Genome-wide analysis of in vivo-evolved Candida auris reveals multidrug-resistance mechanisms.

Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris.

Species distribution and antifungal susceptibilities of clinical isolates of Penicillium and Talaromyces species in China.

Morphologically identified Penicillium (n = 103) and Talaromyces marneffei (n = 8) isolates were collected from various clinical sources between 2016 and 2017 at a medical centre in Beijing, China. Identification to species level was confirmed by sequencing of the internal transcribed spacer (ITS) region, ß-tubulin gene (benA) and RNA polymerase II second largest subunit (RPB2) gene. Of the 111 isolates, 56 (50.5%) were identified as Penicillium spp. and 55 (49.5%) as Talaromyces spp. Eleven species of Penicillium were detected, of which Penicillium oxalicum was the commonest, accounting for 51.8% (29/56), followed by Penicillium rubens (10.7%; 6/56) and Penicillium citrinum (10.7%; 6/56). Among the 55 Talaromyces isolates, nine species were identified, with Talaromyces funiculosus (36.4%; 20/55), Talaromyces stollii (27.3%; 15/55) and Talaromyces marneffei (14.5%; 8/55) being the most common. Of note, 89.3% (50/56) of the Penicillium isolates and 98.2% (54/55) of the Talaromyces isolates exhibited growth at 37°C. The isolates were mainly recovered from patients with pulmonary disorders (56.8%; 63/111), autoimmune disease (12.6%; 14/111) and AIDS (5.4%; 6/111). The azoles and amphotericin B exhibited potent activity against T. marneffei, while various levels of activity were observed against Penicillium and other Talaromyces species The echinocandins had the lowest MECs (MEC90, ≤0.12 mg/L) against most Penicillium and Talaromyces species, with the exception of T. marneffei whose MEC90 (4 mg/L) was five or more dilutions higher than that of the other species tested. These data on the species distribution and antifungal susceptibility expand the current clinical knowledge of Penicillium and Talaromyces species.

Bufalin inhibits ovarian carcinoma via targeting mTOR/HIF-α pathway.

Ovarian cancer is a severe health threat for women with increased incidence and stymied development in diagnosis and therapy. Drug resistance is still a big challenge. Bufalin is a multi-functional steroid-like compound extracted from natural product Chansu and has been tested as antitumour agent recently. The application and mechanism of bufalin in ovarian cancer remain unclear yet. Bufalin was first examined in ovarian epithelial cancer cell as well as primary ovarian tissue to evaluate its inhibitory activity in cell growth and migration, followed by the validation in xenograft tumour model and the patient samples. Bufalin is well tolerated by normal ovarian tissue at up to 40 µM and suppresses the cell growth and migration at 10 µM and xenograft tumour growth at 0.1mg/kg dosage. Bufalin inhibits the mammalian target of rapamycin (mTOR) activation and subsequently decreases hypoxia-induced factor 1 alpha (HIF-1α) level. Overexpression of HIF-1α could abolish the pro-apoptotic and antimigration activity of bufalin in cell culture. Strikingly, low HIF-1α level was correlated with improved responsiveness to cisplatin treatment in ovarian cancer patients. Bufalin was a potent inhibitor of cell growth and migration in ovarian cancer cells through suppression of mTOR activation and HIF-1α induction. Bufalin could be used to enhance the efficacy of cisplatin in ovarian cancer patients.

Correlation of MACC1/c-Myc Expression in Endometrial Carcinoma with Clinical/Pathological Features or Prognosis.

BACKGROUND Endometrial carcinoma (EC) is a type of female reproductive malignant tumor, the incidence of which is generally 20~30%. Multiple factors and genes are involved in the regulation of EC occurrence and progression. This study aimed to measure the expressions of MACC1 and c-Myc in EC patients to analyze their correlation with pathological features of EC. MATERIAL AND METHODS A total of 60 EC patients were recruited in the experimental group, while another cohort of 30 people with endometrial inflammatory hyperplasia was enrolled in the control group. The levels of serum MACC1 and c-Myc were measured by ELISA, and the protein expressions in EC cancer tissues, tumor-adjacent tissues, and controlled endometrial tissues were detected by immunohistochemistry (IHC). The correlation between gene expression and clinical/pathological features was then determined. RESULTS Our data indicate that the level of serum MACC1 and c-Myc in the experimental group was 1.67±0.08 ng/ml and 1.78±0.07 ng/ml, respectively, both of which were significantly higher than that of the control group (p<0.05). However, no significant difference was found among levels of serum MACC1 or c-Myc at different TNM stages (p>0.05). In cancer tissues, the positive rate of MACC1 or c-Myc was 73.3% and 78.3%, respectively, which were significantly higher than that in adjacent or control tissues (p<0.05). MACC1/c-Myc expression was correlated with TNM stage, primary infiltration grade, lymph node metastasis, and distal metastasis (p<0.05). CONCLUSIONS MACC1 and c-Myc are highly expressed in serum and tumor tissues of EC patients. Both are correlated with TNM stage, primary infiltration, and lymph node or distal metastasis, which provides a scientific basis for the development of new biomarkers for the diagnosis of endometrial carcinoma.

Isomalto-oligosaccharides ameliorate visceral hyperalgesia with repair damage of ileal epithelial ultrastructure in rats.

BACKGROUND: Treatment of irritable bowel syndrome (IBS) with probiotics has achieved effectiveness to a certain extent. Whether prebiotics will work is still unclear. This study aimed to investigate the therapeutic effects of the prebiotic isomalto-oligosaccharides (IMO) on visceral hypersensitivity (VHS) in rats and to explore potential mechanism. METHODS: Water avoidance stress (WAS) was used to induce VHS in rats. The score for the abdominal withdrawal reflex (AWR) was determined while colorectal distension and compared between VHS group and control group in order to validate VHS preparation. Rats with VHS were then divided into an IMO-treated group (intragastric 5% IMO, 2 mL/d, 14 days) and a water-control group (intragastric water). After treatment, AWR score and intestinal transit rate (ITR) were determined, stool culture was performed, the ultrastructure of the ileum epithelium was observed with scanning electron microscopy (SEM), and serum cytokines were measured. RESULTS: WAS significantly increased AWR score responding to colorectal distension, and lowered the pain threshold. IMO treatment improved VHS with a reduction in AWR score on graded colorectal distension and an increase in pain threshold. SEM showed damages on the ileal epithelial ultrastructure in VHS rats, which was attenuated by IMO treatment. ITR, fecal microbiota and serum cytokine levels were comparable among control group, water-control group, and IMO-treated rats. CONCLUSION: In this randomized placebo-controlled study, the results showed that IMO ameliorated WAS-induced visceral hyperalgesia in rats, this effect may be attributed to the repair of damages on intestinal epithelial ultrastructure.

Molecular characterization of Cryptococcus neoformans isolated from the environment in Beijing, China.

The molecular type of environmental Cryptococcus neoformans in Beijing was not clear. Our study aims to reveal the molecular characterization of C. neoformans complex from environment in Beijing, China. A total of 435 samples of pigeon droppings from 11 different homes in Beijing were collected from August to November in 2015. Pigeon droppings were inoculated onto caffeic acid cornmeal agar (CACA) to screen C. neoformans complex. Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for species identification. Serotype and mating type was determined by specific primers. Restriction fragment length polymorphisms of URA5 (URA5-RFLP) were applied to genotype. Multi-locus sequence typing (MLST) was done for further identification and sequence type (ST) determination. Altogether, 81 isolates of C. neoformans AFLP1/VNI were recognized from 435 pigeon droppings in this study. The positive rate for C. neoformans AFLP1/VNI from pigeon droppings in different homes varied from 5.0% to 52.6%, the average was 20.2%. All of these cryptococcal strains were serotype A, MATα. They were genotyped as VNI by URA5-RFLP and were confirmed by MLST. No other molecular types of C. neoformans and Cryptococcus gattii isolates were isolated. Their STs were identified as ST 31 (n = 54, 66.7%), followed by ST 53 (n = 10), ST 191 (n = 8), ST 5 (n = 5), ST 57 (n = 3), and ST 38 (n = 1). We concluded that not only clinical but also environmental isolates of C. neoformans need to be investigated more deeply and more extensively. The virulence difference between ST 5 and ST 31 need to be explored in the future.

Application of the DiversiLab system for tracing the source of the mixed infections caused by Cryptococcus neoformans var. grubii from a patient with systemic lupus erythematosus.

Two strains of Cryptococcus neoformans (PU 66 and PU112) were simultaneously isolated from a patient with systemic lupus erythematosus. We aimed to trace the source of the mixed infections. Multi-locus sequence typing (MLST) and the DiversiLab system analyses were performed on the 2 clinical and 23 environmental C. neoformans from pigeon droppings, 11 from the home (H1) the patient visited, 12 from another home (H2) as control. All the strains were uniformly genotyped as C. neoformans var. grubii VNI. Clinical strain PU66 and all the H1 isolates had the same sequence type (ST) - ST5, while for PU112 a new ST was observed - ST265. However, there was only one single base of 7 MLST loci difference between PU66 and PU112. Sequence types of the H2 strains were ST31 and ST297. DiversiLab analysis showed that strain similarity between the two clinical strains was 96.7%. In relation to environmental samples, the highest strain similarity (99.3%) was observed for PU66 and PU70 (H1). However, none of the environmental isolates had similarity over 98.6% comparing to PU112. One source of the mixed infections has been detected, but another needs further investigation.

Lentivirus-mediated knockdown of Krüppel-like factor 9 inhibits the growth of ovarian cancer.

PURPOSE: Ovarian cancer is among the top diseases in the list of malignant gynaecologic tumors. In the present study, we aim to investigate the effect of lentivirus-mediated knockdown of Krüppel-like factor 9 (KLF9) on cell viability and tumor growth in ovarian cancer. METHODS: Firstly, the expression of KLF9 was determined by real-time PCR and western blot in human ovarian cancer tissues. Then, endogenous KLF9 expression was silenced by lentivirus in SKOV3 and OVCAR3 ovarian cancer cells, and followed by MTT and BrdU incorporation assays, cell cycle analysis and tumor xenografts in nude mice. RESULTS: Our results found that the expression of KLF9 is up-regulated in human ovarian cancer. As expected, KLF9 knockdown significantly inhibited cell proliferation and resulted in cell cycle arrest in the G0/G1 phase. Besides, KLF9 deficiency significantly inhibited tumor growth in nude mice. CONCLUSION: Therefore, our data reveal that lentivirus-mediated KLF9 silencing might be promising in the treatment of human ovarian cancer.

Three-locus identification, genotyping, and antifungal susceptibilities of medically important Trichosporon species from China.

Three reference and 45 clinical isolates of Trichosporon were analyzed by conventional phenotypic and molecular methods to determine the species and genotypes of Trichosporon isolates from China. Target loci for molecular methods included the internal transcribed spacer (ITS) region, the D1/D2 domain of the 26S rRNA gene, and the intergenic spacer 1 (IGS1) region. Identification of eight Trichosporon species was achieved, of which Trichosporon asahii was the most common. Of the sequence-based molecular methods, the one targeting the D1/D2 domain assigned 97.9% (47/48) of isolates (seven species) correctly, while tests targeting both the ITS and IGS1 regions correctly identified all 48 isolates. The commercial API 20C AUX and Vitek 2 Compact YST systems correctly identified 91.9% and 73% of isolates when their biochemical profiles were queried against those of species contained in the databases, respectively, and misidentified 63.6% and 36.4% of isolates of species that were unclaimed by the databases, respectively. The predominant genotype among T. asahii clinical isolates, genotype 4 (51.4%), is rarely found in other countries. Voriconazole and itraconazole were the most active drugs in vitro against all the Trichosporon species tested, while caspofungin and amphotericin B demonstrated poor activity.

Accurate and practical identification of 20 Fusarium species by seven-locus sequence analysis and reverse line blot hybridization, and an in vitro antifungal susceptibility study.

Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 µg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria.