Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

OBJECTIVES: We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. METHODS: Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. RESULTS: Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1-29 months. Clusters harbouring Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2-8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. CONCLUSIONS: This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.

Draft Genome Sequence of Candida saopaulonensis from a Very Premature Infant with Sepsis.

The rare fungus Candida saopaulonensis has never been reported to be associated with human infection. We report the draft genome sequence of the first clinical isolate of C. saopaulonensis, which was isolated from a very premature infant with sepsis. This is the first genome assembly reaching the near-complete chromosomal level with structural annotation for this species, opening up avenues for exploring evolutionary patterns and genetic mechanisms of pathogenesis.

Complete Genome Sequence of Candida mucifera from an Otitis Media Patient.

We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. This pathogen exhibited reduced azole susceptibility, similar to its close relatives within the Trichomonascus ciferrii species complex.

The first established microsatellite markers to distinguish Candida orthopsilosis isolates and detection of a nosocomial outbreak in China.

The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration.

ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

Zeb1 Is a Potential Regulator of Six2 in the Proliferation, Apoptosis and Migration of Metanephric Mesenchyme Cells.

Nephron progenitor cells surround around the ureteric bud tips (UB) and inductively interact with the UB to originate nephrons, the basic units of renal function. This process is determined by the internal balance between self-renewal and consumption of the nephron progenitor cells, which is depending on the complicated regulation networks. It has been reported that Zeb1 regulates the proliferation of mesenchymal cells in mouse embryos. However, the role of Zeb1 in nephrons generation is not clear, especially in metanephric mesenchyme (MM). Here, we detected cell proliferation, apoptosis and migration in MM cells by EdU assay, flow cytometry assay and wound healing assay, respectively. Meanwhile, Western and RT-PCR were used to measure the expression level of Zeb1 and Six2 in MM cells and developing kidney. Besides, the dual-luciferase assay was conducted to study the molecular relationship between Zeb1 and Six2. We found that knock-down of Zeb1 decreased cell proliferation, migration and promoted cell apoptosis in MM cells and Zeb1 overexpression leaded to the opposite data. Western-blot and RT-PCR results showed that knock-down of Zeb1 decreased the expression of Six2 in MM cells and Zeb1 overexpression contributed to the opposite results. Similarly, Zeb1 promoted Six2 promoter reporter activity in luciferase assays. However, double knock-down of Zeb1 and Six2 did not enhance the apoptosis of MM cells compared with control cells. Nevertheless, double silence of Zeb1 and Six2 repressed cell proliferation. In addition, we also found that Zeb1 and Six2 had an identical pattern in distinct developing phases of embryonic kidney. These results indicated that there may exist a complicated regulation network between Six2 and Zeb1. Together, we demonstrate Zeb1 promotes proliferation and apoptosis and inhibits the migration of MM cells, in association with Six2.

Residue Degradation and Risk Assessment of Difenoconazole and Its Metabolite during Tea Growing, Processing and Brewing by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Determination.

Residue dissipation and risk assessment of difenoconazole and its metabolite difenoconazole-alcohol during tea growing, processing, and brewing was first investigated by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The limits of quantification for both difenoconazole and difenoconazole-alcohol were 0.001 mg/kg in fresh tea leaves and tea, and 0.0002 mg/L in tea infusion. In field trials, the dissipation half-lives of difenoconazole in fresh tea leaves was 1.77 days. After spraying, the residues of difenoconazole-alcohol increased and then gradually dissipated like difenoconazole. After 14 days, the dissipation rates of difenoconazole and difenoconazole-alcohol reached 99%. When fresh tea leaves were harvested on different days, the total processing factors (PFs) of difenoconazole and difenoconazole-alcohol for green tea were 0.86-1.05 and 0.78-0.85, respectively, while the total PFs for black tea were 0.83-1.13 and 0.82-1.66, respectively. Metabolism of difenoconazole was accelerated during tea processing. When brewing black tea, the leaching rates (LRs) of difenoconazole and difenoconazole-alcohol were 8.4-17.9% and 31.8-38.9%, respectively, while when brewing green tea, the LRs were 15.4-23.5% and 30.4-50.6%, respectively. The LRs of difenoconazole and difenoconazole-alcohol in black tea were higher than those in green tea. The potential threat to human health for dietary intake of difenoconazole and difenoconazole-alcohol residues from tea consumption is negligible. However, the dietary risk of difenoconazole in fruits and vegetables that are essential for daily diets is concerning, with a risk probability of 158%.

Antifungal heteroresistance causes prophylaxis failure and facilitates breakthrough Candida parapsilosis infections.

Breakthrough fungal infections in patients on antimicrobial prophylaxis during allogeneic hematopoietic cell transplantation (allo-HCT) represent a major and often unexplained cause of morbidity and mortality. Candida parapsilosis is a common cause of invasive candidiasis and has been classified as a high-priority fungal pathogen by the World Health Organization. In high-risk allo-HCT recipients on micafungin prophylaxis, we show that heteroresistance (the presence of a phenotypically unstable, low-frequency subpopulation of resistant cells (~1 in 10,000)) underlies breakthrough bloodstream infections by C. parapsilosis. By analyzing 219 clinical isolates from North America, Europe and Asia, we demonstrate widespread micafungin heteroresistance in C. parapsilosis. Standard antimicrobial susceptibility tests, such as broth microdilution or gradient diffusion assays, which guide drug selection for invasive infections, fail to detect micafungin heteroresistance in C. parapsilosis. To facilitate rapid detection of micafungin heteroresistance in C. parapsilosis, we constructed a predictive machine learning framework that classifies isolates as heteroresistant or susceptible using a maximum of ten genomic features. These results connect heteroresistance to unexplained antifungal prophylaxis failure in allo-HCT recipients and demonstrate a proof-of-principle diagnostic approach with the potential to guide clinical decisions and improve patient care.

Decreased echinocandin susceptibility in Candida parapsilosis causing candidemia and emergence of a pan-echinocandin resistant case in China.

Candida parapsilosis is becoming a predominant non-albicans cause of invasive candidiasis (IC). Echinocandins are the preferred choice for IC treatment and prophylaxis. Resistance to echinocandins in C. parapsilosis has emerged in several countries, but little is known about the susceptibility profile in China or about mechanisms of resistance. Here, we investigated the echinocandin susceptibilities of 2523 C. parapsilosis isolates collected from China and further explored the resistance mechanism among echinocandin-resistant isolates. Anidulafungin exhibited the highest MICs (MIC50/90, 1 and 2 µg/mL; GM, 0.948 µg/mL), while caspofungin showed better activity (0.5 and 1 µg/mL; 0.498 µg/mL). Significantly higher echinocandin MICs were observed among blood-derived isolates compared to others, especially for caspofungin (GM, 1.348 µg/mL vs 0.478 µg/mL). Isolates from ICU and surgical wards also showed higher MICs. Twenty isolates showed intermediate phenotypes for at least one echinocandin. One was resistant to all three echinocandins, fluconazole and voriconazole, which caused breakthrough IC during long-term exposure to micafungin. WGS revealed this isolate carried a mutation S656P in hotspot1 region of Fks1. Bioinformatics analyses suggested that this mutation might lead to an altered protein conformation. CRISPR Cas9-mediated introduction of this mutation into a susceptible reference C. parapsilosis strain increased MICs of all echinocandins 64-fold, with similar results found in the subspecies, C. orthopsilosis and C. metapsilosis. This is the first report of a multi-azole resistant and pan-echinocandin resistant C. parapsilosis isolate, and the identification of a FKS1S656P conferring pan-echinocandin resistance. Our study underscores the necessity of rigorous management of antifungal use and of monitoring for antifungal susceptibility.

Simultaneously Achieving Colossal Permittivity, Ultralow Dielectric Loss Tangent, and High Insulation Resistivity in Er-Doped SrTiO3 Ceramics via Oxygen Vacancy Regulation.

High-performance Sr1-xErxTiO3 (x = 0-0.014) ceramics were sintered in different atmospheres using the conventional solid-state reaction method. The phase structure and micromorphology of ceramics were analyzed using X-ray diffraction and scanning electron microscopy. Meanwhile, the Sr1-xErxTiO3 (when x = 0.012) ceramic sintered in hydrogen attains a colossal permittivity (132 543 @1 kHz, 157 650 @1 MHz) and ultralow tan δ (0.009 @1 kHz, 0.03 @1 MHz) and has good frequency stability (20 Hz to 2 MHz) and temperature stability (-180 to 425 °C). X-ray photoelectron spectroscopy, electron paramagnetic resonance, and impedance analysis show that the colossal permittivity and ultralow dielectric loss are attributed to the defect dipoles and defect clusters [TiTi'-VO••-TiTi'], [ErSr•-TiTi'], [2ErTi'-VO••], and [ErSr•-ErTi']. The insulation resistivity is determined by the grain boundary. The dielectric properties of samples sintered in hydrogen are excellent, and then, the oxidation method is used to backfill the oxygen vacancy (VO••), thus improving the insulation resistivity (2.8 × 1014 Ω cm) of the grain boundary. In addition, the diffusion mechanism of ceramic VO•• from low, medium, and high temperatures was studied by monitoring VO•• behavior in real time. The results reveal that the diffusion coefficient of VO•• in the grain boundary is greater than that in the grain; as a result, as the external oxygen partial pressure rises, the VO•• escapes first from the grain boundary. When the external oxygen partial pressure decreases, oxygen atoms enter the grain boundary region first and backfill oxygen vacancies.

Proteomic Analysis of Copper Toxicity in Human Fungal Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an invasive human fungal pathogen that causes more than 181,000 deaths each year. Studies have demonstrated that pulmonary C. neoformans infection induces innate immune responses involving copper, and copper detoxification in C. neoformans improves its fitness and pathogenicity during pulmonary C. neoformans infection. However, the molecular mechanism by which copper inhibits C. neoformans proliferation is unclear. We used a metallothionein double-knockout C. neoformans mutant that was highly sensitive to copper to demonstrate that exogenous copper ions inhibit fungal cell growth by inducing reactive oxygen species generation. Using liquid chromatography-tandem mass spectrometry, we found that copper down-regulated factors involved in protein translation, but up-regulated proteins involved in ubiquitin-mediated protein degradation. We propose that the down-regulation of protein synthesis and the up-regulation of protein degradation are the main effects of copper toxicity. The ubiquitin modification of total protein and proteasome activity were promoted under copper stress, and inhibition of the proteasome pathway alleviated copper toxicity. Our proteomic analysis sheds new light on the antifungal mechanisms of copper.

Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS.

Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.

Molecular Characterization of Candida parapsilosis by Microsatellite Typing and Emergence of Clonal Antifungal Drug Resistant Strains in a Multicenter Surveillance in China.

Candida parapsilosis is an important species causing invasive candidiasis (IC) in China. The present survey was a national multicenter study of the molecular epidemiology and antifungal susceptibility profiles of C. parapsilosis. Non-duplicate C. parapsilosis isolates were collected from 10 hospitals across China in the CHIF-NET program 2016-2017. Isolates were genotyped using four highly polymorphic microsatellite markers, and susceptibility profiles determined using Sensititre YeastOneTM YO10. A total of 319 C. parapsilosis from separate patients with IC were studied; 49.2, 17.9, and 10.3% isolates were from patients in surgical departments, general intensive care units (ICUs) and neonatal ICUs (NICU), respectively. C. parapsilosis showed good susceptibility to nine antifungal drugs. Microsatellite analysis identified 122 microsatellite (MT) types. Most MT types had sporadic distribution. However, we identified 32 clusters across 10 hospitals; seven clusters were caused by seven endemic genotypes involving five or more isolates in hospitals designated as H01, H02, H06, and H10. These clusters mainly affected surgical departments and ICUs, except for genotype MT42 which was seen in 22 patients from NICU (hospital H06). Of 16 fluconazole-resistant isolates, seven from hospital H02 shared the same genotype MT70, and three from hospital H04 were of genotype MT47. For 37 isolates with non-wild type MICs to 5-flucytosine, 29 were from hospital H01 (genotype MT48). Here we present the first nationwide molecular epidemiology study of C. parapsilosis in China, identified several previously unrecognized clusters, which included antifungal drug resistant isolates. These findings provide important data for control of IC in China.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China.

Invasive aspergillosis (IA) due to Aspergillus lentulus is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by A. lentulus in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive A. lentulus infection. Phenotypic differences were observed for A. lentulus isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with Aspergillus fumigatus complex. ITS sequencing was unable to distinguish A. lentulus from A. fumigatus, but sequencing of the benA, CaM, and rod A loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the Aspergillus section Fumigati while the benA gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All A. lentulus isolates showed in vitro resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 µg/ml), itraconazole (MIC 2 µg/ml), voriconazole (MIC of 4-16 µg/ml), and posaconazole (MIC of 0.5-1 µg/ml). However, MECs for the echinocandin drugs ranged from 0.03-0.25, ≤0.008-0.015, and ≤0.015-0.03 µg/ml for caspofungin, micafungin, and anidulafungin, respectively. A. lentulus is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.

Epidemiology And Antifungal Susceptibility Patterns Of Invasive Fungal Infections From 2012 To 2014 In A Teaching Hospital In Central China.

INTRODUCTION: As participants of the national China Hospital Invasive Fungal Surveillance Net program, we sought to describe the epidemiology and antifungal susceptibility patterns of yeast isolates obtained from patients with invasive fungal infection at the First Affiliated Hospital of Zhengzhou University, China. METHODS: A total of 434 yeast isolates recovered from blood and other sterile body fluids were identified to species by matrix-assisted laser desorption ionization -time of flight mass spectrometry with or without supplementation by DNA sequencing. Antifungal susceptibilities were determined by Sensititre YeastOneTM YO10 methodology. RESULTS: Candida albicans was the most common causative species (33.9% of isolates) but significantly decreased in frequency from 37.2% to 27.7% from 2012 to 2014. C. tropicalis was the next most common pathogen (25.1%), followed by C. parapsilosis complex (17.3%), C. glabrata (9%), and C. pelliculosa (6.7%), with other species comprising 8% of isolates. Caspofungin, micafungin, and anidulafungin exhibited potent in vitro activities against the majority of Candida isolates. Azoles demonstrated in vitro activities against C. albicans with a susceptibility rate of >95% and against C. parapsilosis complex, >95% isolates were susceptible. Among C. tropicalis and C. glabrata isolates, resistance rates to fluconazole and voriconazole were 11.9%, 9.1% and 7.7%, 28.2%, respectively. Of note, C. pelliculosa had a high incidence rate in newborns and high rates of resistance to fluconazole and voriconazole of 55.2% and 41.4%, respectively. CONCLUSION: The present study provided valuable local surveillance data on the epidemiology and antifungal susceptibilities of invasive yeast species, which is essential for guiding antifungal treatment protocol development.