| Objective:1.The clinically collected and identified Candida albicans strains were subjected to in vitro drug susceptibility tests,and itraconazole-resistant and sensitive strains were screened out,and the resistance status of clinically isolated Candida albicans to itraconazole was analyzed.2.The biological information of virulence factor SAP2 and transcription factor GCN4 genes of itraconazole single resistant strains and fluconazole,itraconazole and voriconazole sensitive strains isolated from clinical Candida albicans were analyzed by PCR method,and the relationship between SAP2、GCN4 gene mutations and itraconazole resistance was preliminarily confirmed..3.To prepare an in vitro biofilm model of Candida albicans to explore the relationship between the resistance of Candida albicans to itraconazole and the ability of biofilm formation.4.The m RNA expression levels of SAP2 and GCN4 in clinical isolates of Candida albicans under free and biofilm conditions were detected by RT-PCR method,and the role of SAP2 and GCN4 in itraconazole resistance was analyzed.By comparing the m RNA expression levels of SAP2 and GCN4,it was preliminarily confirmed whether GCN4 could regulate SAP2 in clinically isolated itraconazole-resistant Candida albicans..Methods:1.Seventy-five suspected clinical strains of Candida albicans screened by the Drug sensitivity Laboratory of the Second Hospital of Shanxi Medical University from April to July 2021 were collected,which were subjected to in vitro drug susceptibility test with the M27-A4 micro-broth dilution method recommended by the American Clinical and Laboratory Standards Institute(CLSI),and the MIC50 values of the strains were determined and recorded to obtain itraconazole resistance strains and susceptible strains.2.Ten strains of itraconazole-resistant Candida albicans and ten strains of fluconazole,itraconazole and voriconazole sensitive Candida albicans were selected and divided into drug-resistant strain group and sensitive strain group.Preliminary extracted DNA from the two groups of strains and standard strain,amplified the SAP2 and GCN4 gene fragment by Polymerase Chain Reaction(PCR)and sequenced to find missense mutations,and make statistical analysis.3.Select the same strains as the second part of the experiment,and the strains were isolated and purified to establish an in vitro biofilm model,the morphological structure of the strains in different periods was observed under the inverted microscope,the biofilm forming ability of the strains was measured by enzyme labeling instrument,and the OD mean values of the two groups of strains were statistically analyzed.4.The total m RNA of two groups of strains and standard strains in free state and biofilm state was extracted and reverse transcribed into c DNA.The relative expression of SAP2 and GCN4 m RNA in free state and biofilm state was determined by real-time quantitative PCR(RT-PCR),and the corresponding statistical analysis was made.Results:1.There were 70 strains of Candida albicans identified from 75 clinical strains collected,including 24 itraconazole-resistant strains and 46 sensitive strains.34.3% of the strains in this experiment were resistant to itraconazole.2.Sequencing results of SAP2 and GCN4 genes of Candida albicans in drug-resistant strains and sensitive strains:no mutation site was found in SAP2 gene in both groups,and a missense mutation site A106T(T36S)was found in GCN4 gene.Missense mutation only appeared in 4 drug-resistant strains,but no missense mutation was found in sensitive strains.3.The morphology and structure of biofilm: with the increase of time,Candida albicans cells and hyphae increased,and the cells gathered along the hyphae to form a complete membrane-like structure.The results of biofilm forming ability showed that the OD value of drug-resistant strain group(0.42 ±0.07)was higher than that of sensitive strain group(0.23 ±0.04),and the difference was statistically significant(P<0.05).4.In free state,the expression level of SAP2 m RNA in drug resistant strain group was significantly higher than that in sensitive strain group,and the expression level of GCN4 m RNA in drug resistant strain group was significantly higher than that in sensitive strain group.There was a significant positive correlation between SAP2 and GCN4 at the transcriptional level(r=0.693,P<0.05).Under the biofilm state,the SAP2 m RNA expression in the resistant strain group(8.12±5.68)was higher than that in the sensitive strain group(4.75±2.58),but the difference was not statistically significant(P>0.05);The expression level of GCN4 m RNA in the resistant strain group was significantly higher than that in the sensitive strain group(P<0.05),and there was a significant positive correlation between SAP2 and GCN4 at the transcriptional level.The level of SAP2 m RNA in biofilm state was significantly higher than that in free state(P<0.001),and the expression level of GCN4 m RNA in biofilm state was significantly higher than that in free state(P<0.001).Conclusion:1.The resistance rate of patients with clinical Candida albicans infection to itraconazole is relatively high,;Therefore,the selection of drugs for the treatment of Candida albicans infection should be guided by the results of drug sensitivity test.2.No mutation site ware found in the SAP2 gene of Candida albicans clinically isolated,which may not be related to the drug resistance of Candida albicans;the resistance to itraconazole of Candida albicans may be related to the GCN4 gene mutation(T36S),but it need to further study.3.The formation of Candida albicans biofilm may increase its resistance to itraconazole.4.High expression of GCN4 m RNA in clinically isolated Candida albicans may increase itraconazole resistance in free state and biofilm state;In free state,high expression of SAP2 m RNA in clinically isolated Candida albicans may increase itraconazole resistance;The expression levels of SAP2 and GCN4 m RNA in clinically isolated Candida albicans were positively correlated under different conditions. |
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