| 1.AimsThe research is mainly determined the effect of azoles antifungal agents combined with phenylbutyric acid against resistant Candida species planktonic cells,biofilms,and in vivo.2.Methods2.1 The combination phenylbutyric acid with azoles against the planktonic cells of Candida species in static conditionsAccording to the CLSI M27-A3 program of the determination the antifungal drugs sensitivity,the selecition of resistant Candida species by the microdilution method.In addition,the antifungal effect of the comination azoles with phenylbutyric acid against resistant Candida species was evaluated in static conditions.The working concentration was adjucted to 2.5×103 CFU/mL.The plates were incubated for 24 h at 35?.The reduction assay of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(pheny-lamino)carbonyl]-2H-tetrazolium hydroxide(XTT)menadiol colorimetric method was used to assess metabolically active fungal cells.The plates were incubated in the dark for 2h.The microplate reader was used to read the OD values at 492 nm?And the fractional inhibitory concentration index(FICI)method and?E model was used to evaluate the antifungal effect against Candida species.2.2 The dynamic antifungal effect of the combination phenylbutyric acid with azoles against resistant Candida albicansTime-killing method was used to evaluate the effect of the combination phenylbutyric acid with azoles.The working concentration of suspensions was 5×10~3 CFU/mL.The tubes was incubated at 35?for 48 h.At the set time point,samples were aspirated at 0 h,12 h,18 h,24 h,36 h,and 48 h.The reduction assay of XTT-menadiol colorimetric method was used to assess metabolically active fungal cells.The plates were incubated in the dark for 2 h.The microplate reader was used to read the OD values at 492 nm.The time-kill curves were drawed according to OD values.2.3 The effect of the combination phenylbutyric acid with fluconazole against resistant Candida albicans biofilms2.3.1 The determination of sessile minimum inhibitory concentrations(SMICs)According to the CLSI M27-A3 program of the determination the antifungal drugs sensitivity,the SMICs were determined.96-well plates were used to study the efficacy of the combination phenylbutyric acid with fluconazole against resistant Candida albicans biofilms.The working concentration was adjucted to 2.5×10~3 CFU/mL.The 200?L suspensions were aspirated to add into the plates and the plates were incubated for 8 h and12 h to prepare the biofilms.PBS was used to wash the biofilms three times.Then,PBA and fluconazole were added,and the plates were incubated at 35?for 24 h.The reduction assay of XTT-menadiol colorimetric method was used to assess metabolically active fungal cells.The plates were incubated in the dark for 2 h.The microplate reader was used to read the OD values at 492 nm.And FICI method and?E model were used to evaluate the antifungal effect against Candida albicans biofilms2.3.2 The different concentration of phenylbutyric acid combined with fluconazole against andida albicans biofilmsThe suspension concentration was 2.5×10~3 CFU/mL,which was cultured for 12 h to prepare biofilms.Then,the suspension was aspirated and PBS was used to wash the biofilms three times.The concentration of phenylbutyric acid was 4?g/mL,32?g/mL,and128?g/mL.And the combination phenylbutyric acid with FLC was added to the biofilms.The biofilms were incubated for 24 h at 35?.The crystal violet staining method was used to assess the antif-ungal effect and the OD values were read at 570 nm.2.4 The combination phenylbutyric acid with fluconazole against resistant Candida albicans in vivoGalleria mellonella model is frequently used in the laboratory to assess C.albicans infection in vivo.2.4.1 The selection of fluconzole concentrationGalleria mellonella(approximately 0.25±0.2g)was used in the research.10 larvaes were in each group.And the concentration of FLC was ranged from 640?g/mL to 40?g/mL.4×10~6 CFU resistant Candida albicans wad injected into larvaes.After 2 h,10?L of fluconazole was injected into larvaes.The concentraton of FLC was determined according to the high survival rate of the larvaes into 4 d incubation.2.4.2 The determination of DMSO concentrationThe concentraton of phenylbutyric acid was ranged from 1280?g/mL to 40?g/mL.Galleria mellonella(approximately 0.25±0.2 g)was used in the research and 10 larvaes were in each group.10?L of FLC was injected into larvaes.The concentraton of FLC was determined according to the high survival rate of the larvaes into 4 d incubation.2.4.3 The selection concentration of phenylbutyric acidThe concentraton of phenylbutyric acid was ranged from 320?g/mL to 40?g/mL,and that of fluconazole was 320?g/mL.This research was divided into 4 groups and 10larvaes(approximately 0.25±0.2 g)was used.4×10~6 CFU resistant Candida albicans was injected into larvaes.After 2 h,10?L of mixture solution of phenylbutyric acid and FLC was injected into larvaes.The concentraton of phenylbutyric acid was determined according to the high survival rate of the larvaes into 4 d incubation.2.4.4 The combination phenylbutyric acid with fluconazole against resistant Candida albicans in vivoThe concentration of fluconazole was 320?g/mL,and that of phenylbutyric acid was80?g/mL.The study was individed into 4 groups(including control group,FLC group,phenylbutyric acid group,and the fluconazole combined with phenylbutyric acid group)and 16 larvaes(approximately 0.25±0.2 g)was in eahch group.4×10~6 CFU resistant Candida albicans was injected into larvaes.After 2 h,10?L of PBS,fluconazole,phenylbutyric acid and the combination solution was injected into larvaes.The survival rate curve was darwed according to the state of each larve into 4 d.2.4.5 The histopathology of larvaesIn the 2 d,2 larvaes was picked out from the above 3.4.4 experiment.Then,the embedding liquid was used to embed the larvaes and periodic acid-schiff stain method was used to stain the tissue.The freezing microtome was used to cut the larvae,and microscope was used to obsearve the histopathology and photograph2.4.6 Determination of fungal tissue-burden researchThree larvaes was picked out from the above 3.4.4 experiment in each day.Three larvaes(Including control group,fluconazole group,and the fluconazole combined with phenylbutyric acid group)were used to homogenize in 3 ml sterile PBS.PBS suspensions were aspirated and dropped on sterile YPD agar.The plates were incubated for 24 h at35°C and we counted the colonies to confirm the CFU/larvae.3.Result3.1 Effect of combination of phenylbutyric acid and azoles against the plantonic cells of Candia albicans speciesThe static effect showed that phenylbutyric acid in combination with fluconazole,for non-Candida albicans,the combinations presented indeference or additive effect;for the resistant Candida albicans showed a stronger synergistic antifungal effect,when phenylbutyric acid was 32?g/mL,the MICs of fluconazole decreased from 512?g/mL to 1?g/mL,the MICs of itraconazole ranged from 8?g/mL to 0.125?g/mL,and when phenylbutyric acid was 16?g/mL,the MICs of voriconazole from 8?g/mL reduced to 0.25?g/mL.In addition,the concentrations of the immobilized azoles were added into a series of phenylbutyric acid concentration,and the effect of the combinations increased with the drugs concentration increasing.The dynamic antifungal effect of the combination phenylbutyric acid with azoles against resistant Candida albicans showed that the effect of the combinations were synergisy against resistant Candida albicans.the effect of azoles or the combinations was similar before 12h,but the antimicrobial effects were stronger than phenylbutyric acid alone and the control;and the effect of the combination is the strongest than other groups after 24 h.3.2 Effect of combination phenylbutyric acid and fluconazole against resistant Candida albicans biofilmsThe results showed that the effect of the combination against Candida albicans biofilms(8 h and 12 h)was synergy;when phenylbutyric acid was 32?g/mL,SMICs for CA10 ranged from more than 512?g/mL to 2?g/mL,and that of CA16,SMICs for CA10reduced from more than 512?g/mL to 4?g/mL.The value of ?SYN and ?ANT was the evaluation criterion to assess the effect of the combination of fluconazole and phenylbutyric acid.And the?SYN values of the combination against C.albicans(CA10)biofilms(8 h and 12 h)were 2122.77%and3253.34%,and the?ANT values of CA10 were-21.73%and 0%,respectively.And the sum of?SYN and?ANT showed that the effect of the combination synergy against C.albicans.When the concentration of FLC was fixed,and that of phenylbutyric acid was ranged from 128?g/mL to 4?g/mL,the combinations showed that when the concentration of phenylbutyric acid was more,the antifungal effect was stronger.And this resultt is consistent with FICI method and the?E method.3.3 The combination phenylbutyric acid with fluconazole against resistant Candida albicansin vivoThe result showed the higher survival rate than other groups,when the concentration of FLC was 320?g/mL;When the concentration of DMSO used as solvent was<5%,DMSO was no effect on the final antifungl efficacy;And FLC concentration was fixed at320?g/mL,the higher surivival rate was achieved when phenylbutyric acid was at 80?g/mL;phenylbutyric acid can enhance the antifungal effect of fluconazole when the concentration of fluconazole or phenylbutyric acid was fixed at 320?g/mL or 80?g/mL,respectively(P<0.01);The histopathology and fungal tissue-burden research was further confirmed that phenylbutyric acid can enhance the antifungal effect of FLC against resistant Candida albicans4.Conclusion(1)Fluconazole in combination with phenylbutyric acid exerts synergistic effects against resistant Candida albicans.(2)Fluconazole in combination with phenylbutyric acid exerts synergistic effects against resistant Candida albicans biofilms.(3)Phenylbutyric acid can enhanced the antifungal activity of fluconazole against Candida albicans in the model of Galleria mellonella... |
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