The In Vitro And In Vivo Effects And Mechanisms Of Proton Pump Inhibitors In Combination With Fluconazole Against Drug-resistant Candida Albicans

Background:The incidence of invasive fungal infections has increased continuously in recent decades,especially the infection caused by Candida species.In candidiasis,C.albicans is the most common strain.Antifungal drugs used in the treatment of candidiasis are limited and some of them cause serious side effects to host tissues.Owing to the great efficacy and low toxicity,azoles have been extensively used in clinical practice to treat candidiasis.However,along with the increase of infection and extensiv application of azoles,drug-resistant strains have frequently emerged.In order to overcome fungal resistance,the researches on antifungal sensitizers have attracted considerable attention.Proton pump inhibitors(PPIs)have been found to inhibit Candida albicans hyphae formation,and in combination with fluconazole in clinical can cure acute esophageal necrosis and Candidal esophagitis.Although some studies found that PPIs combined with fluconazole in vitro have no synergistic effects on sensitive Candida albicans,studies have found that D-octapeptide as a kind of PPIs can enhance the efficacy of fluconazole in resistance Candida albicans and Candida dubliniensis,suggesting that PPIs may be used as sensitizers of fluconazole.At present,there is no study on the combination of PPIs and fluconazole against resistant Candida albicans.Objective:The objective of this study was to determine the in vitro combined effects of PPIs and fluconazole against the planktonic and preformed biofilms of resistant Candida albicans,establish thewax moth larvae infection model to evaluate the in vivo interactions of drug combinations,and explore the possible mechanisms of drug interactions.Results:1.PPIs significantly increased the sensitivity of resistant Candida albicans to fluconazole in vitro.The PPIs tested have weak intrinsic antifungal activitis against resistant Candida albicans,but when combined with fluconazole,could significantly decrease the minimum inhibitory concentration of fluconazole,which can be decreased from 512 to 0.5-4>g/mL,the FICI values are 0.03-0.13.The ∑SYN values are much more than∑ANT values,and are greater than 200%.The results of the two models are consistent,and all drug combinations have strong synergistic effects.2.PPIs enhance the efficacy of fluconazole against resistant Candida albicans biofilms at different stages.PPIs were demonstrated to significantly decrease the minimum inhibitory concentration of fluconazole against resistant C.albicans biofilms pre-formed over 4,8 and 12 h(FICIs of 0.06-0.25),showing a strong synergistic antibiofilm effect.The minimum inhibitory concentration of fluconazole was not significantly decreased when the biofilms was preformed at 24 h,indicating the indifference with the FICI values were 2.3.PPIs combined with fluconazole prolonged the survival rate of Galleria mellonella and reduced the tissue damage.The survival rate of Galleria mellonella in the control group was 25%,and that of Galleria mellonella in fluconazole and PPIs monotherapy groups was between 20%and 35%.The survival rate of Galleria mellonella in the drug combination groups was between 70%-85%.Compared with the fluconazole monotherapy group,the drug combinations were significantly different(P<0.05).In addition,in the control group and drug monotherapy groups,there were large numbers of melanised nodules and the areas of melanised nodules were large in the tissue,while the larval tissue of the drug combination group was relatively fine and complete,and the melanised nodules were smaller and scarcely observed.4.PPIs could not inhibit the efflux pump activity of resistant Candida albicans.Compared to the control group,there was no difference in the fluorescence intensity of the OME,LAN,PTP,RAB and ESO groups(P>0.05),indicating that these PPIs could not exert effects on the efflux pump activity of CA10.The decreasing trend of fluorescence intensity in the experimental group with ILA was faster than that in the control group(P<0.001),indicating that ILA may enhance efflux pump protein activity of CA10 or due to its solubility,its generated drug particles resulted in fluorescence interference.5.PPIs combined with fluconazole reduce the extracellular phospholipase activity and morphologic switch of resistant C.albicans.The Pz values of the control group and the drug monotherapy groups was<0.69 and the Pz value of the drug combination group was 0.87±0.01.The drug monotherapy did not inhibit the phospholipase activity,when compared with the control group,(P>0.05).Compared with other groups,the drug combination significantly exert an inhibitory effects on the phospholipase activity(P<0.0001).In the growth of hypha,the length of hypha in the drug combination group was shorter than that in growth control group and drug monotherapy groups,and round or oval yeast cells can be observed in the drug combination group.Conclusion:PPIs can enhance the efficacy of fluconazole against resistant Candida albicans both in vitro and in vivo,and increased the susceptibility of resistant C.albicans biofilms to fluconazole.The mechanism study found that the synergistic antifungal effects of the drugs combinations may be related to the inhibition of the phospholipase activity and the switch of morphology.This study will provide new ideas and experimental data for the development of new antifungal drugs and the overcoming of fungal resistance.