Study On Synergistic Effect And Mechanism Of Rifapentine With Fluconazole Against FLC-resistance Candida Albicans In Vitro

Background and aims:Canidia albicans is one type of pathogenicity fungus when the conditions are suitable.In recent years,the patients having impaired immunity are increasing on account of massive and unreasonable application of broad-spectrum antibiotics,abuse of hormone,and massive application of immunosuppressive agents after chemotherapy of tumor and organ transplantation,which significantly increase the probability of fungal infection,among which Candida albicans infection is the most common phenomenon.Fluconazole,is usually used as preferred clinical drug for treating Candida albicans infection due to better oral absorption effect,higher bioavailability,and less side effect,however,with the massive and unreasonable application of it,the incidence rate of fungal drag resistance is increasing year by year,resulting in the failure of clinical treatment on fungal infection.According to the earlier drug sensitivity assay of our research group,it can be found that Rifapentine,Sertraline and others may have synergistic effect on FLC-resistance Candida albicans when they combine with Fluconazole.Rifapentine,as the rifamycinoid antibiotics,is the first chosen to treat Tuberculosis,having better effect of anti-mycobacterium tuberculosis and less adverse reactions than Rifampicin.So far,the synergistic effect of antifungal activity in vitro of Rifapentine has seldom been studied,therefore,the effect studies on Rifapentine combing with Fluconazole against Candida albicans in vitro will be conducted by us,so as to explore effective drug for clinical treatment on Candida albicans infection.Experimental Methods:Part I:Referring to M27-A2 scheme of America Clinical and Laboratory Standards Institute（CLSI）,the minimal inhibitory concentration（MIC）of Rifapentine and Fluconazole against Candida albicans was determined;and the effect of Rifapentine combing with Fluconazole against candida albicans in vitro was also determined by use of checkerboard broth microdilution testing.Meanwhile,the clinically isolated FLC-resistance Candida albicans 100 is selected as research object,and these two drugs have greatest synergistic effect in it among the strains studied in earlier chemosensitivity test,so Canidia albicans 100 is selected for further experimental studies.Through time-growth curve experiment and Spot assay experiment,the synergistic effect of Rifapentine on Fluconazole against drug-resistant Candida albicans in vitro was verified,and the drug concentration needed by subsequent researches was also explored.Part II:The co-culture of Candida albicans 100 with Rifapentine,Fluconazole and the combination of these two drugs was conducted respectively for 8h,meanwhile,the blank control group without drugs was established,then,the influence of blank control group,Rifapentine group,Fluconazole group and combining group on ultrastructure of Candida albicans 100 were observed by transmission electron microscope.Part III:The co-culture of Candida albicans 100 with Rifapentine,Fluconazole and the combination of these two drugs was conducted respectively for 12h and 24h,meanwhile,the blank control group without drugs was established,then,the DNA content of Candida albicans of blank control group,Rifapentine group,Fluconazole group and combining group was detected by using flow cytometry instrument,meanwhile,the influence of drugs on cell cycle was also observed.Experimental Results:Part I:There is no antimicrobial activity of Rifapentine on 12 Candida albic ans selected in experiment when used solely(MIC₈₀>1024μg/m L).In fluconazole-susceptible Candida albicans,Rifapentine has no synergistic effect after combining with Fluconazole（FICI>0.05）;while in Fluconazole-resistance Candida albicans,Rifapentine has significant synergistic effect after combining with Fluconazole（FI CI<0.01）,since Rifapentine has the greatest synergistic effect on Fluconazole agai nst Candida albicans 100 in vitro,this strain was selected as the study object in subsequent researches.Growth curve showed that the phase of logarithmic growth of Candida albicans 100 was 4¹2h,which entered into stable phase and declini ng phase of growth afterwards.Compared with control group of growth,single dr ug group has no obvious inhibiting effect on Candida albicans 100,while combin ing group has distinct inhibiting effect.When Rifapentine（125ug/m L）was applied combining with Fluconazole in different drug concentration（4,16,64ug/mL）,the inhibiting effect of Rifapentine on Candida albicans 100 was not in direct propo rtion to the dosage of Fluconazole;however,when Rifapentine in different drug c oncentration（15.63,31.25,125ug/m L）was applied combining with Fluconazole（4ug/m L）,the inhibiting effect of Rifapentine in higher drug concentration on Candi da albicans 100 was greater.Part II:The results of transmission electron microscopy showed that the thallus of growth control group had regular morphology,intact and clear round or oval cellular structure,smooth cell wall,continuous and clear cell membrane structure,and normal nuclei and mitochondria;Rifapentine group had normal cellular morphology basically,clear boundary,while partial cells had swelling and irregular nuclei,without obvious vacuoles;besides,most cells of Candida albicans group had almost normal cellular morphology,with sprouting,while the cell wall of partial cells had uneven electron density,stratum lucidum had focal thickened phenomenon,moreover,the cell membrane of it was rough,with swelling mitochondria,while the nucleoplasm of is was relatively clear,with few vacuoles;in addition,most cells of combining group had irregular cellular morphology,the electron transparent layer of cell wall was uneven,cell membrane and cell wall were detached,among which the cell membrane was discrete and deficient,with unclear layer and boundary,meanwhile,mitochondria was swelling,and the nucleoplasm had unclear circumscription,a large amount of unequal lumpy substance gathered around cell membrane,cytoplasm had uneven density and obscure organelle,with much vacuoles in it,nuclear membranes were broken and dissolved,and the nuclei was gathered irregularly and clumped.Part III:The DNA content of different cell cycle of FLC-resistance Candida albicans 100 was detected by flow cytometry after drug application for 12h and 24h respectively.At 12h,the cell count percentage at control group,Rifapentine group,Fluconazole group and combining group at G0/G1 phase respectively:（76.41+1.52）%,（75.48+1.41）%,（72.36+1.01）%,（8.11+0.22）%,S phase:（5.46+0.73）%,（8.46+0.23）%,（6.86+0.56）%,（12.52+0.54）%,G2/M phase:（18.14+1.11）%,（16.06+1.37）%,（20.78+1.48）%,（79.37+0.52）%;At 24h,the cell count percentage at control group,Rifapentine group,Fluconazole group and combining group at G0/G1 phase respectively:（82.36+1.25）%,（70.52+3.11）%,（60.45+1.07）%,（8.11+0.22）%,S phase:（6.03+0.92）%,（12.15+1.92）%,（15.19+1.23）%,（7.79+1.45）%,G2/M phase:（11.61+0.86）%,（17.33+1.32）%,（24.36+0.40）%,（86.26+0.94）%.Through comparison between two time points,it could be concluded that the influence of drugs on cell cycle of Candida albicans 100 was largely consistent,however,compared with growth control group and single drug group,combining group had less cell population in G0/G1 phase（p<0.01）,and more cell population in G2/M phase（p<0.01）,showing that the cell cycle of combining group was blocked in G2/M phase.Conclusion:When used in isolation,Rifapentine has no anti-candida albicans activity,while it can significantly enhance the activity of Fluconazole against FLC-resistance Candida albicans,which indicates that Rifapentine has synergistic effect on Fluconazole against drug-resistant Candida albicans in vitro.Through observation of ultrastructure of cell in each group by transmission electron microscopy,it can be speculated that Rifapentine can impact normal reproduction of cell DNA or RNA transcription,and protein translation through blocking synthesis of RNA and DNA,and it may also inhibit the activity of some enzymes on cell membrane directly,in order to exert its synergistic effect on Fluconazole against drug-resistant Candida albicans in vitro.Through the detection of flow cytometry instrument,it can be found that cell cycle can be blocked in G2/M phase,and normal division and proliferation of fungal cells can be inhibited when these two drugs are used together,so as to exert antifungal effect.Our research group first find that Rifapentine has synergistic effect on Fluconazole against drug-resistant Candida albicans in vitro,due to complicated antifungal mechanism of action of drug combination,the mechanisms of synergistic effect of Rifapentine on Fluconazole against drug-resistant Candida albicans in vitro are explored preliminarily and partially.In sequential studies,the synergistic effect of Rifapentine on Fluconazole against drug-resistant Candida albicans in vitro will be further explored through detecting sterol content of cell membrane,intracellular drug content,related gene expression and other experiments,so as to provide new approach for clinical treatment,and new drug research and development of drug-resistant Candida albicans infection.