Fluconazole Resistance Mechanisms In Penicillium Marneffei

Objective To explore the mechanisms of Fluconazole(FLC) Resistance in Penicillium marneffei(PM).Methods Nine strains of PM were used in this research, including two fluconazole-resistant clinical strains、six fluconazole-resistant induced strains in vitro and one wild type strain FRR2161.We analysed this strains according to: (1)Susceptibility to antifungal agents in vitro were assessed by a microdilution method according to the guidelines presented in the CLSI document M38-A 2with minor modifications. And established the judgement of Fluconazole resistance for PM. (2)Study of phenotype characteristics.(3)To observe the conidia by copperband slide culture.(4)Analysis of ITS(Internal Transcribed Spacer) sequence and Random amplification polymorphic DNA(RAPD) assay.(5)Sequence analysis of cyp51B gene.(6)RNA isolation and Real-time quantitative polymerase chain reaction(RT-qPCR). (7) The significance of the differences in the data about relative expression was determined by Student’s t test.Statistical analysis was done with the SPSS packatge (version 16.0).AP value of<0.05 was considered significant.Results (1)the MICs of fluconazole-resistant strains were 128～256μg/ml, according with the judgement of PM FLC resistance. (2)The growth rate of fluconazole-resistant isolates was slower than the strain FRR2161.(3)Fluconazole-resistant strains producting less conidias compared with the strain FRR2161.(4)A11 experimental strains shared the same ITS sequence which was the same with the data of GenBank for PM.(5)The bands of DNA pattern of six fluconazole-resistant induced strains were of the same mobility with the female parent, and distinct from fluconazole-resistant clinical strains.(6)The sequence of the cyp51B gene from experimental isolates showed different point mutations:01 and C4 had a point mutation at T1583C, which led to the substitution of tyrosine 440 for histidine(Y440H); B4 and B5 had a point mutation at G1587T, which led to the substitution of glycine 441 for valine(G441V); C3 and B3 didn’t have the point mutation; Al had a point mutation at T1618C, but no amino acid substitution; A3 had two point mutation at G1587A and T1618C, which led to the substitution of glycine 441 for asparagine(G441D). (7) Compared with the strain FRR2161, the expression of AtrF、Mdr1、PMFCZ in the fluconazole-resistant group increased(P<0.05); The expression of cyp51B、ABC、MFS didn’t show obvious difference(P> 0.05).Conclusion The major reason of FLC resistance in PM seemed to be related to point mutations in cyp51B; Increased expression of efflux pumps had also been proposed to anticipate the mechanisms of FLC resistance.