胺碘酮与唑类药物的联合抗真菌作用及机制研究
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摘要
近半个世纪以来,随着医药科技的迅猛发展,临床上抗生素的使用日趋广泛,很容易引起人们发生菌群失调;而及免疫抑制剂、激素、抗肿瘤化疗和细胞毒性药物等的大量应用,可以降低机体的免疫机能;再加上留置导管、血液透析、消化道内窥等的侵入性操作增加了病源菌感染的机会;这些风险因素单独或相互叠加起作用使条件致病菌—尤其是真菌感染的发生率正在不断上升,念珠菌属特别是白色念珠菌的临床分离率最高。真菌感染发展迅速,如果得不到及时治疗,可导致患者死亡。与此同时,随着抗真菌药物的大量应用,其选择压力可将耐药菌变为优势菌,并诱导真菌发生基因突变,从而使真菌耐药率不断增加,真菌感染的治疗越来越困难。联合用药作为解决耐药真菌感染的主要方法之一,成为近年来的研究热点,受到广泛关注。
抗心律失常药物胺碘酮(amiodarone,AMD)是治疗心房颤动的一线用药,是预激综合症伴顽固性心律失常病人的首选药物。最新文献报道胺碘酮具有新奇的广谱的杀真菌活性,且与小剂量氟康唑联用时具有协同抗真菌作用。本实验在全面测定了胺碘酮与唑类药物单用或联合应用的静态和动态抗真菌作用的基础上,进一步研究了胺碘酮对唑类抗真菌药物的增敏作用机制,并观察了药物作用对真菌细胞形态学的影响。
一、胺碘酮与唑类药物的静态联合抗真菌作用
参照修订后的CLSI M27-A2方案,设置胺碘酮浓度范围0.5～256μg/ml,氟康唑浓度范围0.25～256μg/ml,伊曲康唑和伏立康唑浓度范围0.008～64μg/ml进行药物的抗真菌作用测定。结果表明CA5,CA8,CA12,CA14,CA129为敏感白色念珠菌,CA10,CA15,CA16,CA135,CA137为唑类交叉耐药白色念珠菌;胺碘酮对所有菌株的抗真菌作用都很弱。
应用棋盘法测定胺碘酮与唑类药物对上述白色念珠菌的静态联合抗真菌作用,设置胺碘酮浓度范围0.125～8μg/ml,氟康唑浓度范围0.125～128μg/ml,伊曲康唑和伏立康唑浓度范围0.008～8μg/ml,以分数抑菌浓度指数(FICI法)、反映面模型(RSM法)、非参数反应面(△E法)三种方法进行全面评价。结果显示药物联合对白色念珠菌耐药株的FICI的平均值为0.010:RSM法的协同-拮抗参数ICα均大于0,且其对应的95%致信区间也大于0;△E法的协同百分数和(∑SYN)的平均值为1250%,拮抗百分数和(∑ANT)的平均值为-18.7%,即联合用药对耐药菌表现协同作用。同时,实验测得药物联合对白色念珠菌敏感株的FICI的平均值为1.84;RSM法各组的协同参数α均大于0,但其对应的95%致信区间均包含0;△E法的∑SYN的平均值为59%,∑ANT的平均值为-138.5%,即联合用药对敏感菌表现为无关作用。对三种评价方法进行比较发现,非参数法更适用于评价抗真菌药物与非抗真菌药物的联合作用效果。
二、胺碘酮与唑类药物的动态联合抗真菌作用
应用时间-杀菌曲线法测定胺碘酮与唑类药物对耐药白色念珠菌CA10、敏感白色念珠菌CA14的动态联合抗真菌作用。筛选出胺碘酮药物浓度4μg/ml,氟康唑药物浓度8μg/ml,伊曲康唑和伏立康唑药物浓度0.5μg/ml进行测定。在体系培养的第0、6、12、24、48h的时间点,通过活菌计数和XTT(二甲氧唑黄法)两种方法获得真菌细胞数量,并对第48h菌液浓度的常用对数值(log_(10)CFU/ml)进行分析比较。结果显示动态联合抗真菌作用测定证实了胺碘酮本身弱的抗真菌活性;证实了胺碘酮对唑类药物抗耐药菌的协同作用,即与唑类药物相比,联合用药菌浓度常用对数下降值≥2;证实了胺碘酮对唑类药物抗敏感菌的无关作用,即与唑类药物相比,联合用药菌浓度常用对数下降值≤1。与此同时,我们发现活菌计数法与XTT法具有良好的一致性,相关系数r=0.9622。
三、细胞外的钙调节与药物作用
应用平板划线法测定胺碘酮与唑类药物对耐药白色念珠菌CA10、CA137,敏感白色念珠菌CA8、CA14的联合抗真菌作用以及细胞外的钙调节对联合作用的影响。设置4.5×10~5CFU/ml的菌液划线接种于含有一定浓度胺碘酮、氟康唑、伊曲康唑、伏立康唑以及各种钙调节剂的YPD琼脂培养基上,35℃恒温培养48h。结果显示,胺碘酮显著增强了唑类药物对耐药菌的作用,稍稍增强了唑类药物对敏感菌的作用;钙通道激活剂BayK8644不影响联合用药的效果;低浓度钙离子对耐药菌敏感菌都有生长促进作用,可以减弱联合用药的效果;高浓度钙离子、钙离子络合剂EGTA、EDTA对耐药菌敏感菌都有生长抑制作用,可以增强联合用药的效果;钙通道阻滞剂贝尼地平可显著增强联合用药对耐药菌的作用,稍稍增强联合用药对敏感菌的作用。
四、胺碘酮增敏作用与胞内钙离子的关系
Fluo-3/AM是跨膜的钙荧光指示剂,可以迅速与细胞内钙离子结合,经可见光激发后发出荧光,定性反映细胞内的钙离子脉冲变化。应用流式细胞术测定药物联合作用对耐药白色念珠菌CA10细胞内钙离子的影响。菌株在YPD液体培养基中培养至对数生长期,以D-Hanks缓冲液清洗细胞,重悬并调节菌浓度约为10~7CFU/ml,37℃120rpm下3μM的Fluo-3/AM避光负载30min,流式细胞仪测定药物加入后细胞内钙离子的变化。结果显示,胺碘酮可增加基础钙的双相升高(第一相和第三相),这与胺碘酮的弱抗真菌作用一致;氟康唑对细胞内钙离子浓度的影响较小,但可以将基础钙的低水平双相升高逆转为单相升高(第二相);胺碘酮对唑类药物抗耐药菌的增敏作用在于促进了细胞内钙库的钙的释放,极大的提高了胞浆内钙离子浓度。
五、胺碘酮对细胞内唑类药物蓄积的影响
罗丹明6G(Rhodamine 6G,R6G)是一种荧光物质,可被动扩散进入真菌细胞,与氟康唑、伊曲康唑、伏立康唑同为白色念珠菌主动外排泵CDR(CandidaDrug Resistance)的底物。我们应用荧光法测定胺碘酮对白色念珠菌蓄积罗丹明6G的影响,并进一步应用流式细胞术研究药物对白色念珠菌吸收、泵出罗丹明6G的影响。耐药菌CA10、CA135,敏感菌CA8和CA14在YPD液体培养基中培养至对数生长期,采用YPD液体培养液测定罗丹明6G在细胞内的蓄积情况,采用不含糖PBS体系测定罗丹明6G在细胞内的吸收情况,采用含5%糖的PBS体系测定罗丹明6G在细胞内的泵出情况。结果显示,胺碘酮对耐药菌的增敏作用机制在于增加了药物在细胞内的蓄积,这一作用是由药物吸收增加而不是泵出减少引起的;胺碘酮对敏感菌细胞蓄积、吸收、泵出罗丹明6G的过程均无明显影响。
六、药物作用对白色念珠菌细胞形态的影响
应用光学显微镜,在1000倍油镜下观察胺碘酮与唑类药物对白色念珠菌耐药株CA10、敏感株CA14形态学的影响。药物作用6h的结果表明,细菌及组织染色方法大多数也可用于真菌细胞形态观察。药物作用24、48h后的结果表明,生长对照组细胞的细胞壁与细胞核清晰可辨,细胞大量出芽,生成菌丝;胺碘酮单用组与生长对照相比,真菌细胞数量无显著差别,细胞壁与细胞核不可辨,菌体大小不一;唑类药物单用组与生长对照相比,真菌细胞数量明显减少,细胞染色暗淡,菌体大小不一;药物联用组与唑类药物单用体系相比,敏感白色念珠菌无显著差异,耐药白色念珠菌中前者比后者的真菌细胞数量明显减少,出现无法着色的细胞及细胞碎片类似物。
In the past decades,with the rapid development of medicine technology,the widespread application of antibiotics,the extensive use of immunosuppressant、hormone、antitumor chemotherapy and cytotoxic drugs,the prevalence of conditioned pathogen—especially fungi infections have increased significantly,Candiada albicans has the highest isolating rate among them.Given no promptly cure,fungi infection would progress quickly with a high mortality rate.Meanwhile,following by the frequent administration of antifungals,resistant rate is increasing continuously,as a result of selected pressure of chemotherapeutics,which made fungi infection more difficult to treat.Now,drugs combinations method has become one of the hottest and most effective strategies to solve this problem.
Antiarrhythmic drug amiodarone(AMD)is the first line medication in treating atrial fibrillation,and the choice drug for patients with pre-excitation syndrome and refractoriness arrhythmia.Recent literatures reported amiodarone had a novel,broad fungicidal activity,that can acted synergistic with low dosage of fluconazole.Static and dynamic antifungal activity of AMD and azoles acted alone or in combinations were fully examined;mechanisms of sensitization of amiodarone on azoles against resistant Candida albicans were futher studied;and the cellular morphology changes after drugs actions were observed as well.
ⅠStatic antifungal activity of amiodarone combined with azoles
Referring to the revised CLSI M27-A2 document,susceptibility testing was performed by amiodarone within 0.5～256μg/ml、fluconazole within 0.25～256μg/ml、itraconazole and voriconazole within 0.008～64μg/ml.CA5,CA8,CA12,CA14 and CA129 were tested to be susceptible,while CA10,CA15,CA16,CA135 and CA137 resistant.We also found that amiodarone had nearly no antifungal acitivity on all strains.
Static combined antifungal effect testing was carried according to checkerboard method,concentrations ranged 0.125～8μg/ml for amiodarone、0.125～128μg/ml for fluconazole and 0.008～8μg/ml for itraconazole and voriconazole.FICI、RSM and AE methods were employed for overall assessment.The combinations of amiodarone and azoles were synergistic against resistant isolates with a average FICI of 0.010,a average ICαand its 95%confidence interval(CI)exceed zero,a averageΣSYN of 1250%andΣANT of -18.7%.While The combinations of amiodarone and azoles displayed indifferent against susceptible strains with a average FICI of 1.84,a average ICa exceed zero but its 95%CI containing,a averageΣSYN of 59%andΣANT of -138.5%.After comparising all three methods,non-parameter method was better for assessing the combined effect of antifungals and non-antifungal agents.
ⅡDynamic antifungal activity of amiodarone combined with azoles
Time-kill method had been applied to test the dynamic activity of amiodarone combined with azoles against CA10 and CA14.Concentrations of amiodarone (4μg/ml)、fluconazole(8μg/ml)、itraconazole(0.5μg/ml)and voriconazole(0.5μg/ml) were screened.At determined time point(0、6、12、24、48h),viable count and XTT assay were used to obtain the number of cells.After analysis of log_(10)CFU/ml at 48h, weak antifungal activity of amiodarone was confirmed;synergism of amiodarone combined with azoles against resistant strain were confirmed by a≥2 decrement in log_(10)CFU/ml compared to the corresponding azole;indifference of amiodarone combined with azoles against sensitive strain were confirmed by a≤1 decrement in log_(10)CFU/ml compared to the corresponding azole.Finally,we demonstrated that viable count and XTT assay had an excellent coincidence,with r=0.9622.
ⅢExtracellular calcium modulation and drug effect
Plate streaking was occupied to research the influences of calcium modulators on the combined effect of amiodarone and azoles against CA10、CA137、CA8、CA14. After streaking the fungi suspension onto YPD agar medium with certain concentrations of drugs and calcium modulators,plates were incubated at 35℃for 48h.Results displayed that amiodarone strikingly reinforced the effect of azoles against resistant strains,and slightly enhanced the effect of azoles against sensitive strains.BayK8644 had no influence on drugs combinations;low concentration of calcium can promote fungi growth,while high concentration of calcium、EGTA and EDTA can inhibit fungi growth,and strengthened the antifungal actions.Addition of benidipin largly augments the combination activity against resistant fungi and slightly affects the combination activity against sensitive isolates.
ⅣRelationship between sensitization of amiodarone and intracellular calcium
Fluo-3/AM is a trans-membrane fluorescent indicator,which can immediately give out fluorescence after bounding to Ca~(2+),and reflect the square pulse changes of intracellular calcium.Flow cytometry was introduced to test the effect of drugs combinations on intracellular calcium of CA10(resistant).CA10 was incubated in YPD liquid medium until log phase growth,then washed by D-Hanks buffer and resuspended to 10~7CFU/ml.Cells were loaded with isovolumic Fluo-3/AM at 37℃and 120rpm for 30min to a final concentration of 3μM.Results showed that an application of AMD resulted in a biphasic elevation of cytoplasmic calcium,which may relate to its weak antifungal activity;fluconazole has no effect on intracellular [Ca~(2+)]i,but can reversed the biphasic elevation(phase 1 and 3)of cytoplasmic[Ca~(2+)]i of "control" to monophasic elevation(phase 2);sensitization of AMD may due to the release of calcium from calcium store,and extremely raised intracellular calcium.
ⅤEffect of amiodarone on intracellular azoles accumulation
Rhodamine 6G(R6G)can passive diffuse into fungi cells,and has the same substrate of CDR(Candida Drug Resistance)with fluconazole、itraconazole and voriconazole.Fluorometric method and flow cytometry were introduced to test the accumulation、uptake and effluxion of R6G(10μM)with or without amiodarone in CA10、CA135、CAB、CA14.Strains were incubated in YPD liquid medium until log phase growth.Samples of R6G accumulation were carried out in YPD liquid medium, R6G uptake was performed in PBS buffer and R6G effluxion was done in 5% glucose-containing PBS buffer.Results exhibited that sensitization mechanism of amiodarone is the increment of R6G accumulation,which is resulted from increased R6G uptake,not reduced R6G effluxion;and amiodarone had no obvious effect on R6G accumulation、uptake、effluxion in sensitive strains.
ⅥEffect of drugs on cellular morphology of Candida albicans
Immersion objective(×1000)of light microscope was used to overview the effects of amiodarone and azoles alone or in combinations against CA10 and CA14.There are no cellular morphology changes among drugs actions after 6h incubation,which demonstrated that most bacterium and tissue dyeing methods suitable for fungi cells, too.Strains after 24 and 48h incubation with drugs showed:cells of "growth control" have clear wall and nucleus,with pack of budding and filaments;cells of "amiodarone alone" have no clear wall and nucleus,with aniso-size of cell body;"azoles alone" has less cells than "growth control",with aniso-size of cell body and gloomy staining; cells of "drugs combinations" have no significant deviation with "azoles alone " in CA14,while in CA10 there has the least number of cells,with appearance of no staining or debris-liking cells.
抗心律失常药物胺碘酮(amiodarone,AMD)是治疗心房颤动的一线用药,是预激综合症伴顽固性心律失常病人的首选药物。最新文献报道胺碘酮具有新奇的广谱的杀真菌活性,且与小剂量氟康唑联用时具有协同抗真菌作用。本实验在全面测定了胺碘酮与唑类药物单用或联合应用的静态和动态抗真菌作用的基础上,进一步研究了胺碘酮对唑类抗真菌药物的增敏作用机制,并观察了药物作用对真菌细胞形态学的影响。
一、胺碘酮与唑类药物的静态联合抗真菌作用
参照修订后的CLSI M27-A2方案,设置胺碘酮浓度范围0.5～256μg/ml,氟康唑浓度范围0.25～256μg/ml,伊曲康唑和伏立康唑浓度范围0.008～64μg/ml进行药物的抗真菌作用测定。结果表明CA5,CA8,CA12,CA14,CA129为敏感白色念珠菌,CA10,CA15,CA16,CA135,CA137为唑类交叉耐药白色念珠菌;胺碘酮对所有菌株的抗真菌作用都很弱。
应用棋盘法测定胺碘酮与唑类药物对上述白色念珠菌的静态联合抗真菌作用,设置胺碘酮浓度范围0.125～8μg/ml,氟康唑浓度范围0.125～128μg/ml,伊曲康唑和伏立康唑浓度范围0.008～8μg/ml,以分数抑菌浓度指数(FICI法)、反映面模型(RSM法)、非参数反应面(△E法)三种方法进行全面评价。结果显示药物联合对白色念珠菌耐药株的FICI的平均值为0.010:RSM法的协同-拮抗参数ICα均大于0,且其对应的95%致信区间也大于0;△E法的协同百分数和(∑SYN)的平均值为1250%,拮抗百分数和(∑ANT)的平均值为-18.7%,即联合用药对耐药菌表现协同作用。同时,实验测得药物联合对白色念珠菌敏感株的FICI的平均值为1.84;RSM法各组的协同参数α均大于0,但其对应的95%致信区间均包含0;△E法的∑SYN的平均值为59%,∑ANT的平均值为-138.5%,即联合用药对敏感菌表现为无关作用。对三种评价方法进行比较发现,非参数法更适用于评价抗真菌药物与非抗真菌药物的联合作用效果。
二、胺碘酮与唑类药物的动态联合抗真菌作用
应用时间-杀菌曲线法测定胺碘酮与唑类药物对耐药白色念珠菌CA10、敏感白色念珠菌CA14的动态联合抗真菌作用。筛选出胺碘酮药物浓度4μg/ml,氟康唑药物浓度8μg/ml,伊曲康唑和伏立康唑药物浓度0.5μg/ml进行测定。在体系培养的第0、6、12、24、48h的时间点,通过活菌计数和XTT(二甲氧唑黄法)两种方法获得真菌细胞数量,并对第48h菌液浓度的常用对数值(log_(10)CFU/ml)进行分析比较。结果显示动态联合抗真菌作用测定证实了胺碘酮本身弱的抗真菌活性;证实了胺碘酮对唑类药物抗耐药菌的协同作用,即与唑类药物相比,联合用药菌浓度常用对数下降值≥2;证实了胺碘酮对唑类药物抗敏感菌的无关作用,即与唑类药物相比,联合用药菌浓度常用对数下降值≤1。与此同时,我们发现活菌计数法与XTT法具有良好的一致性,相关系数r=0.9622。
三、细胞外的钙调节与药物作用
应用平板划线法测定胺碘酮与唑类药物对耐药白色念珠菌CA10、CA137,敏感白色念珠菌CA8、CA14的联合抗真菌作用以及细胞外的钙调节对联合作用的影响。设置4.5×10~5CFU/ml的菌液划线接种于含有一定浓度胺碘酮、氟康唑、伊曲康唑、伏立康唑以及各种钙调节剂的YPD琼脂培养基上,35℃恒温培养48h。结果显示,胺碘酮显著增强了唑类药物对耐药菌的作用,稍稍增强了唑类药物对敏感菌的作用;钙通道激活剂BayK8644不影响联合用药的效果;低浓度钙离子对耐药菌敏感菌都有生长促进作用,可以减弱联合用药的效果;高浓度钙离子、钙离子络合剂EGTA、EDTA对耐药菌敏感菌都有生长抑制作用,可以增强联合用药的效果;钙通道阻滞剂贝尼地平可显著增强联合用药对耐药菌的作用,稍稍增强联合用药对敏感菌的作用。
四、胺碘酮增敏作用与胞内钙离子的关系
Fluo-3/AM是跨膜的钙荧光指示剂,可以迅速与细胞内钙离子结合,经可见光激发后发出荧光,定性反映细胞内的钙离子脉冲变化。应用流式细胞术测定药物联合作用对耐药白色念珠菌CA10细胞内钙离子的影响。菌株在YPD液体培养基中培养至对数生长期,以D-Hanks缓冲液清洗细胞,重悬并调节菌浓度约为10~7CFU/ml,37℃120rpm下3μM的Fluo-3/AM避光负载30min,流式细胞仪测定药物加入后细胞内钙离子的变化。结果显示,胺碘酮可增加基础钙的双相升高(第一相和第三相),这与胺碘酮的弱抗真菌作用一致;氟康唑对细胞内钙离子浓度的影响较小,但可以将基础钙的低水平双相升高逆转为单相升高(第二相);胺碘酮对唑类药物抗耐药菌的增敏作用在于促进了细胞内钙库的钙的释放,极大的提高了胞浆内钙离子浓度。
五、胺碘酮对细胞内唑类药物蓄积的影响
罗丹明6G(Rhodamine 6G,R6G)是一种荧光物质,可被动扩散进入真菌细胞,与氟康唑、伊曲康唑、伏立康唑同为白色念珠菌主动外排泵CDR(CandidaDrug Resistance)的底物。我们应用荧光法测定胺碘酮对白色念珠菌蓄积罗丹明6G的影响,并进一步应用流式细胞术研究药物对白色念珠菌吸收、泵出罗丹明6G的影响。耐药菌CA10、CA135,敏感菌CA8和CA14在YPD液体培养基中培养至对数生长期,采用YPD液体培养液测定罗丹明6G在细胞内的蓄积情况,采用不含糖PBS体系测定罗丹明6G在细胞内的吸收情况,采用含5%糖的PBS体系测定罗丹明6G在细胞内的泵出情况。结果显示,胺碘酮对耐药菌的增敏作用机制在于增加了药物在细胞内的蓄积,这一作用是由药物吸收增加而不是泵出减少引起的;胺碘酮对敏感菌细胞蓄积、吸收、泵出罗丹明6G的过程均无明显影响。
六、药物作用对白色念珠菌细胞形态的影响
应用光学显微镜,在1000倍油镜下观察胺碘酮与唑类药物对白色念珠菌耐药株CA10、敏感株CA14形态学的影响。药物作用6h的结果表明,细菌及组织染色方法大多数也可用于真菌细胞形态观察。药物作用24、48h后的结果表明,生长对照组细胞的细胞壁与细胞核清晰可辨,细胞大量出芽,生成菌丝;胺碘酮单用组与生长对照相比,真菌细胞数量无显著差别,细胞壁与细胞核不可辨,菌体大小不一;唑类药物单用组与生长对照相比,真菌细胞数量明显减少,细胞染色暗淡,菌体大小不一;药物联用组与唑类药物单用体系相比,敏感白色念珠菌无显著差异,耐药白色念珠菌中前者比后者的真菌细胞数量明显减少,出现无法着色的细胞及细胞碎片类似物。
In the past decades,with the rapid development of medicine technology,the widespread application of antibiotics,the extensive use of immunosuppressant、hormone、antitumor chemotherapy and cytotoxic drugs,the prevalence of conditioned pathogen—especially fungi infections have increased significantly,Candiada albicans has the highest isolating rate among them.Given no promptly cure,fungi infection would progress quickly with a high mortality rate.Meanwhile,following by the frequent administration of antifungals,resistant rate is increasing continuously,as a result of selected pressure of chemotherapeutics,which made fungi infection more difficult to treat.Now,drugs combinations method has become one of the hottest and most effective strategies to solve this problem.
Antiarrhythmic drug amiodarone(AMD)is the first line medication in treating atrial fibrillation,and the choice drug for patients with pre-excitation syndrome and refractoriness arrhythmia.Recent literatures reported amiodarone had a novel,broad fungicidal activity,that can acted synergistic with low dosage of fluconazole.Static and dynamic antifungal activity of AMD and azoles acted alone or in combinations were fully examined;mechanisms of sensitization of amiodarone on azoles against resistant Candida albicans were futher studied;and the cellular morphology changes after drugs actions were observed as well.
ⅠStatic antifungal activity of amiodarone combined with azoles
Referring to the revised CLSI M27-A2 document,susceptibility testing was performed by amiodarone within 0.5～256μg/ml、fluconazole within 0.25～256μg/ml、itraconazole and voriconazole within 0.008～64μg/ml.CA5,CA8,CA12,CA14 and CA129 were tested to be susceptible,while CA10,CA15,CA16,CA135 and CA137 resistant.We also found that amiodarone had nearly no antifungal acitivity on all strains.
Static combined antifungal effect testing was carried according to checkerboard method,concentrations ranged 0.125～8μg/ml for amiodarone、0.125～128μg/ml for fluconazole and 0.008～8μg/ml for itraconazole and voriconazole.FICI、RSM and AE methods were employed for overall assessment.The combinations of amiodarone and azoles were synergistic against resistant isolates with a average FICI of 0.010,a average ICαand its 95%confidence interval(CI)exceed zero,a averageΣSYN of 1250%andΣANT of -18.7%.While The combinations of amiodarone and azoles displayed indifferent against susceptible strains with a average FICI of 1.84,a average ICa exceed zero but its 95%CI containing,a averageΣSYN of 59%andΣANT of -138.5%.After comparising all three methods,non-parameter method was better for assessing the combined effect of antifungals and non-antifungal agents.
ⅡDynamic antifungal activity of amiodarone combined with azoles
Time-kill method had been applied to test the dynamic activity of amiodarone combined with azoles against CA10 and CA14.Concentrations of amiodarone (4μg/ml)、fluconazole(8μg/ml)、itraconazole(0.5μg/ml)and voriconazole(0.5μg/ml) were screened.At determined time point(0、6、12、24、48h),viable count and XTT assay were used to obtain the number of cells.After analysis of log_(10)CFU/ml at 48h, weak antifungal activity of amiodarone was confirmed;synergism of amiodarone combined with azoles against resistant strain were confirmed by a≥2 decrement in log_(10)CFU/ml compared to the corresponding azole;indifference of amiodarone combined with azoles against sensitive strain were confirmed by a≤1 decrement in log_(10)CFU/ml compared to the corresponding azole.Finally,we demonstrated that viable count and XTT assay had an excellent coincidence,with r=0.9622.
ⅢExtracellular calcium modulation and drug effect
Plate streaking was occupied to research the influences of calcium modulators on the combined effect of amiodarone and azoles against CA10、CA137、CA8、CA14. After streaking the fungi suspension onto YPD agar medium with certain concentrations of drugs and calcium modulators,plates were incubated at 35℃for 48h.Results displayed that amiodarone strikingly reinforced the effect of azoles against resistant strains,and slightly enhanced the effect of azoles against sensitive strains.BayK8644 had no influence on drugs combinations;low concentration of calcium can promote fungi growth,while high concentration of calcium、EGTA and EDTA can inhibit fungi growth,and strengthened the antifungal actions.Addition of benidipin largly augments the combination activity against resistant fungi and slightly affects the combination activity against sensitive isolates.
ⅣRelationship between sensitization of amiodarone and intracellular calcium
Fluo-3/AM is a trans-membrane fluorescent indicator,which can immediately give out fluorescence after bounding to Ca~(2+),and reflect the square pulse changes of intracellular calcium.Flow cytometry was introduced to test the effect of drugs combinations on intracellular calcium of CA10(resistant).CA10 was incubated in YPD liquid medium until log phase growth,then washed by D-Hanks buffer and resuspended to 10~7CFU/ml.Cells were loaded with isovolumic Fluo-3/AM at 37℃and 120rpm for 30min to a final concentration of 3μM.Results showed that an application of AMD resulted in a biphasic elevation of cytoplasmic calcium,which may relate to its weak antifungal activity;fluconazole has no effect on intracellular [Ca~(2+)]i,but can reversed the biphasic elevation(phase 1 and 3)of cytoplasmic[Ca~(2+)]i of "control" to monophasic elevation(phase 2);sensitization of AMD may due to the release of calcium from calcium store,and extremely raised intracellular calcium.
ⅤEffect of amiodarone on intracellular azoles accumulation
Rhodamine 6G(R6G)can passive diffuse into fungi cells,and has the same substrate of CDR(Candida Drug Resistance)with fluconazole、itraconazole and voriconazole.Fluorometric method and flow cytometry were introduced to test the accumulation、uptake and effluxion of R6G(10μM)with or without amiodarone in CA10、CA135、CAB、CA14.Strains were incubated in YPD liquid medium until log phase growth.Samples of R6G accumulation were carried out in YPD liquid medium, R6G uptake was performed in PBS buffer and R6G effluxion was done in 5% glucose-containing PBS buffer.Results exhibited that sensitization mechanism of amiodarone is the increment of R6G accumulation,which is resulted from increased R6G uptake,not reduced R6G effluxion;and amiodarone had no obvious effect on R6G accumulation、uptake、effluxion in sensitive strains.
ⅥEffect of drugs on cellular morphology of Candida albicans
Immersion objective(×1000)of light microscope was used to overview the effects of amiodarone and azoles alone or in combinations against CA10 and CA14.There are no cellular morphology changes among drugs actions after 6h incubation,which demonstrated that most bacterium and tissue dyeing methods suitable for fungi cells, too.Strains after 24 and 48h incubation with drugs showed:cells of "growth control" have clear wall and nucleus,with pack of budding and filaments;cells of "amiodarone alone" have no clear wall and nucleus,with aniso-size of cell body;"azoles alone" has less cells than "growth control",with aniso-size of cell body and gloomy staining; cells of "drugs combinations" have no significant deviation with "azoles alone " in CA14,while in CA10 there has the least number of cells,with appearance of no staining or debris-liking cells.
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