Essential Oils with Each Other and with Clotrimazole Prevents the Formation of Candida Biofilms and Eradicates Mature Biofilms.

Fungal infections by Candida spp. are opportunistic and most often occur in individuals with some predisposing factor. Essential oils (EO) have anti-Candida potential, being a therapeutic alternative to be explored, especially for superficial and mucosal candidiasis. The objective was to analyze the synergistic potential between the EO of Citrus limon, Cupressus sempervirens, Litsea cubeba and Melaleuca alternifolia, and each of them with clotrimazole, to inhibit in vitro the formation and eradication of Candida spp. biofilms. Added to this, the survival of Caenorhabditis elegans was evaluated after exposure to EO, clotrimazole and their synergistic combinations. Anti-Candida activity was determined by microdilution for the substances alone and in EO–EO and EO–clotrimazole combinations. The combinations were performed by the checkerboard method, and the reduction in the metabolic activity of biofilms was determined by the viability of MTT/menadione. C. elegans larvae survival was evaluated after 24 h of exposure to EO, clotrimazole and synergistic combinations. The minimum inhibitory concentration (MIC) of EO ranged from 500 to >4000 µg/mL. The lowest MIC (500 µg/mL) was for C. sempervirens and L. cubeba on a C. krusei isolate; for clotrimazole, the MIC ranged from 0.015 to 0.5 µg/mL. Biofilm inhibition and eradication both ranged from 1000 to >4000 µg/mL. The lethal concentration (LC50) of C. limon, L. cubeba and M. alternifolia was 2000 µg/mL for C. elegans, while for C. sempervirens and clotrimazole, it was not determined within the concentration limits tested. In combination, more than 85% of the larvae survived M. alternifolia–clotrimazole, M. alternifolia–L. cubeba, C. sempervirens–clotrimazole and C. sempervirens–C. limon combinations. This study is the first, to our knowledge, to present a synergistic relationship of EO–EO and EO–clotrimazole combinations on Candida spp. biofilms.

Whole genome sequencing data of Candida krusei, pathogen causing Candidaemia, from Department of Parasitology Culture Collection, Faculty of Medicine Universitas Indonesia

Candida krusei is a Candida non-albicans species with a high prevalence, which causes candidaemia. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections; however, it is only a fungistatic against Candida spp., and both inherent and acquired resistance to fluconazole have been reported. C. krusei species is also reported as the only Candida sp. which has an intrinsic resistance factor to fluconazole. Therefore, in dealing with antifungal resistance, it is necessary to develop new antifungal agents that are efficient in the treatment of fungal infections, especially those caused by C. krusei. The purpose of this study was to investigate the genome of clinical C. krusei isolates and correlate the resistant phenotypes with mutations in resistance genes. A total of 16 samples of C. krusei from clinical samples from hospitals in Jakarta were used in the experiment. All colonies were extracted using the QIAamp DNA Mini Kit. The library was prepared using the Illumina DNA Prep Kit. The sequencing process was carried out on the Illumina MiSeq Platform using a 2x301 paired-end configuration. FASTQ raw files are available under the BioProject Accession Number PRJNA819536 and Sequence Read Archive Accession Numbers SRR18739949 and SRR18739964.

Investigation of the Efficacy and Antifungal Drug Resistance of Non-Albicans Candida Species in Mycotic Mastitis

This research aimed to use a conventional and molecular approach to determine Candida species isolated from cattle clinical mastitis and examine their antifungal susceptibility. In this research, 100 milk samples with mastitis were collected from dairy farms in five different Izmir districts. On chromogenic agar, 23 Candida isolates were isolated from milk samples with mastitis, and 20 (87.0%) isolates were identified as C. krusei, two (8.6%) isolates as C. albicans, and one (4.3%) isolate as C. tropicalis. PCR analysis verified that all Candida isolates were C. krusei, C. albicans, and C. tropicalis. According to sequence analysis, it was determined that 11 of the C. krusei isolates had &amp;gt;97% similarity to “Pichia kudriavzeii ZKI117 5.8S ribosomal gene”; six of the C. krusei isolates to “Pichia kudriavzeii isolate L-012 small subunit ribosomal RNA gene”; two C. krusei isolates to “Pichia kudriavzeii isolate 3 internal transcribed spacer 1”; and one C. krusei isolates to “Pichia kudriavzeii LL11_078 18S ribosomal gene”; two C. albicans isolates to “Candida albicans isolate B02 5.8S ribosomal gene”; one C. tropicalis isolate to “Candida tropicalis isolate CTR1201 18S ribosomal gene”. In the antifungal susceptibility test results, all Candida isolates (%100) were resistant to metronidazole and flucytosine. Consequently, it was determined that identifying Candida agents and determining antifungal susceptibility in farms with mycotic mastitis will ensure the application of correct treatment protocols and reduce economic losses due to fungal mastitis.

Potential antifungal impact of citral and linalool administered individually or combined with fluconazole against clinical isolates of Candida krusei

Introduction: Candida krusei is recognized as a major fungal pathogen in patients with immunodeficiency disorders. The present study aimed at investigating the anticandidal activities of citral and linalool combined with fluconazole (FLZ) against FLZ-resistant C. krusei strains. Methods: Antifungal activities were evaluated by the broth microdilution (MD) method to determine the minimum inhibitory and fungicidal concentrations (namely, MICs and MFCs) according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document. The interactions were further evaluated using fractional inhibitory concentration indices (FICIs) for combinations of citral+FLZ and linalool+FLZ, calculated from checkerboard MD assays. Results: The mean ± standard deviation (SD) MIC values of citral, linalool, and FLZ against the C. krusei isolates were 70.23 ± 17, 150 ± 38.73, and 74.66 ± 36.95 μg/mL, respectively. Some fungicidal activities were also observed for citral (2.5) and linalool (1.53) against the C. krusei isolates. The FICI values of citral+FLZ and linalool+FLZ for the C. krusei isolates ranged from 0.4 to 1.00 and 0.19 to 0.63, respectively. The additive and synergistic interactions of linalool + FLZ were further observed in 12 (57.1%) and 9 (42.9%) C. krusei isolates. However, there was an additive interaction for citral + FLZ in 17 (80.9%) isolates. They also showed a synergistic interaction in only four (19.1%) isolates. Moreover, linalool and citral plus FLZ did not have any antagonistic effect on any isolates. Conclusion: The study findings support the possible capabilities of citral and linalool, as anticandidal agents, and FLZ might be supplemented with citral and/or linalool for treating FLZ-resistant C. krusei infections.