In Vitro Effects Of Ambroxol On Cryptococcus Adherence,Planktonic Cells And Biofilm Formation

Background and PurposeCryptococcosis is one of the most important,life-threatening fungal infections in humans,mainly caused by Cryptococcus neoformans and C.gattii.Cryptococcus is a capsuled yeast-like fungus,widely distributed in nature.The gold standard antifungal therapeutic choices for the management of Cryptococcal Disease recommend by the Infectious Diseases Society of America are limited.The antifungal properties of azoles makes maintainance therapy for a long time,which has led to the emergence of drug resistant strains.Furthermore,Cryptococcus can form biofilms on medical devices,facilitating virulence and resistance to antifungal agents by shielding itself from physical or chemical harm.Therefore,the treatment of cryptococcal disease is still facing challenges.Ambroxol,the metabolite ? of Bromhexine,is one of the most widely used mucokinetic and expectorant drugs in respiratory diseases.In recent years,off-label use for ambroxol has caused much attention.Studies have shown that the drug has inhibitive effect on many pathogens.We investigated the antifungal effects of ambroxol on Cryptococcus planktonic cells and mature biofilms.MethodsWe performed a determination of internal transcribed spacer(ITS)region and genetic diversity within clinical Cryptococcus species complex isolates collected from our hospital.C.neoformans var.grubii strain H99(serotypes A);C.neoformans var.neoforoans strains B3501,ATCC 34874,RV 43185,RV59939(serotypes D)and C.gattii ATCC 34880(serotypes C)and BLS24(serotypes B)were included as standard strains.According to the current Clinical Laboratory Standards Institute(CLSI)method of susceptibility testing(CLSI M27-A3),we investigated the the growth inhibition of ambroxol against Cryptococcus strains by broth microdilution method.For combination effect,we studied the potentially synergetic effect of ambroxol and fluconazole.Ambroxol influence on adhesion of Cryptococcus planktonic cells was measured by flow cytometry.Furthermore,mature bio films were establishedin vitro to observe metabolic activity changes.Results1.ITS and multi focus sequencing typing determinationNine clinical isolates were identificated by ITS molecular biological identification as Cryptococcus strains.Five of all were C.neoformans var.grubii(VN I genotype).Two were C.neoformans var.neoformans(VN ? genotype).Two were C.gattii(VG ?genotype).2.In Vitro effects of ambroxol on Cryptococcus strainsThe MIC results were read according to standard M27-A3.For FLU and Amb,the MIC value is 80%growth inhibition compared to the growth controL The MIC values ranged from 0.5 to 4?g/mL for fluconazole and 125 to 500?g/mL for ambroxo1.Planktonic cells MIC of fluconazole in combination with ambroxol decreased in comparison with fluconazole alone,and MIC of ambroxol in combination with fluconazole did not show antagonistic effect.We evaluated whether ambroxol has the ability to prevent adhesion and found this drug was able to reduce the adhesion of all stains tested.Adhesion in the presence of ambroxol was reduced to 13.13±4.63.All Cryptococcus strains could produce biofilm by crystal violet staining.We evaluated the susceptibility of nature bio films formed by stains with smooth phenotype to FLU and Amb,and metabolic activity was measured by the XTT reduction assay.Bio films incubated with fluconazole displayed no significant change of metabolic activity when compared to the control.Biofilms treated with ambroxol alone showed a colorimetric change and a significant reduction in metabolic activity in comparison with fluconazole alone.However,the drug combination yielded no synergistic effect on mature biofilm.ConclusionThe most of clinical isolates used in our study were C.neoformans var.grubii strains(genotype VN ?,serotype A),and showed sensitivity to fluconazole in their planktonic cells.There were no significant differences in susceptibilities between different serotype species for fluconazole.Based on antifungal drug susceptibility testing,ambroxol showed antifungal activity against planktonic cells and biofilms of C neoformans.Furthermore,ambroxol was found to be synergetic with fluconazole against planktonic cells and was able to reduce cell adherence to polystyrene.Our results suggest that ambroxol was able to inhibit cryptococcal cells and biofilms,indicating a potential role in the prevention and treatment of cryptococcosis.