The Antif?ngal Effects And Mechanisms Of Galli?m Compo?nds Against Candida Species

AimsThe research is mainly determined the antifungal effect of Gallium Compounds,Fluc-onazole and Gallium Compounds combined with Fluconazole against Candida species in vitro and in vivo,both FLC resist ant strains and susceptive strains were tested.And to explore its underlying mechanism.Methods1.The antifungal effects of Gallium Compounds against Candida species in vitro.To explore the antifungal effect of Gallium Compounds,Fluconazole and Gallium Compounds combined with Fluconazole against the planktonic cells of Candida spec-ies?Candida albicans,Candida glabrata,Candida krusei?in static conditions according to the checkerboard method described in CLSI M27-A3.The 96-well plates were incubated at35?for 24h.Obtain the number of cells use the XTT assay.The 96-well plates were incu-bated for 2h in the dark.Read the OD values at 492 nm use the microplate reader.And the Fractional Inhibitory Concentration Index?FICI?method and?E model were used to eval-uate the antifungal effect of Gallium Compounds,Fluconazole and synergy of Gallium Compounds combined with Fluconazole against Candida species.Time-killing method was used to evaluate the antifungal effect of Gallium Compounds.Read the OD values at492nm by the microplate reader,samples were aspirated at 0h,12h,24h,36h and 48h.The samples were incubated at 35?for 24h.Obtain the number of cells use the XTT assay.The samples were incubated for 2h in the dark.Read the OD values at 492 nm use the microplate reader.The time-kill curves were drawed according to OD values.96-well pla-tes were used to determine the SMICs of the Gallium Compounds against resistant Candi-da biofilms according to the checkerboard method described in CLSI M27-A3.The biofi-lms formed over4h,8h,12h,24h and 48h.The 96-well plates were incubated at 35?for24h.Obtain the number of cells use the XTT assay.The 96-well plates were incubated for2h in the dark.Read the OD values at 492 nm use the microplate reader.?E model were used to evaluate the effect of the Gallium Compounds against resistant Candida biofilms.2.The effect of the Gallium Compounds against resistant Candida in vivo.Galleria mellonella model was used to evaluate the antifungal effect of Gallium Compounds in vivo.5×10⁶CFU resistant Candida albicans wad injected into Galleria mellonella.10?L of Ga?NO₃?₃ was injected into Galleria mellonella After three hours.Galleria mellonella model 20 larvaes per group,and the concentration of Ga?NO₃?₃ was ranged from 64?g/ml to 16?g/ml.The survival rate curve was darwed using SPSS statistics17.0 according to the state of each Galleria mellonella into 4 days.Two Galleria mello-nella models was picked out in the 2d from the above experiment.The embedding liquid was used to embed the Galleria mellonella model and the Galleria mellonella was cut using the freezing microtome.Hematoxylin Stain method was used to stain the tissue,and microscope was used to obsearve the histopathology.3.The mechanisms of Gallium Compounds against resistant C.albicans.As to mechanism study,the activity of extracellular phospholipases have been etected in vitro by egg yolk agar method and CAS siderophore detection plate was used to analysis protein expression of resistant C.albicans.The activity of extracellular phospholipases of resistant C.Albicans have been detected in vitro by egg yolk agar method.And PZ value were used to evaluate the antifungal effect of Gallium Compounds.Ga?NO3?3 were added into CAS siderophore detection plate to detect siderophore content and Ga?NO₃?₃were added into mediun to analysis protein expression of resistant C.albicans.Result1.The antifungal effects of Gallium Compounds against resistant Candida species in vitro.The static effect showed that Gallium Compounds for sensitive Candida albicans,resistant Candida albicans,resistant Candida glabrata and Candida krusei has a stronger antifungal effect,showed that Gallium Compounds in combination with FLC,for sensitive Candida albicans,resistant Candida albicans,resistant Candida glabrata and Candida krusei,the combinations presented indeference or the static effect.The MICs of FLC for sensitive Candida albicans range of 0.125?g/ml-0.5?g/ml,the MICs of Ga?NO₃?₃ for sensitive Candida albicans range of 8?g/ml-16?g/ml,the MICs of GaCl₃ for sensitive Candida albicans range of 4?g/ml-8?g/ml.The MICs of FLC for resistant Candida albi-cans was 512?g/ml,the MICs of Ga?NO3?3 for resistant Candida albicans range of16?g/ml-32?g/ml,the MICs of GaCl₃ for resistant Candida albicans range of 8?g/ml-16?g/ml.The MICs of FLC for resistant Candida glabrata was 64?g/ml,the MICs of Ga?NO₃?₃ for resistant Candida glabrata was 16?g/ml,the MICs of GaCl₃ for resistant Candida glabrata range of 8?g/ml-16?g/ml.The MICs of FLC for Candida krusei was64?g/ml,the MICs of Ga?NO₃?₃ for Candida krusei was 16?g/ml,and the MICs of GaCl₃for Candida krusei range of 8?g/ml-16?g/ml.The dynamic antifungal effect showed that Gallium Compounds for resistant Candida albicans,resistant Candida glabrata and Cand-ida krusei has a stronger antifungal effect,the time-killing curves confirmed the dynamic antifungal effects studies agreed with the static effect analysis.The results showed that the effect of Gallium Compounds against resistant Candida species biofilms?4h,8h and 12h?was strongly and the effect of Gallium Compounds against resistant Candida species biof-ilms?24h and 48h?was slightly.The SMICs of FLC for resistant Candida species biofilms?4h,8h and 12h?was more than 512?g/ml.The SMICs of Ga?NO3?3 for CA10 biofilms?4h,8h and 12h?was 16?g/ml,32?g/ml and 32?g/ml;the SMICs of GaCl₃ for CA10 biofilms?4h,8h and 12h?was 8?g/ml,16?g/ml and 32?g/ml.The SMICs of Ga?NO₃?₃ for CG2biofilms?4h,8h and 12h?was 16?g/ml,32?g/ml and 64?g/ml;the SMICs of GaCl₃ for CG2biofilms?4h,8h and 12h?was 16?g/ml,16?g/ml and 64?g/ml.The SMICs of Ga?NO₃?₃ for CK8 biofilms?4h,8h and 12h?was 16?g/ml,32?g/ml and 64?g/ml;the SMICs of GaCl₃ for CK8 biofilms?4h,8h and 12h?was 16?g/ml,16?g/ml and 64?g/ml.2.The effect of the Gallium Compounds against resistant Candida in vivo.The survival rate curve was darwed using SPSS statistics 17.0 according to the state of each Galleria mellonella into 4 days.The result showed the higher survival rate of treated group than the observation group,when the concentration of Ga?NO3?3 was64?g/ml,the difference was statistically significant?P<0.05?.And the concentration of Ga?NO₃?₃ was more,the survival rate was higher.The embedding liquid was used to embed the Galleria mellonella model and the Galleria mellonella was cut using the freezing microtome.Hematoxylin Stain method was used to stain the tissue,and microscope was used to obsearve the histopathology.The treated group which Ga?NO3?3were added weaken the damage of the histopathology of Galleria mellonella.3.The mechanisms of Gallium Compounds against resistant C.albicans.In the aspect of mechanism study,we observed that Ga?NO₃?₃ could weaken the activity of extracellular phospholipases of resistant C.albicans.The Pz value of the observation group was 0.7,meaning high Phospholipase activity of resistant C.albicans?CA10?.The Pz value of the observation group was 0.7,meaning the high Phospholipase activity of resistant C.albicans?CA10?.When the concentration of Ga?NO₃?₃ was 8?g/ml,the Pz value was 0.81,meaning the low Phospholipase activity of resistant C.albicans?CA10?.When the concentration of Ga?NO3?3 was more,the activity of extracellular phospholipases was weaker.Ga?NO3?3 was added into CAS siderophore detection plate to analysis protein expression of resistant C.albicans.We observed that Ga?NO₃?₃ could cause a up-regulation of siderophore expression of CA10.Ga?NO₃?₃ probably interdict the metabolism of Iron and kill the resistant C.albicans by competing with Iron for entering the C.Albicans.ConclusionGallium Compounds exerts a stronger antifungal effect against sensitive Candida albicans,resistant Candida albicans,resistant Candida glabrata and Candida krusei.Gallium Compounds in combination with FLC exerts indeference or the static effect against Candida.Gallium Compounds exerts a strong effect against resistant Candida albicans,resistant Candida glabrata and Candida krusei biofilms?4h,8h and 12h?.Gall-ium Compounds exerts a slight effect against resistant Candida albicans,resistant Candida glabrata and Candida krusei biofilms?24h and 48h?.The result showed the higher survival rate of treated group than the observation group,and the concentration of Ga?NO3?3 was more,the survival rate was higher.Ga?NO3?3 could weaken the activity of extracellular phospholipases of resistant C.Albicans and up-regulation the expression of siderophore.The mechanisms of Gallium Compounds against resistant Candida species probably is to weaken the activity of extracellular phospholipases interdict the metabolism of Iron and kill the resistant C.albicans by competing with Iron for entering the C.Albicans.This study could provide deep insight into antifungal agent discovery and clinical therapy.