High-concentration COD wastewater treatment with simultaneous removal of nitrogen and phosphorus by a novel Candida tropicalis strain: Removal capability and mechanism.

Aerobic and anaerobic continuous stirred-tank reactor (CSTR), up-flow anaerobic sludge blanket (UASB) were set up and inoculated with newly isolated Candida tropicalis. Reactors were operated at high concentrations of chemical oxygen demand (COD) (8000 mg/L), the modified UASB expressed better COD removal rate simultaneously removal of nitrogen and phosphate than other two reactors. Notably, under both aerobic or anaerobic conditions, large amounts of organic acids and alcohol were generated. Transcriptomic analysis showed that carbon metabolism under anaerobic conditions shared the same pathway with aerobic conditions by regulating and inhibiting some functional genes. Experiments utilizing different carbon sources proved that our strain has excellent performances in utilizing organic materials, which were verified by transcriptomic analysis. Finally, the strain was applied to treat four types of sugar-containing wastewaters. Among them, our strain exerts the best removal capability of COD (90%), nitrogen (89%), and phosphate (82%) for brewery wastewater.

Molecular epidemiology of Candida tropicalis isolated from urogenital tract infections.

Candida tropicalis is a common human pathogenic yeast, and its molecular typing is important for studying the population structure and epidemiology of this opportunistic yeast, such as epidemic genotype, population dynamics, nosocomial infection, and drug resistance surveillance. In this study, the antifungal susceptibility test and multilocus sequence typing (MLST) analysis were carried out on C. tropicalis from central China. Among 64 urogenital isolates, 45 diploid sequence types (DST) were found, of which 20 DSTs (44.4%) were new to the central database. The goeBURST analysis showed that CC1 (clonal complex) was the only azole-resistant (100%, 10/10) cluster in Wuhan, which was composed of DST546, DST225, DST376, and DST506, and most of the strains (90%, 9/10) were isolated from the urinary tract. Potential nosocomial infections were mainly caused by CC1 strains. The azole resistance rate of urinary isolates (50.0%, 21/42) was higher than that of vaginal isolates (27.3%, 6/22). The genotype diversity and novelty of vaginal isolates were higher than those of urinary isolates. C. tropicalis population in Wuhan was genetically diverse and divergent from that seen in other countries. In this study, there were significant differences in genotype and azole susceptibility between urine and vaginal strains. The azole-resistant cluster (CC1) found in urine is of great significance for the clinical treatment and prevention of nosocomial infection. The newly discovered DSTs will contribute to further study the similarity, genetic relationship, and molecular epidemiology of C. tropicalis worldwide.

Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment.

Candida tropicalis arises as one of the predominant non-Candida albicans Candida (NCAC) species causing invasive candidiasis in Asian countries. A rise in reports of C. tropicalis with a parallel increase in fluconazole resistance has also been observed. The genes and underlying pathways associated with azole antifungal resistance in C. tropicalis is still not properly understood. The RT-qPCR is the most promising approach for expression analysis of target genes to understand the mechanisms of resistance. The reliability and reproducibility of this technique depend on the selection of suitable reference genes for the normalization in expression study. The present study investigated the expression stability levels of ten genes including ACT1, EF1, GAPDH, PGK1, RDN5.8, RDN18, RDN28, SDHA, TUB1, and UBC13 for their suitability in fluconazole treated/untreated C. tropicalis. The stability levels of these genes were examined by the ∆∆CT, ΔCT, Pfaffl methods and five independent software including hkgFinder, geNorm, NormFinder, BestKeeper, and RefFinder software. We report, the EF1 and ACT1 were the most stable reference genes for normalization and can be used for the gene expression analysis in C. tropicalis. To the best of our knowledge, our study is the first to select and validate the reference genes in C. tropicalis for RT-qPCR based expression analysis.

Clonality of Fluconazole-Nonsusceptible Candida tropicalis in Bloodstream Infections, Taiwan, 2011-2017.

Candida tropicalis is the leading cause of non-C. albicans candidemia in tropical Asia and Latin America. We evaluated isolates from 344 patients with an initial episode of C. tropicalis candidemia. We found that 58 (16.9%) patients were infected by fluconazole-nonsusceptible (FNS) C. tropicalis with cross resistance to itraconazole, voriconazole, and posaconazole; 55.2% (32/58) of patients were azole-naive. Multilocus sequence typing analysis revealed FNS isolates were genetically closely related, but we did not see time- or place-clustering. Among the diploid sequence types (DSTs), we noted DST225, which has been reported from fruit in Taiwan and hospitals in Beijing, China, as well as DST376 and DST505-7, which also were reported from hospitals in Shanghai, China. Our findings suggest cross-boundary expansion of FNS C. tropicalis and highlight the importance of active surveillance of clinical isolates to detect dissemination of this pathogen and explore potential sources in the community.

Repurposing of Ribavirin as an Adjunct Therapy against Invasive Candida Strains in an In Vitro Study.

The use of antifungal agents in clinical settings is limited by the appearance of drug resistance and adverse side effects. There is, therefore, an urgent need to develop new drugs to strengthen the treatment of invasive fungal diseases. The aim of this study is to describe the potential repurposing of ribavirin as an adjunct therapy against Candida spp. Primary screening of a Prestwick Chemical library against Candida albicans ATCC 90028 and fluconazole-resistant Candida albicans strains was performed. Subsequently, we evaluated the responses of 100 Candida sp. strains to ribavirin, an antiviral agent, using the broth microdilution method as recommended by CLSI. We checked the involvement of efflux pump activity in the development of ribavirin resistance. We studied time-kill curves and performed a checkerboard assay for a ribavirin-antifungal combination study. Twenty-one nonstandard antifungal compounds were identified, including ribavirin. Ribavirin had antifungal activity in vitro against 63 Candida strains, including strains of C. albicans, C. parapsilosis, and C. tropicalis, with MICs ranging from 0.37 to 3.02 µg/ml, while MICs for C. krusei, C. glabrata, C. lusitaniae, and some C. albicans strains remained high (≥24.16 µg/ml). No relation was observed between efflux pump activity and ribavirin resistance. Ribavirin exhibited fungistatic activity against multidrug-resistant (MDR) C. albicans and fungicidal activity against a C. parapsilosis strain. In addition, ribavirin acted synergistically with azoles against Candida strains for which ribavirin MICs were <24.4 µg/ml. This study highlights the potential clinical application of ribavirin, alone or in association with other antifungal agents, as an adjunct anti-Candida drug.

Delay in effective therapy in anidulafungin-resistant Candida tropicalis fungaemia: Potential for rapid prediction of antifungal resistance with whole-genome-sequencing.

OBJECTIVES: This study investigated the feasibility of using whole-genome sequencing (WGS) for the prediction of antifungal resistance in anidulafungin-resistant Candida tropicalis candidaemia isolates. METHODS: Next-generation sequencing was performed for three anidulafungin-resistant C. tropicalis isolates on an Illumina MiSeq system with in-house bioinformatics analysis. RESULTS: Mutations in Fks1p associated with anidulafungin resistance were identified. Other mutations associated with varying levels of phenotypic resistance to fluconazole were also identified. CONCLUSIONS: These data demonstrate the potential to predict antifungal resistance using WGS. With improving technology, real-time WGS may be used for tailoring effective antifungal therapy in patients with candidaemia.

Development of mazF-based markerless genome editing system and metabolic pathway engineering in Candida tropicalis for producing long-chain dicarboxylic acids.

Candida tropicalis can grow with alkanes or plant oils as the sole carbon source, and its industrial application thus has great potential. However, the choice of a suitable genetic operating system can effectively increase the speed of metabolic engineering. MazF functions as an mRNA interferase that preferentially cleaves single-stranded mRNAs at ACA sequences to inhibit protein synthesis, leading to cell growth arrest. Here, we constructed a suicide plasmid named pPICPJ-mazF that uses the mazF gene of Escherichia coli as a counterselectable marker for the markerless editing of C. tropicalis genes to increase the rate of conversion of oils into long-chain dicarboxylic acids. To reduce the ß-oxidation of fatty acids, the carnitine acetyltransferase gene (CART) was deleted using the gene editing system, and the yield of long-chain acids from the strain was increased to 8.27 g/L. By two homologous single exchanges, the promoters of both the cytochrome P450 gene and the NADPH-cytochrome P450 reductase gene were subsequently replaced by the constitutively expressed promoter pGAP, and the production of long-chain dicarboxylic acids by the generated strain (C. tropicalis PJPP1702) reached 11.39 g/L. The results of fed-batch fermentation showed that the yield of long-chain acids from the strain was further increased to 32.84 g/L, which was 11.4 times higher than that from the original strain. The results also showed that the pPICPJ-mazF-based markerless editing system may be more suited for completing the genetic editing of C. tropicalis.

Development of Echinocandin Resistance in Candida tropicalis following Short-Term Exposure to Caspofungin for Empiric Therapy.

Isolation of two echinocandin-resistant Candida tropicalis strains from endotracheal secretions of a patient following short-term exposure to caspofungin is described. Both strains exhibited resistance to echinocandins by Etest and reference broth microdilution, showing a homozygous S645P mutation within the hot spot 1 (HS-1) region of FKS1 and belonging to a unique multilocus sequence type. Other C. tropicalis isolates collected from patients in the same intensive care unit within a 60-day period were susceptible to echinocandins and contained wild-type FKS1 sequences.

Hemolytic capability and expression of a putative haem oxygenase-encoding gene by blood isolates of Candida tropicalis are influenced by iron deprivation and the presence of hemoglobin and erythrocytes.

Although hemolytic activity is known to be a putative virulence factor contributing to candidal pathogenesis, its production by Candida tropicalis, a species closely related to Candida albicans, is poor understood. The present study was undertaken to evaluate the hemolytic activity and the expression level of a putative haem oxygenase encoding gene by blood isolates of C. tropicalis following growth in iron deprivation, and in the presence of hemoglobin and erythrocytes. The lowest values of hemolytic activity were observed in cell-free culture supernatants of isolates growing in iron-restricted medium (RPMI medium and RPMI medium supplemented with iron chelator bathophenanthrolindisulphonic acid). Hemolysis was increased in the presence of either hemoglobin or erythrocytes. Reverse transcriptase PCR analysis showed that the putative haem oxygenase encoding gene (CtHMX1), potentially related with iron uptake, was up-regulated (p < 0.001) following growth in iron deprivation and in the presence of hemoglobin; CtHMX1 was repressed in the presence of human erythrocytes (p < 0.001). Our data suggest that hemoglobin had positive effect in the production of hemolytic factor and gene expression related to iron uptake in C. tropicalis.

[Inhibitory effects of butyl alcohol extract of Baitouweng decoction on virulence factors of Candida tropicalis].

OBJECTIVE: To investigate the effects of butyl alcohol extract of baitouweng decoction (BAEB) on the fungal cell surface hydrophobicity (CSH), filamentation and biofilm formation of Candida tropicalis. METHOD: Gradual dilution method was used to determine the MIC. XTT assay was applied to determine the SMIC80. Time-Kill assay was employed to draw the Time-Kill curve. The water-hydrocarbon two-phase assay was used to measure the cell surface hydrophobicity. Scanning electron microscopy (SEM) was applied to observe the morphological changes of the biofilm. Confocal laser scanning microscopy (CLSM) was applied to determine the thickness of the biofilm. The quantification real-time PCR (qRT-PCR) was used to detect expression changes of releated genes (UME6, ALST3 and NRG1). result: The MICs of BAEB against C. tropicalis strains are determined as 64-128 mg x L(-1). The SMIC80 s of BAEB against the biofilm of Candida tropicalis strains are determined as 256-512 mg x L(-1). Time-Kill curve results indicate that BAEB has a promise fungicidal effect at 256 and 512 mg x L(-1). SEM results shows that 512 mg x L(-1) BAEB can inhibit the formation of C. tropicalis biofilm on Silicone catheter, and the morphology of biofilm is also affected by BAEB. The thickness of C. tropicalis biofilm is reduced by BAEB according to CLSM results. Furthermore, qRT-PCR results indicate that expression of UME6 and ALST3 are significantly down-regulated by BAEB 256,512 mg x L(-1), and NRG1 is not affected by BAEB. CONCLUSION: BAEB inhibits effectively the CSH, filamentation and biofilm formation of VVC strains of C. tropicalis.

[Subtractive gene cloning and gene-disruption for elucidation of pseudohyphal formation in Candida tropicalis].

The dimorphic transition from yeast to pseudohyphae in the petroleum-assimilating yeast Candida tropicalis occurs following the addition of ethanol to glucose semi-defined medium. Subtractive gene cloning was performed on the cDNA from the yeast-growing control culture and on that from the ethanol-supplemented one (the ethanol culture). A homologue of Schizosaccharomyces pombe nmt1+ or Saccharomyces cerevisiae THI5 was isolated from the cDNA fraction as a preferentially expressed gene for the ethanol culture. This homologue was tentatively called Ctnmt1+, since exogenous thiamine repressed its expression in C. tropicalis growth media. The ethanol culture showed a biphasic pattern of growth phases and the expression of Ctnmt1+ occurred at the first growth phase. The supplementation of thiamine to the ethanol culture at the first phase was followed by repression of Ctnmt1+ expression and also delay of pseudohyphal growth: filamentous growth was inhibited and chains of yeast cells were formed. A Ctnmt1+ disruptant of this organism did not show thiamine auxotrophy and produced pseudohyphal filaments even in the control culture. The supplementation of oxythiamine, an analog of thiamine, to the control culture was followed by the appearance of pseudohyphal filaments, indicating the participation of thiamine during the process of pseudohyphal growth in this organism.