【作者】 刘萍；

【导师】 孙淑娟； 侯应龙；

【作者基本信息】 山东大学 ， 药学， 2012， 硕士

【摘要】 近年来随着医疗技术的进步,医疗植入体如导管、人工瓣膜和关节置换物等用于临床治疗的几率增加,病原菌粘附在医疗植入体表面形成生物膜(biofilm,BF),造成BF相关的感染发病率上升。白色念珠菌(Candida albicans, CA)、金黄色葡萄球菌(Staphylococcus aureus, SA)是导管相关BF感染的常见分离菌,且有时呈混合感染。BF的形成使BF内的菌极强的耐药性和抵抗机体免疫系统作用的能力,耐药性大大增强,治疗难度增大。目前,由于研究新型抗菌药物耗资巨大,研究周期较长,联合用药成为解决BF相关的耐药问题的研究热点之一。目的：通过测定万古霉素、米诺环素、利福平、左氧氟沙星、磷霉素和阿奇霉素与氟康唑(FLC)联合应用对浮游与成膜的CA与SA混合生长的体外联合抗菌效果,以及钙调节剂对联合用药的影响,寻找能够抗混合BF感染的药物组合,并进行机制探讨。方法：选取耐药CA与耐药SA(R-R),耐药CA与敏感SA (R-S)、敏感CA与耐药SA(S-R)及敏感CA与敏感SA(S-S)形成的混合BF为研究对象,采用微量稀释法分别测定FLC与抗菌药物万古霉素、米诺环素、利福平、左氧氟沙星、磷霉素和阿奇霉素联合对混合菌的浮游及成膜状态的作用,以肉眼观察法与XTT (OD值)法两种方法判定作用效果,利用非参数法(FICI与ΔE)分析实验数据,并以FLC及抗菌药物单用抗CA-SA混合BF的作用、联合用药抗CA及SA单一菌的作用和药物单用抗单一菌的效果为对照组,综合评价联合用药的作用效果。以平板画线法筛选对联合用药抗不同敏感程度CA与SA混合生物膜作用有增效作用的钙离子调节剂,并用微量稀释法测定钙调节剂与FLC及抗菌药物三药合用抗混合浮游菌及其对混合BF的作用效果。通过对比钙离子调节剂与FLC、钙离子调节剂与抗菌药物合用以及抗菌药物与FLC合用抗混合BF的作用,综合评价钙离子调节剂对FLC联合抗菌药物抗混合BF作用的影响。利用transwell膜将药物作用后的混合菌液分离,分别评价药物对混合BF中CA及SA的作用；通过干重法测定FLC与万古霉素等抗菌药物合用后混合BF基质含量,对比生长对照、FLC单用及抗菌药物单用时基质含量的变化,联合用药对混合BF细胞外基质影响；以“三明治”法评价联合用药时FLC及抗菌药物透过耐药及敏感混合BF的量,通过对比FLC及抗菌药物单用时药物透过混合BF的量,以及联合用药透过CA或SA单BF的量,分析联合用药产生的协同作用是否与影响药物透过生物膜的量有关；应用PCR技术评价混合BF中菌丝编码蛋白Hwp1在不同用药组(混合BF生长对照组、单CABF对照组、FLC单用组、抗菌药物单用组、FLC与抗菌药物合用组)的表达改变,观察联合用药协同作用机制是否是通过影响CA菌丝生长从而抑制混合BF形成。并综合上述结果分析联合用药协同抗混合BF相关的机制。结果：FLC单用和抗菌药物单用时没有明显的抗浮游混合菌及混合BF的作用；以FICI法评价联合用药作用时发现：对于浮游的CA-SA混合菌,氟康唑与米诺环素合用时对于敏感和耐药的CA及SA混合菌具有较强的抗菌作用；FLC与万古霉素合用时,对于敏感和耐药的混合浮游菌均具有良好的协同作用,合用时万古霉素的最低有效浓度(MIC)高于单用时抗单一SA菌的MIC；当与FLC合用时,磷霉素和左氧氟沙星抗混合菌的作用结果相似,只对敏感的CA所形成的混合菌起效；而利福平与FLC合用时,只对敏感的SA所形成的混合菌起效；阿奇霉素与氟康唑合用时,抗混合菌的作用不明显,无协同作用。利用ΔE法评价联合用药抗混BF时发现,对于不同敏感程度的混合BF抗菌药物与FLC合用时各个浓度点的ΔE均大于0,当FLC和抗菌药物的浓度高于MIC值时,ΔE显著增大,并且联合用药抗R-R组的ΔE明显高于其他组。FLC与抗菌药物合用对不同成膜时间的CA-SA混合BF作用效果不同,2h、4h和8h形成的BF与浮游菌作用效果相近,但对于成膜时间为24h的混合BF联合用药几乎没有作用。对比联合用药抗成膜时间为8h和12h的CA-SA混合BF的结果,发现米诺环素与FLC合用具有最强的作用效果,对敏感及耐药的混合BF都具有良好的抗菌作用；万古霉素、左氧氟沙星与FLC合用时,对于耐药CA与敏感SA、敏感CA与耐药SA及敏感CA与敏感SA形成的混合BF具有良好的协同作用,但对于耐药菌形成的混合BF作用微弱；当与FLC合用时,阿奇霉素的作用弱于万古霉素和左氧氟沙星,仅对敏感CA形成的混合BF(S-R,S-S)有效；磷霉素和利福平与FLC合用对混合BF的作用较差,仅对敏感菌形成的混合BF (S-S)有效。对比联合用药抗混合BF与浮游菌的作用发现,BF的形成可使药物作用减弱,生物膜越成熟作用效果越弱。然而,利用ΔE法评价联合用药抗混BF时发现,抗菌药物与FLC合用时的各个浓度点的AE均大于0,当FLC和抗菌药物的浓度升高,ΔE显著增大。平板划线法研究表明,钙通道阻滞剂贝尼地平、钙离子螯合剂EDTA对不同敏感程度的CA与SA混合生长均具有抑制作用,可以增强FLC与米诺环素联合用药抗混合BF的作用；EDTA的作用明显强于贝尼地平。当贝尼地平的浓度为4gg/ml,EDTA为0.25mM时,可以显著增加联合用药的抗浮游混合菌和混合BF的作用,降低联合用药中米诺环素和FLC的最低有效浓度。分别评价FLC与米诺环素用药对混合BF中CA和SA的作用,通过对比R-R与R-S组实验结果,发现SA的耐药性可以影响混合生物膜中CA的耐药性,即在相同浓度的FLC与抗菌药物作用下与耐药SA所形成的混合BF中的CA数量多于与敏感SA所形成的混合BF中CA的量；比较相同药物组合浓度作用后的R-R与S-R组中SA的数量发现,混合BF中SA的量主要与抗菌药物浓度有关,CA的量越多对SA的量也增加,但是影响不显著。当联合用药中抗菌药物的浓度大于MIC时,才具有良好的抗SA的作用；而CA在药物合用浓度较低(小于FLC抗单菌的MIC)时生长明显被抑制；在同一浓度的抗菌药物作用后,混合BF中的SA数量随着CA的数量增大(FLC浓度降低)而增多。FLC单用对混合BF细胞外基质有一定的抑制作用,但作用弱于FLC与抗菌药物合用效果。单用米诺环素细胞外基质的形成增多,但联合用药时FLC与米诺环素合用可明显降低细胞外基质的含量；利福平与FLC合用,抗细胞外基质形成的作用较为显著,但利福平单用对细胞外基质的形成也有一定的抑制作用；万古霉素与FLC合用抗细胞外基质形成的作用明显强于万古霉素单用,但相比于生长对照及FLC单用万古霉素与FLC合用的协同作用较弱；磷霉素与阿奇霉素的作用效果相似,单用抗菌药物时混合BF细胞外基质与生长对照相比含量减少,并且与FLC合用时,可以增强抗细胞外基质形成的作用,但联合用药的作用效果并不显著。联合用药药物透过后所产生的抑菌圈大小主要与含药平板中的药物浓度有关,而与混合生物膜的敏感程度无密切相关,不同敏感程度的混合BF间药物透过量差别较小；并且药物透过单一BF的作用结果与药物透过混合BF所产生的抗CA抑菌圈大小相近。米诺环素、利福平与FLC合用时可以观察到联合用药与FLC单用相比增加了抗CA抑菌圈；对于SA铺就的菌板,抗菌药物单用及与FLC合用时透膜后产生的抑菌圈大小无肉眼可见的明显区别,与抗菌药物单用抗SA的作用相似。对比FLC单用、米诺环素单用及FLC与米诺环素合用组与生长对照间混合菌总RNA中菌丝编码蛋白HWP1的表达量没有显著差异；联合用药的不同作用时间(6h、12h和24h)之间不同药物作用的HWP1的表达量差别较小结论：1.万古霉素、米诺环素、利福平、左氧氟沙星、磷霉素及阿奇霉素与FLC合用可以产生协同抗CA-SA混合浮游菌和混合BF生长的作用。其中,米诺环素与FLC合用效果最为显著,可以显著降低药物抗混合BF的SMIC。阻断细胞钙离子通道及降低细胞外钙离子浓度均可增加米诺环素与FLC联合抗混合菌的作用。2.混合BF的形成会增加CA和SA的耐药性,联合用药通过杀灭混合BF中的CA,破坏BF的屏障作用增加药物对SA的作用；联合用药抗混合菌可以减少混合BF细胞外基质的含量,但不增加药物透过BF的能力,也不影响CA菌丝编码蛋白HWP1的表达量。联合用药的协同作用机制并不是通过抑制CA菌丝生成而起到减少BF细胞外基质的作用,也不是通过增加药物透膜量起到协同抗混合BF的作用；联合用药的协同作用可能与影响细胞内钙离子平衡有关,但具体机制有待进一步研究。总之,抗菌药物万古霉素、利福平、米诺环素、左旋氧氟沙星、磷霉素和阿奇霉素与FLC合用联合时对浮游状态及形成BF混合菌均表现出协同作用,对敏感性不同的CA和SA所形成的混合菌均具有良好的协同效果,并且钙离子通道拮抗剂贝尼地平和钙离子螯合剂EDTA对其有增效作用。完成本实验为为临床治疗CA和SA混合BF感染提供药物选择的理论依据。通过联合用药途径,增强药物对CA和SA混合BF感染的治疗效果,这对于克服致病菌的耐药性,提高对导管相关BF耐药菌感染的治疗,具有重大的现实意义、经济效益和社会效益。更多还原

【Abstract】 Polymicrobial biofilms represent as an understudied and clinically relevant health problem with the potential to serve as an infectious reservoir for a variety of microorganisms, including bacteria and fungi. As a natural member of the mucosal microbiota, C. albicans readily forms biofilms on a wide variety of polymers such as indwelling medical devices. C. albicans is the4th leading cause of bloodstream infections and the3rd most common organism isolated from intravascular catheters and is associated with the highest incidence of mortality. It is estimated that27%of nosocomial C. albicans bloodstream infections are actually polymicrobial, and Staphylococcus aureus was the third most common organism isolated in conjunction with C. albicans. Since the therapies against polymicrobial infections are not well characterized, combination therapy was consented to be one of the feasible methods for alternative treatment. Previous work from our laboratory demonstrated that the combination of fluconazole (FLC) and minocycline (MINO) has synergistic antifungal effects against plantonic and sessile C.albicans. Whether the combinations of FLC and antibacterial agents have synergistic effctes against the mixed strains were explored in this study, and also the potential mechanisms.Objective:In order to find new therapies against mixed bioflims, the combined effects of antibacterial agents and FLC against plantonic and sessile C.albicans mixed with Staphylococcus aureus was studied, including vancomycin(VAN), Minocycline (MINO), Rifampin (RIF), Ofloxacin (OFLX), Azithromycin(AMZ) and Fosfomycin (FOM). And also the effects of calcium regulation agents on the combinations were explored. The potential mechanisms was investigated by evaluating the roles of drugs on the two strains and testing quantitative changes of extracellular matrix, as well as the expression of the gene Hwpl(encoding hypha of Candida albicans). MethodsAccording to the method described in CLSI M27-A3, the microdilution method was used to investigate the combined antimicrobial effects of fluconazole with antibacterial agents (rifampin, minocycline, ofloxacin, fosfomycin, vancomycin and azithromycin) against Candida albicans mixed with Staphylococcus aureus both in plantonic and in biofilm.The mixed suspension was separated by transwell membrane to respectively evalueate drug effects against Candida albicans and Staphylococcus Both sensitive and resistant C. albicans and S. aureus were tested in mixed planktonic cells and mixed biofilms; the results were evaluated both by visual observation and XTT (OD value) method, and two non-parametric methods (FICI and ΔE) were used to analyze the results.We tested the quantitative changes of extracellular matrix after givn drugs by weighting the bioflim dry weight, which was carried out by the palts-glass slide model; the cell counting method was used to evaluate the combined effect against the two strains respectively after dividing the two strains in the mixed bioflim by transwell membrane; the "sandwich" method was used in order to find out the effect of antibacterial agents on FLC penetrating the mixed biofilms of C. albicans and S.aureus; the expression of the gene Hwpl (encoding hypha of Candida albicans) was studied by PCR. All of these studies were to investigate the mechanism of synergistic drug-interaction and of biofilm resistance.ResultsThe combination of FLC and antibacterial agents had synergistic effects against plantonic C.albicans mixed with S. aureus, and also the mixed bioflims.MiNO had the strongest synergistic effects with FLC, and this combination can destroy the mixed BF in low concentrations which were lower than the MIC of the strains when disposed alone. There were no significant difference between plantonic and2h-BF,4h-BF or8h-BF when disposed with drug combinations, but the MIC of the combinations were higher when against12h-BF. There was no significant antibacterial effects when against24-BF.Compared to the growth group, the FLC inhibited the fungal growth slightly. There were no significant differences between BEN or EDTA disposing alone group and the control group. No significant antifungal effect was found when MINO used alone or combined with those calcium regulators. When FLC group was taken as control, the addition of MINO enhanced the antifungal effect of FLC. When calcium channel blocker BEN and calcium chelator EDTA were combined with FLC separately, slight antifungal effect was observed, while when BEN/EDTA was combined with FLC and MINO, a obvious enhancement was displayed, especially when EDTA was combined with FLC and MINO, with the MIC of BEN and EDTA4ug/ml and0.25mM, respectively.The quantity extracellular matrix was decreased in the combination group which suggests that the combination therapy can inhibit the extracellular matrix; Transwell membranes can divide the two strains in the mixed biofilm and after counting those two strains, we found that the SA was related to the concentrations of the antibacterial agents. Only when MINO combined with FLC can cause a significant larger inhibition zone against CA than using FLC alone, but there were no such changes when against SA; the polymerase chain reaction (PCR) evaluated the impact of drug combinations on mycelium encoded protein HWP1expression, there were no great disparities between drug combination group, drug alone group and growth control.Conclusion1. In this study, microdilution method was used to study the combiantion effects of the antibacterial agents and FLC, and the resualts were evaluated in FICI and ΔE method. We found that the combinations of FLC and MINO, VAN, FOM, OLFX, RIF, or AMZ have synergistc antibacterial effects against plantonic and mixed bioflim. The time of forming mixed biofilm can impact the antibacterial effects greatly, there were no significant difference between plantonic cells and2h-BF,4h-BF or8h-BF when disposed with drug combinations, but the MIC of the combinations were higher than that against12h-BF. There were no significant antibacterial effects when against24-BF.The calcium regulation agents could enhance combination effects of FLC and MINO, and the EDTA have stronger effects than BEN.2. The mechanism study found that combination drug can reduce the amount of the mixed biofilm cells, mainly due to the suppression of Candida albicans. Although drug combinations can decrease the dry weights of the extracellular matrix, the drug combination can not enlarge the amount of FLC which can penatrate the biofilm, and not reduce the expression of the Candida albicans hypha protein encoded by HWP1, which suggest that the combined drugs can inhibit the growth of the mixed strains not by reducing the formation of the biofilm, and that C. albicans play the role of anti-mixed biofilm growth.In short, the drug combinations of FLC and antibacterial agents showed synergistic effects against the mixed strains of the different sensitivity of Candida albicans and Staphylococcus aureus. Minocycline and FLC have the strongest synergy. The calcium channel blocker benidipine and calcium chelator EDTA can enhance the combined effects. C. albicans play the role of anti-mixed biofilm growth.Combined drugs can inhibit the growth of the mixed strans not by reducing the formation of the biofilm.更多还原