Analysis Of Fluconazole Resistance Induced By Candida Glabrata In Vitro And Its Related Mechanisms

PART I Fluconazole induction experiment in vitro to develop drug-resistant strains of Candida glabrata and the mutation analysis of drug-resistant PDR1 and ERG11 genesObjective To explore the changes of antifungal susceptibility of Candida glabrata isolated from clinical blood culture after long-term exposure to fluconazole in vitro,and analyze the mutations of fluconazole resistance related genes after induction experiment.Methods Five non-repetitive Candida glabrata strains isolated from the blood culture of the Laboratory Department of the First Affiliated Hospital of Anhui Medical University were collected and identified by MALDI-TOF MS mass spectrometry analysis.Using ATB Fungus 3 antifungal drug sensitivity test identified them as fluconazole sensitive strains.Through long-term exposure test of fluconazole in vitro,they developed into fluconazole resistant strains and then determined the stability of drug resistance.The sequences of fluconazole-resistant genes PDR1 and ERG11 of Candida glabrata were analyzed by DNA-Sequencing,and the sequence changes of fluconazole-resistant and fluconazole-resistant strains were compared with DNAMAN software,and the base and protein mutation sites were found by comparing with the sequence in Gen Bank.Results After the induction of fluconazole in vitro,five strains of Candida glabrata bloodstream infection not only developed fluconazole resistance,but also resulted in cross-resistance with triazole antifungal drugs itraconazole and voriconazole.Three missense mutations(G1127A/R376 Q,G2315A/R772 K,G4681A/E1083K)occurred in PDR1 gene and one missense mutation(C405A/F135L)occurred in ERG11 gene,all of which were newly found mutation sites,which have not been reported before.Conclusion Candida glabrata exposed to fluconazole for a long time in vitro could lead to cross-resistance between fluconazole and triazoles,and the drug resistance characteristics remain stable.After Candida glabrata acquired triazole resistance,mutations in PDR1 and ERG11 genes related to drug resistance can occur.In the future,it is necessary to further determine the relationship between these mutation types and triazole resistance of Candida glabrata.PART II Analysis of the m RNA relative expression levels and efflux pump function of fluconazole-resistant genes in Candida glabrataObjective To analyze the changes of the m RNA relative expression of the drug-resistance related genes PDR1,ERG11 and the efflux pump protein coding genes CDR1,CDR2 and SNQ2 of Candida glabra after the fluconazole induction experiment in vitro,and explore the changes of the efflux pump function of Candida glabra,and determine the relationship between them and the characteristics of triazole resistance.Methods Five fluconazole-sensitive strains screened in the part I and five drug-resistant strains obtained after induction were analyzed by real-time fluorescent quantitative PCR for the changes in the m RNA relative expression levels of the drug-resistant genes PDR1,ERG11 and the efflux pump coding genes CDR1,CDR2,SNQ2 of Candida glabrata,and R-6G fluorescent dye was used to analyze the changes in the function of the efflux pump of Candida glabrata.Results The relative m RNA expression levels of drug resistance related genes PDR1,ERG11,CDR1,CDR2 and SNQ2 in the fluconazole-induced drug resistance group of Candida glabrata were significantly up-regulated(p<0.05),among which the relative m RNA expression level of PDR1 and ERG11 genes were(7.525 ± 1.239)and(4.004 ± 1.720)respectively.,while the m RNA expression level of efflux pump protein coding gene CDR1(6.902 ± 2.087)was significantly higher than that of CDR2(2.925 ± 0.866)and SNQ2(2.325 ± 0.815).The results of flow cytometry showed that compared with the fluconazole sensitive group of Candida glabrata,The R6 G uptake of the fluconazole resistant group induced by Candida glabrata decreased and the efflux efficiency increased(p<0.05).Conclusion Long-term fluconazole exposure of Candida glabrata isolates in vitro could significantly increase the m RNA expression of drug-resistant genes PDR1,ERG11 and efflux pump coding genes CDR1,CDR2,SNQ2 of Candida glabrata,and the initiatived efflux function of Candida glabrata was strengthened,thus promoting the occurrence of drug resistance.