Ethanol tolerance is related to the expression of multiple genes, and genome-based engineering approaches are much more efficient than manipulation of single genes. In this study, ultraviolet (UV) mutagenesis, dielectric barrier discharge (DBD) air plasma mutagenesis, and artificial transcription factor (ATF) technology were adopted to treat an industrial yeast strain S. cerevisiae Sc4126 to obtain mutants with improved ethanol tolerance. Mutants with high ethanol tolerance were obtained, and the ratio of positive mutants was compared. Among the three approaches, the rate of positive mutation obtained by ATF technology was 10- to 100-folds of that of the two other methods, with highest genetic stability, suggesting the ATF technology promising for rapid alteration of phenotypes of industry yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; methods ; Mutagenesis ; Saccharomyces cerevisiae ; drug effects ; genetics ; growth & development

OBJECTIVETo investigate the distribution and drug resistance spectrum of clinical bacterial and Candida isolates.

METHODSMost of the bacterial isolates were identified using automated BD Phoenix, and a few with K-B method carried out manually. Candida isolates were identified by color-display plate and K-B method.

RESULTSThe most common isolates in the 2478 strains were P. aeruginosa (15.6%), E. coli (11.5%), C. albicans (9.6%), K. pneumoniae (9.3%), S. aureu (8.2%), and S. epidermidis (7.5%). In gram-negative isolates, the antibiotics with the lowest resistance rate were meraopenem (14.4%), cefoperazone/Sulbactam (14.8%), Imipenem (21.9%), piperacillin/tazobactam (27.4%), ceftazidime (30.0%), amikacin (31.1%), and cefepime (33.1%). The detection rate of E.coli and K. pneumoniae isolates producing extended spectrum beta-lactamase (ESBLs) were 47.4% and 37.3% respectively. In gram-positive isolates, the antibiotics with the lowest resistance rate were vancomycin (0.9%), teicoplanin (1.1%), nitrofurantoin (6.9%), amikacin (20.1%), chloramphenicol (30.7%), and cefoperazone/sulbactam (31.5%). The methecillin-resistant rates of S. aureu , S. epidermidis, and S. haemolyticus were 57.1%, 65.0%, and 66.0%. For Candida isolates, the most sensitive antibiotics were amphotericin B (0.3%), nystain (0.3%), itraconazole (5.6%), fluconazole (9.4%), and fluorocytosine (9.4%).

CONCLUSIONThe results suggest high rate of ESBL production and oxacillin resistance of the bacteria isolated in the hospital. More rational use of antimicrobial agents is crucial for reducing the drug-resistance of the bacteria, and effective measures must be taken to reduce dissemination of multidrug-resistant bacteria.

Anti-Infective Agents ; pharmacology ; Bacteria ; drug effects ; isolation & purification ; Candida ; drug effects ; isolation & purification ; Drug Resistance, Bacterial ; Drug Resistance, Fungal ; Microbial Sensitivity Tests ; Oxacillin ; pharmacology ; beta-Lactamases ; biosynthesis

OBJECTIVETo investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida.

METHODSResistance to fluconazole was induced in a Candida albicans isolate 435 from vagina by culturing in YEPD broth with increasing fluconazole concentration in vitro, and the resistant isolate 435-2 (MIC=128 microg/ml ) was obtained after 80 days of incubation. Comparisons between 435 and 435-2 either in fluconazole-containing medium or in drug-free medium were performed with the modified DD-PCR including amplification with long primers, silver staining, reverse dot blot and non-radiographic labeling techniques.

RESULTSThree differential displayed bands were found which showed high homology to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1, respectively. The up-regulating expression of ADH1 and CDR1 associated with fluconazole resistance was further identified by RT-PCR.

CONCLUSIONThe up-regulating expression of ADH1 and CDR1 was associated with fluconazole resistance in Candida albicans, ADH1 might be a candidate of novel fluconazole resistant gene.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Drug Resistance, Fungal ; genetics ; Fluconazole ; pharmacology ; Fungal Proteins ; genetics ; Membrane Transport Proteins ; genetics ; Oxidoreductases ; genetics ; Polymerase Chain Reaction ; methods

Breeding of robust industrial Saccharomyces cerevisiae strains with high ethanol tolerance is of great significance for efficient fuel ethanol production. Zinc finger proteins play important roles in gene transcription and translation, and exerting control on the regulation of multiple genes. The sequence and localization of the zinc finger motif can be designed and engineered, and the artificial zinc finger protein can be used to regulate celluar metabolism. Stress tolerance of microbial strains is related to multiple genes. Therefore, it is possible to use artificially-designed zinc finger proteins to breed stress tolerant strains. In this study, a library containing artificial zinc finger protein encoding genes was transformed into the model yeast strain S288c. A recombinant strain named M01 with improved ethanol tolerance was obtained. The plasmid in M01 was isolated, and then transformed into the industrial yeast strain Sc4126. Ethanol tolerance of the recombinant strain of Sc4126 were significantly improved. When high gravity ethanol fermentation using 250 g/L glucose was performed, comparing with the wild-type strain, fermentation time of the recombinant strain was decreased by 24 h and the final ethanol concentration was enhanced by 6.3%. The results of this study demonstrate that artificial zinc finger proteins are able to exert control on stress tolerance of yeast strains, and these results provide basis to construct robust industrial yeast strains for efficient ethanol fermentation.
Adaptation, Physiological ; drug effects ; Drug Resistance, Fungal ; genetics ; Ethanol ; pharmacology ; Fungal Proteins ; genetics ; metabolism ; Industrial Microbiology ; Mutation ; genetics ; Peptide Library ; Saccharomyces cerevisiae ; genetics ; growth & development ; Zinc Fingers

BACKGROUNDThe cytochrome P450 lanosterol 14alpha-demethylase (Erg11p) encoded by ERG11 gene is the primary target for azole antifungals. Changes in azole affinity of this enzyme caused by amino acid substitutions have been reported as a mechanism of azole antifungal resistance. This study aimed to investigate the relationship between amino acid substitutions in Erg11p from fluconazole resistant Candida albicans (C. albicans) isolates and their cross-resistance to azoles.

METHODSMutations in ERG11 gene were screened in 10 clinical isolates of fluconazole resistant C. albicans strains. DNA sequence of ERG11 was determined by PCR based DNA sequencing.

RESULTSIn the 10 isolates, 19 types of amino acid substitutions were found, of which 10 substitutions (F72S, F103L, F145I, F198L, G206D, G227D, N349S, F416S, F422L and T482A) have not been reported previously. Mutations in ERG11 gene were detected in 9 isolates of fluconazole resistant C. albicans, but were not detected in 1 isolate.

CONCLUSIONSAlthough no definite correlation was found between the type of amino acid substitutions in Erg11p and the phenotype of cross-resistance to azoles, the substitutions F72S, F145I and G227D in our study may be highly associated with resistance to azoles because of their special location in Erg11p.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; genetics ; Cytochrome P-450 Enzyme System ; genetics ; Drug Resistance, Fungal ; Fluconazole ; pharmacology ; Fungal Proteins ; genetics ; Microbial Sensitivity Tests ; Mutation

OBJECTIVETo investigate the effects of ethyl acetate extract of Huanglian Jiedu decoction (EAHD) , alone and in combination with fluconazole (FLZ) on FLZ-resistant Candida albicans.

METHODThe minimum inhibitory concentrations (MIC) and sessile MIC80 (SMIC80) of EAHD and FLZ to FLZ-resistant C. albicans were determined by CLSI M27-A3 microdilution method, and the synergy of EAHD combined with FLZ were examined by the checkerboard microdilution assay. Agar plate-method was adopted to observe the rate of antifungal activity according to time-kill curve. HPLC and qRT-PCR were utilized to evaluate the changes of ergosterol content and expressions of related genes, respectively.

RESULTMICs of EAHD ranged from 156 to 1,250 mg · L(-1), those of FLZ from 256 to above 2,048 mg · L(-1) with FICI approximate 0.066 in combination; SMIC80 of EAHD were higher than 1,250 mg · L(-1), SMIC80 of FLZ were higher than 512 mg · L(-1) and up to above 2,048 mg · L(-1). Combination group also showed synergy effect except one group showing addition effect. The results of T-K experiment also confirmed obviously fungicidal effect when treated for 12 h. When compared with control groups, the ergosterol was reduced 85% and 50% in the treatments of combination and EAHD alone by HPLC, respective- ly. The expressions of ERG1, ERG2, ERG6, ERG7 and ERG11 were upregulated, and ACS1, ACS2, MET6 were downregulated when exposed to FLZ. The expressions of the above genes were downregulated by treatment of EAHD. The expressions of ERG2, ERG6, ERG11 were upregulated, while ERG1, ERG7, ACS1, ACS2, MET6 were downregulated in combination group.

CONCLUSIONThe combination of EAHD and FLZ exhibited synergy against FLZ-resistant C. albicans through decreasing the synthesis of ergosterol, and resulting in the breakage of cell membrane.

Antifungal Agents ; pharmacology ; Candida albicans ; drug effects ; growth & development ; metabolism ; Drug Resistance, Fungal ; drug effects ; Drug Synergism ; Drugs, Chinese Herbal ; pharmacology ; Ergosterol ; biosynthesis ; Fluconazole ; pharmacology ; Microbial Sensitivity Tests

Due to substantial morbidity and high complications, diabetes mellitus is considered as the third "killer" in the world. Medicinal fungal polysaccharides, as water-soluble macromolecular substances with low toxicity, exhibit diversified pharmacological actions such as immune regulation, anti-tumor, antivirus, antioxidant, anti-aging, hypoglycemic effect and improving liver and kidney function. In recent year, a number of investigators reported medicinal fungal polysaccharides showed good anti-diabetes and hypoglycemic activity, and their acting mechanisms involved in glycometabolism and β cell function, e. g. promoting glycogen synthesis, promoting glycolysis, inhibiting the activity of α-glucosidase, promoting insulin secretion, increasing insulin sensitivity, enhancing antioxidation. Therefore, the hypoglycemic activity and its mechanisms of action of medicinal fungal polysaccharides showed characteristics of multiple effects, multi-target, and multi-pathway regulation. These finding suggest that medicinal fungal polysaccharides are a promising source for the development of discovery of anti-diabetic agent.
Animals ; Carbohydrate Metabolism ; drug effects ; Fungal Polysaccharides ; pharmacology ; Humans ; Hypoglycemic Agents ; pharmacology ; Insulin Resistance ; Insulin-Secreting Cells ; drug effects ; Oxidative Stress ; drug effects

BACKGROUND: At present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species. METHODS: We performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs. RESULTS: Of the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole. CONCLUSIONS: The EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.
Antifungal Agents/*pharmacology ; Candida/*drug effects/isolation & purification ; Candidiasis/epidemiology/microbiology ; Drug Resistance, Fungal/drug effects ; Fluconazole/*pharmacology ; Humans ; Microbial Sensitivity Tests ; Pyrimidines/*pharmacology ; Republic of Korea ; Triazoles/*pharmacology

BACKGROUND: At present, the clinical breakpoints (CBPs) of both fluconazole and voriconazole are available only for 3 common Candida species in the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Epidemiological cutoff values (ECVs) were recently applied to both methods to detect the emergence of acquired resistance (i.e., non-wild-type isolates) among 5 common Candida species. METHODS: We performed a nationwide study to determine the fluconazole and voriconazole susceptibility of Candida bloodstream isolates (BSIs) using both the CLSI and EUCAST methods. A total of 423 BSIs of 5 Candida species were collected from 8 hospitals. The azole susceptibilities were assessed on the basis of the species-specific CBPs and ECVs. RESULTS: Of the 341 BSIs of 3 common Candida species (i.e., C. albicans, C. tropicalis, and C. parapsilosis), 0.3% and 0.9%, 0.0% and 1.5% of isolates were categorized as fluconazole and voriconazole resistant according to the CLSI and EUCAST CBPs, respectively. Of 423 total BSIs, 1.4% and 2.6% had fluconazole minimum inhibitory concentrations (MICs) exceeding the ECVs according to the CLSI and EUCAST, respectively; 1.0% and 2.1% had voriconazole MICs exceeding the ECVs according to the CLSI and EUCAST, respectively. Categorical agreement between the methods using ECVs was 98.3% for fluconazole and 98.3% for voriconazole. CONCLUSIONS: The EUCAST and CLSI methods using ECVs provide highly concordant results. Moreover, non-wild-type isolates with possibly acquired azole resistance were rare among the BSIs of 5 common Candida species in Korea.
Antifungal Agents/*pharmacology ; Candida/*drug effects/isolation & purification ; Candidiasis/epidemiology/microbiology ; Drug Resistance, Fungal/drug effects ; Fluconazole/*pharmacology ; Humans ; Microbial Sensitivity Tests ; Pyrimidines/*pharmacology ; Republic of Korea ; Triazoles/*pharmacology

OBJECTIVETo establish a model of Candida biofilm and to explore its characteristics, ultrastructure, influences by saliva and serum, and sensitivity to antifungal agents.

METHODSEvaluations of the in vitro growth kinetics, influences by saliva and serum, and sensitivity to antifungal agents of Candida biofilm were performed with the abated tetrazolium salt XTT method on a 96-well microtire petri dish. The ultrastructure of Candida biofilm was observed under Confocal Laser Scanning Microscope (CLSM).

RESULTSThe bioactivity of Candida biofilm increased with culturing time and serum could obviously increase the action of biofilm. The Candida biofilm was significantly resistant to routine antifungal agents.

CONCLUSIONThe Candida cells adhered in biofilms are significantly different in morphology from those in suspension and are resistant to routine antifungal agents such as Amphotericine B, Fluconazole and Itraconazole.

Amphotericin B ; pharmacology ; Antifungal Agents ; pharmacology ; Biofilms ; drug effects ; Candida ; drug effects ; ultrastructure ; Drug Resistance, Fungal ; Fluconazole ; pharmacology ; Itraconazole ; pharmacology ; Microbial Sensitivity Tests ; Microscopy, Confocal