| Obsjective The purpose of this study was to investigate the sensitizing effect of berberine(BBR)to fluconazole(FLC)-resistant Candida albicans and its possible mechanism,and to obtain resistance-related genes and virulence factors through genome-wide sequencing analysis inhibition of related resistance genes by the combination of berberine and fluconazole and analysis of possible signaling pathways and metabolic pathways.Methods Twenty-four strains in hospital specimens of Affiliated General Hospital of Ningxia medical University were collected from January 2015 to June 2016.The resistant strains were screened by NCCLS M44-A diffusion method using microbial detector and Komagica identification strains.The drug resistance test and crystal violet staining method were used to determine the diameter of bacteriostasis circle and the ability of producing membrane.MIC value of in vitro antibacterial activity of berberine and fluconazole was determined by microdate plate dilution method.Bactericidal Effect and Dynamic Bactericidal Activity of Berberine and Fluconazole with Time-Kill Curve Method.The growth of the biofilm was observed by fluorescence staining.The expression of CDR1,MDR,ALS3,ERG11,TUP1 and HWP1 mRNA was detected by qRT-PCR.The KEGG metabolism Pathological changes of ergosterol ERG gene under different drug treatment.Results A middle-membrane form of FLC-resistant Candida albicans isolated from specimens of patients with clinical catheterization.After the crystal violet staining,it can be observed that the weak-producing bacteria exist in a yeast-like form with a small amount of mycelial growth.The middle-producing bacteria show moderate amounts of myceliumformation,while the strong-producing strains mostly grow in the mycelial phase.According to the micro-chessboard dilution assay,the inhibition rate of this drug-producing strain was very low when treated with FLC alone.The inhibition rate was still <40% at the concentration of 64 ?g / ml,while the treatment of MIC60 with FLC alone was 512 ?g / ml.The addition of BBR decreased the FLC drug concentration by 16 points,which was significantly different from that of FLC alone(P <0.05).The mRNA expression of CDR1,MDR,ALS3,ERG11,TUP1 and HWP1 were detected by qRT-PCR.Compared with the combination of FLC and BBR,the expression of mRNA of CDR1,MDR,ALS3,ERG11,TUP1 and HWP1 were significantly decreased(P <0.05).According to the sequencing results,6276 genes were found,including 2384 small satellite sequences,144 tRNAs,32 snRNAs and 763virulence-related genes.The KEGG metabolic map shows that the metabolic genes of this strain are mainly derived from amino acid and carbon metabolism.The metabolic pathway map shows that a large number of ergosterol ERG genes are involved in the synthesis of cell membrane sterols,which may be related to drug resistance.After drug combination treatment of ERG1,ERG3,ERG4,ERG5,ERG6,ERG24,ERG25 and ERG9 genes were increased or decreased in different degrees compared with fluconazole alone(P<0.05).Conclusion(1)Candida albicans was cultured to form a biofilm and with the extension of time the number of cells and hyphae increased.The formation of biofilm was obvious.(2)Biofilm of Candida albicans maturation increased the susceptibility of fluconazole,while the addition of berberine drug can enhance the biofilm drug-resistant bacterial beads susceptibility to fluconazole.(3)Berberine enhances the possible mechanism of fluconazole in the treatment of Candida albicans with membrane-forming drug resistance decreases the expressions of CDR1,MDR1 and adhesion genes ALS3 and HWP1 in efflux pump,and increases the concentration of drug entering the intracellular and thus inhibits the target enzyme activity decreases ERG11 gene expression overexpression of mycobacterial repressor TUP1 reduces biofilm formation.(4)Berberine can enhance the fluconazole reduce theCandida albicans cell membrane sterol protein gene expression,improve the sensitivity of the strains to drugs. |
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