| Objective:Make yeast form of Penicillium marneffei(PM) to fluconazole-resistance in vitro, screening the same parent drug-resistant strains and sensitive strains of PM, by the use of molecular biology methods to know the level of FCZ resistant gene sequence (GenBank: AL684103.1) expression, compared resistance gene expression of FCZ resistant strains and sensitive strains of PM at the transcription level differences between strains, in order to analyze the resistance and resistance gene expression pattern of PM, enhance research on PM resistance and for the Penicillium marneffei disease (Penicilliosis marneffei, PSM) and provide reference to clinical drug monitoring.Method:By gradually increasing the concentration of FCZ from the PM per plant 1/2 of minimum inhibitory concentration (MIC value) beginning of the induction, and gradually increase the FCZ concentration 2MIC,4MIC......, the FCZ is set to the highest concentration induced by 32μg/ml, make 11 strains yeast form of PM in vitro induction to resistance, while according to the Committee for Clinical Laboratory Standards (CLSI, formerly NCCLS) M38-A program issued by micro-dilution MIC values measured changes in drug-resistant strains and the corresponding filter sensitive strains by real-time Quantitative reverse transcription-polymerase chain reaction (Real-time PCR) determination the drug-resistant strains and sensitive strains PM FCZ expression of resistance genes, analyze the relationship of MIC value changes between gene expression.Results:1. By fluconazole in vitro induced by gradually increasing the concentration,11 strains yeast form of PM, the MIC values were increased,8 strains PM meet to resistance criteria, of which 5 strains MIC value of 64μg/ml,3 strains MIC value of 128μg/ml. In addition, H2,H5,H9 min to the state after induction.2. FCZ resistance gene expression in all both of 8 strains PM induced by the FCZ, increased from 2.56±1.84 to 7.50±3.00, P＜0.05, statistically significant differences,8 stains PM induced by the FCZ resistance gene expression group was higher than before induction group; PM per plant induced by fluconazole FCZ resistance gene were increased to different extents, H3 was the most significant rise, gene expression after the induction of resistance to FCZ before the induction of was 16.4 times, resistance gene of 8 stains PM rise in the range 1.8-16.4 after the induction of FCZ. A group (MIC values 4μg/ml PM group) and C group (MIC values 16μg/ml PM group), D group (MIC values 64μg/ml PM group) FCZ resistance compared gene expression, B group (MIC values 8μg/ml PM group) and the D group compared FCZ resistance gene expression, E group (MIC values of 128μg/ml) and A,B,C,D group compared with the amount of FCZ resistance gene, using analysis of variance, P value between the groups were<0.05, the difference was statistically significant, while the A group and B group, B group and C group, C group and D group was statistically no significant difference.Conclution:1. By using gradually increasing concentrations of fluconazole can successfuly induce PM resistance in vitro, provide a reliable theoretical basis to the reseach of resistance mechanism for laying the foundation and for the clinical use of drugs.2. PM resistance to FCZ may be associated with high expression of FCZ resistance gene.3. High expression of FCZ resistance gene can increase drug efflux capacity, leading PM to FCZ less sensitive or resistance, while there may be other mechanisms involved in the generation of clinical resistance to PM.
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