【作者】 周亚滨；

【导师】 贾继辉；

【作者基本信息】 山东大学 ， 医学微生物学， 2013， 博士

【摘要】 研究目的念珠菌感染能够引起诸多感染性疾病,其中白假丝酵母菌和近平滑念珠菌是主要致病性念珠菌菌株。生物被膜是一种不可逆粘附于底板、交界面以及细胞间互相粘结的、以包埋在细胞外基质中的菌细胞为结构特征的微生物群体。生物被膜在自然界中广泛存在,并以对抗微生物药物的耐药性为主要特征。生物被膜形成后,念珠菌的耐药性大幅增加,使念珠菌引起感染的治疗愈加困难。因此,寻找新的有效防治念珠菌生物被膜感染的方法是亟待解决的问题。研究发现,阿司匹林能够明显的减少白假丝酵母菌生物被膜的形成。Alem等人发现阿司匹林对正在生长和完全成熟的生物被膜有效,然而,该效应具有剂量依赖性,阿司匹林只有在超过人体耐受的浓度下才能显示出抗念珠菌生物被膜的药理效应,由此产生的毒理作用极大地限制了它的临床应用。两性霉素B,一种多烯大环内酯类药物,对浮游状态的念珠菌感染极为有效,是治疗危重深部真菌感染的重要药物。然而,其副作用和毒性较强,经常因此而中断和延误治疗。另外,生物被膜形成后对两性霉素B具有很强的耐药性。我们设想将具有抑制真菌生物被膜作用的阿司匹林与传统抗真菌药物两性霉素B联合应用,通过两药物协同效应的研究,以期达到降低毒理作用,增强抗菌效应,为难治性真菌感染的治疗开辟新途径。研究方法1.体外实验：目前研究体外联合用药的评价标准不一,如何科学地评估联用用药效应成为挑战。为此,我们采用棋盘法对联合用药的浓度进行评估,并通过部分抑菌浓度指数法(the fractional inhibitory concentration index, FICI),即联合用药时各药的最低抑菌浓度分别除以各药单用时MIC值商的和,当FICI≤0.5时为协同作用,0.5<FICI≤1时为相加作用,1<FICI≤4时为无关作用,FICI>4为拮抗作用；以及新近开发的ΔE模型,全面直观的反映联合用药的效果。AE模型与只采用MIC值评估的FICI法不同,是对整个浓度范围内的剂量效应进行评估,使结果分析更加可信。针对棋盘实验的局限性,我们又设计了针对生物被膜药动学特点的时间杀菌曲线,探究两药在时间和浓度上的协同作用。同时,为提高实验结果的准确性和再现性,在数据读取上采用XTT比色法代替了传统的肉眼目测和CFU菌落计数。最终在形成方法上以棋盘实验和时间杀菌曲线实验相结合,在结果评价上以FICI法和ΔE模型相结合的思路,共同保证实验结果的严谨性和准确性。由此,建立了基于真菌生物被膜联合用药评价的技术体系。我们采用棋盘法和微量稀释法,测定了阿司匹林和两性霉素B对白假丝酵母菌标准株YEM30,白假丝酵母菌临床株CCA10和近平滑念珠菌标准株ATCC22019浮游细胞和生物被膜细胞的单药MIC以及两药间的联合作用,并比较了不同情况下药物效能的差异,采用FICI法和ΔE进行结果分析,实现优势互补。此外,我们通过评估时间杀菌曲线实验得出每种药物基于时间和浓度的杀菌效能,以此来衡量药物的药动学参数和联合用药效果。2.机制研究：检测了阿司匹林作用前后念珠菌生物被膜相关基因的表达情况,初步探讨了其抑制生物被膜形成的分子生物学机制。3.动物实验：构建小鼠假孕模型,接种白假丝酵母菌,构建小鼠念珠菌阴道炎模型。进行不同药物组合处理,取阴道灌洗液CFU计数并计算菌负荷,通过t检验比较阿司匹林、两性霉素B单独或联合使用时对小鼠阴道念珠菌的抑制效果。研究结果1.体外实验:已发表在Antimicrob Agents Chemother.2012Jun;56(6):3250-601.1药物单用和联合用药MIC值测定：单独用药时,对于浮游状生长的真菌,阿司匹林具有弱的抗菌活性,两性霉素B具有很强的杀菌活性；而在生物被膜细胞中,可观察到两性霉素B具有强耐药性,对应菌株的MIC(IC50)在生物被膜形成后分别上升到原来的64到128倍。然而阿司匹林对生物被膜细胞的抑菌效能与浮游细胞相比变化很小,这表明了阿司匹林具有潜在的抗生物被膜效应。浮游细胞状态下,联合用药时单药MIC浓度仅降低了1-2个梯度；而生物被膜状态下,两药联用后发现单药MIC浓度明显降低。根据来自FICI指数的结果,两性霉素B和阿司匹林的MIC值分别降低了32、16倍,降低到1～2μg/ml和0.031-0.063mg/ml。1.2棋盘实验中,在生物被膜状态下,FICI和ΔE法测定的三株实验菌均表现为强烈的协同作用,且两种方法具有很好的一致性：所有的FICI指数小于0.5,所有受测菌株的ΣSYNs均超过900%。而在浮游细胞状态下,协同作用在ATCC22019和YEM30中被观察到,在CCA10中则表现为无相互作用。1.3棋盘实验结果表明的阿司匹林和两性霉素B浓度依赖性的联用效果同样在时间杀菌曲线实验中所证实。此外,该曲线还反映出两种药物之间时间依赖性的协同效应。阿司匹林单药作用时,随药物浓度升高,抑菌效果增强,显示出浓度依赖性杀菌效应。此外,杀伤速率和程度是随时间逐渐变化,且其变化速度随时间不同而不同。随着药物浓度的升高,达到抑菌浓度所需的时间缩短。联合作用中,在不同浓度的阿司匹林中加入低剂量的两性霉素B,可使杀菌效能明显增强,到达杀菌终点的时间明显缩短。2.机制研究：我们研究了阿司匹林作用后,与念珠菌生物膜形成相关的几个主要分子的表达状况,实验结果显示GCA1表达升高,CDC35、CSR1、EFG1、HWP1均有不同程度下降。3.动物实验：CFU计数测定不同组合用药后小鼠阴道灌洗液的菌负荷,通过t检验表明,联用组在用药后第4、6、9、15天及停药后第3和第6天均表现出明显的抑菌效果,具有显著统计学差异(p<0.05),且联用组抑菌率显著高于单药组。研究结论1、建立了基于FICI和AE法评价阿司匹林和两性霉素B联合应用对抗念珠菌生物被膜协同效应的技术方法。2、证实阿司匹林通过上调GCA1分子表达,下调CDC35、CSR1、EFG1、HWP1分子表达干预念珠菌生物被膜,实现其与两性霉素B联合应用对念珠菌生物被膜发挥协同抑菌效应。3、发现阿司匹林和两性霉素B联合应用对小鼠阴道体内念珠菌有较强的协同抑菌效应。以上研究对难治性真菌感染的治疗提供了新的思路。更多还原

【Abstract】 OBJECTIVEInfections caused by Candida species manifest in a number of diseases. For the past few years, Candida albicans and Candida parapsilosis are still two of the leading Candida species causing infections worldwide. Microbial biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances. Biofilms are ubiquitous in nature and are characterized by their recalcitrance towards antimicrobial treatment. The increase of drug resistance and invasion caused by biofilm formation brings enormous challenges to the management of Candida infection. Therefore, there is a continuous need for the discovery of new antimicrobial agents that are effective against biofilms.The previous resμlts show that aspirin, one of the oldest and most widely used anti-inflammatory drugs, dramatically decreases biofilm formation by C. albicans. Alem et al found aspirin was active against growing and fully mature (48h) biofilms and its effect was dose related. However, the significant effects of aspirin on growth and biofilm formation of Candida spp. were achieved only with suprapharmacological concentrations of the drug, which limits its clinical application. Amphotericin B (AMB), a polyene macrolide agent, used as "the gold standard" antifungal drug since1960s, is a crucial agent in the management of serious systemic fungal infections. In spite of its proven track record, its well-known side effects and toxicity will sometimes require discontinuation of therapy despite a life-threatening systemic fungal infection. Aspirin, which possesses a weak and broad-spectrum antimicrobial activity towards some planktonic and biofilm cultures, may be useful in combined therapy with conventional antifungal agents to reduce their dose and improve their efficacy, while amphotericin B, needs to be combined with agents that are cheaper, more effective, more tolerable, and less toxic, particularly less nephrotoxic than AMB deoxycholate. Considering these, combination between aspirin and amphotericin B is an excellent choice for clinical use.Vaginal candidiasis is a frequent and common distressing disease affecting up to75%of the women of fertile age; most of these women have recurrent episodes. It has long been appreciated that mucosal infections are in essence biofilms. Common themes have emerged between the biofilm in vitro and in vivo. Unique features have emerged as well, such as the influence of host interactions on mucosal biofilms, complex interactions with bacterial flora, and biofilm-associated echinocandin resistance.The first part of our work was needed to illustrate whether the combianation of aspirin and AMB have a strong synergistic action to candidiasis in vitro or not. The second part of our work was aimed at assessing the anti-fungal activity and the combined effects of aspirin and AMB in an experimental infection of vaginal candidiasis. The final part of our work was to investigate the underlying mechanism of the synergistic action.METHODS1. In vitro As there have been controversies over assessing the nature and intensity of drug interactions, The combined use of the most commonly used methods, the fractional inhibitory concentration index (FICI) and a newly developed method, the ΔE model, which uses the concentration-effect relationship over the whole concentration range instead of using the MIC index alone enables the interpretation of results more reliable. As an attractive tool for studying the pharmacodynamics of antimicrobial agents, time-kill curves can provide detailed information about antimicrobial efficacy as a function of both time and concentration. In the meantime, the XTT reduction assay used for data generation instead of the traditional methods of visual reading and colony counting make it possible to evaluate fungal biofilm growth accurately.We investigated the minimal inhibitory concentration (MIC) and combined effects of aspirin and amphotericin B against the planktonic cells and biofilm cells of C. albicans and C. parapsilosis by microdilution method and the checkerboard microdilution method, and compared the differences of drug efficacy in different states. Furthermore, we obtained the detailed information about antimicrobial efficacy as a function of both time and concentration by time-killing test to assess the pharmacodynamics of each antimicrobial agent and the combined effects. New methods and interpretation models such as ΔE model were employed in comparison with FICI.2. Mechanism:We study the role of aspirin against candida biofilms related gene expression, and the mechanism of biofilm inhibition was discussed preliminarily3. In vivo:Mice were maintained under pseudoestrus condition prior to infection, and then candida albicans was inoculated and colonization on the vaginal mucosa. Treatment with different combination drug, The fungus burden was quantitated (CFU) by cultural a vaginal lavage fluid. Differences between asprin treated, AMB treated and ASA-AMB treated mice were evaluated by viable count data of the fungus burden in the vagina and the inhibitory effect of vaginal candida in mice were compared using the Student’s t-test (two-tailed).RESULTS1. In vitro1.1By testing the drug alone, in planktonic cells, aspirin has weak effect on the tested strains and AMB has strong fungicidal effect, while in biofilm cells, the highest level of resistance to AMB is observed, with the MIC (IC50) to the corresponding strain increased up to64and128-fold after biofilm formation, respectively, based on MICs by XTT. However, aspirin’s fungistatic activity in biofilm cells seems to change little in comparison to planktonic cells, which indicates dramatic antibiofilm activity. In terms of planktonic cells, the MICs of either individual agent were reduced by one to two dilutions against the tested strains, while remarked reductions were observed for AMB against biofilm cells when combined with aspirin. The geometric mean (GM) of the MIC to AMB and aspirin decreased up to32and16-fold, respectively, based on FIC indices.1.2In the checkerboard microtiter plate format, strong synergism was observed in biofilm cells of all three strains analyzed by FICI and ΔE. The two models correlated very well. The FICI indexes were far less than0.5, while all the ΣSYNs of the three tested strains were beyond900%, which indicate a super strong synergistic action between aspirin and amphotericin B. As to planktonic cells, synergisms were observed in ATCC22019and YEM30, Indifference was observed in CCA10.1.3The concentration-dependent synergistic action of aspirin and amphotericin B against biofilm cells that proved by checkerboard microdilution assay was confirmed by time-kill curves. Furthermore, the curves also revealed the time-dependent synergistic action between them. In aspirin alone, discernible improvement in the extent of fungistatic activity and the slope function of the time-kill curve to each strain was noted as the amount of drug in solution increased. Marked concentration-dependent fungistatic activity was also observed, what’s more, the rate and extent of fungistatic activity varied over time and the time to achieve a fungistatic endpoint was shortened as the dose increased. In combination, the addition of amphotericin B in low dose to various concentrations of aspirin resulted in strikingly improvement in extent of activity and trend toward a shorter time to the fungistatic endpoint versus single agents.2. We studied how the aspirin influences the expression of gene which related to biofilm formation. The experimental results show that the expression of GCA1is raised, and CDC35, CSR1, EFG1, HWP1decreased.3. In vivoWe determinated the fungus load of vaginal lavage fluid after different combination drugs treated in mice, the fungal load in the vagina were measured at the9,11,13,16and24days post-infection (2,4,6,9,15days after treatment), there is a significant reduction of Candida load in mice treated with ASA-AMB with respect to asprin and AMB treated alone or diluent saline treated mice starting from11days post infection(4days after treatment), and this beneficial effect as maintained until22days post infection (15days after treatment), even6days after therapy discontinued (p <0.05). CONCLUSION1. Established a method to evaluate synergistic effect of aspirin and amphotericin B combined use against candida biofilm based on FICI and delta E method2. We demonstrated that aspirin can intervene in candida biofilm by raising GCA1gene expression and cut CDC35, CSR1, EFG1, HWP1gene expression when aspirin combined application with amphotericin B against candida biofilm play a synergistic antibacterial effect.3. Found that a strong synergistic antimicrobial effect of aspirin and amphotericin B combined application against vaginal candida in mice.Based on the above study,a new way of thinking have provided for the treatment of refractory fungal infection.更多还原