【作者】 姚琦；

【导师】 周红；

【作者基本信息】 第三军医大学 ， 药理学， 2011， 博士

【摘要】 目的:在临床上,抗生素在细菌感染性疾病的治疗中发挥着重要的作用。然而,当长时间暴露于抗生素后,细菌通过改变其代谢通路、产生灭活酶、以及调节相关外排泵蛋白的表达而导致对一些抗生素产生一定程度的耐药。一项关于革兰氏阴性菌耐药的研究发现,最常见的革兰氏阴性细菌如铜绿假单胞菌、鲍氏不动杆菌、以及大肠埃希菌对临床上广泛使用的抗生素如头孢他啶、头孢哌酮、环丙沙星、庆大霉素等产生耐药。到目前为止,除了β-内酰胺酶抑制剂外,还没有一项成功的策略可以恢复抗生素的抗菌活性。在前期研究中,我们发现青蒿琥酯,一种广泛用于恶性疟及脑型疟治疗的青蒿素的衍生物,在其浓度达到256μg/ml时可将大肠埃希菌ATCC35218对庆大霉素的最小抑制浓度由4μg/ml降为0.25μg/ml。扩大青蒿琥酯的联用范围后,我们同样发现青蒿琥酯可显著增加大肠埃希菌ATCC35218对舒氨西林与头孢匹胺的敏感性。更令人兴奋的是,这种协同的抗菌效应在大肠埃希菌临床分离株与耐甲氧西林的金黄色葡萄球菌中也被观察到。同时,我们发现青蒿琥酯明显增加了柔红霉素在大肠埃希菌ATCC35218中的聚集。在动物实验中,我们发现青蒿素与舒氨西林合用时明显降低了热灭活大肠埃希菌攻击脓毒症小鼠的死亡率。事实上,青蒿琥酯在体外是如何增敏不同抗生素的抗菌效应的还不是很明确。鉴于此,在本研究中我们首先检测了青蒿琥酯联合不同β-内酰胺类抗生素对大肠埃希菌ATCC35218的抗菌增敏作用。随后我们探讨了主要的外排泵基因在青蒿琥酯所产生的抗菌增敏效应中所发挥的作用。基于此,我们试图阐明青蒿琥酯在大肠埃希菌中所引起的抗菌增敏效应的具体机制,同时,为临床上降低感染性疾病治疗中革兰氏阴性菌的耐药提供一种有效的治疗措施。方法:一、采用二倍微孔稀释法检测青蒿琥酯处理与不处理情况下,大肠埃希菌ATCC35218和临床分离株对不同β-内酰胺类抗生素的敏感性;二、采用动态生长曲线法观察不同β-内酰胺类抗生素单独或与青蒿琥酯联合使用时对大肠埃希菌ATCC35218和临床分离株的抑制作用;三、采用逆转录PCR检测不同浓度青蒿琥酯预处理对相关外排泵基因表达的影响;四、采用实时定量PCR检测合适浓度氨苄西林与青蒿琥酯联合使用时对大肠埃希菌ATCC35218差异外排泵基因表达的影响;五、采用逆转录PCR检测不同浓度青蒿琥酯预处理对差异外排泵基因上游调控子mRNA表达水平的影响;六、采用实时定量PCR检测合适浓度氨苄西林与青蒿琥酯联合使用时对大肠埃希菌ATCC35218差异外排泵基因上游调控子mRNA表达水平的影响;七、采用二倍微孔稀释法检测青蒿琥酯处理与不处理情况下,AcrA、AcrB敲除的大肠埃希菌AG100A不同β-内酰胺类抗生素的敏感性;八、采用反义寡核苷酸干扰技术探讨差异外排泵基因在青蒿琥酯产生的抗菌增敏效应中所处的地位以及发挥的作用;九、采用Western blot技术检测青蒿琥酯对大肠埃希菌ATCC35218中差异外排泵基因蛋白表达水平的影响;十、液相色谱-质谱联用技术检测氨苄西林在细菌体内的聚集情况;结果:一、尽管青蒿琥酯单独使用并没有明显的抗菌作用,但它几乎增加了大肠埃希菌对所有被检测的β-内酰胺类抗生素包括氨苄西林、舒氨西林、青霉素G、苯唑西林、头孢匹胺的敏感性;二、单独的青蒿琥酯对于大肠埃希菌的生长没有明显的抑制作用,但与不同的β-内酰胺类抗生素联合使用后相对于单纯的抗生素处理可明显抑制细菌的生长;三、RT-PCR结果表明,不同浓度的青蒿琥酯并不能显著降低外排泵相关基因MdtA、MdtC与MdtF的mRNA水平,但512μg/ml的青蒿琥酯明显降低了AcrA、AcrB、MdtE的表达水平。此外,512-32μg/ml青蒿琥酯对于共同的外膜通道TolC的表达并没有明显影响;四、qRT-PCR结果表明,相对于单纯的青蒿琥酯或者氨苄西林处理,联合处理明显降低了大肠埃希菌中AcrB mRNA的水平。然而,AcrA、MdtA、MdtC、MdtE、MdtF基因的表达水平并没有明显影响;相类似地,青蒿琥酯对于TolC的表达同样没有明显影响;五、AcrA和AcrB基因双敲除的大肠埃希菌AG100A对β-内酰胺类抗生素的敏感性明显增加,但青蒿琥酯的联合处理并没有增加AG100A对β-内酰胺类抗生素的敏感性;六、针对AcrA和AcrB基因的反义寡核苷酸干扰明显降低了大肠埃希菌对氨苄西林的敏感性。当大肠埃希菌中的AcrA基因被有效沉默时,青蒿琥酯的额外处理使细菌的耐药性进一步降低。然而,当AcrB基因被沉默时,青蒿琥酯的加入并没有进一步地降低细菌的耐药性;七、512μg/ml青蒿琥酯显著地下调了大肠埃希菌ATCC35218中AcrB蛋白的表达水平。然而,其他浓度(32-256μg/ml)青蒿琥酯对于AcrB蛋白的表达并没有抑制作用;八、除了MarA基因,512-32μg/ml青蒿琥酯对于主要的外排泵系统AcrAB-TolC上游的调控子的表达并没有明显影响。然而,相对于单纯的青蒿琥酯或氨苄西林处理,256μg/ml青蒿琥酯联合256μg/ml氨苄西林可明显减少上游正性调控子SoxS、MarA的表达水平。同时,青蒿琥酯联合氨苄西林对于负性调控子AcrR、MppA的表达没有明显影响;九、青蒿琥酯增加了氨苄西林在大肠埃希菌ATCC35218体内的聚集。当青蒿琥酯预处理浓度为128μg/ml时,抗生素在细菌体内的聚集达到最大值。当青蒿琥酯浓度超过此浓度时聚集量反而逐渐减少。十、青蒿琥酯不能增加AcrB基因缺失菌株大肠埃希菌AG100A中柔红霉素的聚集,同时,青蒿琥酯也不能增加AcrB基因过表达的大肠埃希菌AG100A/pQE30-AcrB中柔红霉素的聚集。结论:1.青蒿琥酯广泛地增加了大肠埃希菌对不同β-内酰胺类抗生素的敏感性;2.青蒿琥酯抑制了外排泵系统AcrAB-TolC的表达;3. AcrB和AcrA基因缺失可显著增加细菌对β-内酰胺类抗生素的敏感性;4.对于AcrA基因的反义寡核苷酸干扰不足以减弱青蒿琥酯的抗菌增敏作用,而AcrB的沉默使青蒿琥酯的抗菌增敏作用几乎消失;5. AcrB在青蒿琥酯产生的抗菌增敏效应中起着主要的作用,而AcrA在这一事件中发挥着次要的作用;6.青蒿琥酯单独或联合氨苄西林显著下调了AcrAB-TolC外排泵系统上游某些正性调控子如MarA和SoxS的表达;7. 512μg/ml青蒿琥酯显著地下调了大肠埃希菌ATCC35218中AcrB蛋白的表达水平,但低于此浓度青蒿琥酯对于AcrB蛋白的表达没有影响。8.青蒿琥酯不能增加大肠埃希菌AG100A细菌体内柔红霉素的聚集,表明AcrB缺失时,青蒿琥酯失去了主要的作用环节,其抗菌增敏作用消失;而AcrB过表达时,青蒿琥酯同样不能增加细菌体内柔红霉素的聚集,表明AcrB的过表达同样使有限的青蒿琥酯的效应无法发挥;9.青蒿琥酯增加了氨苄西林在大肠埃希菌ATCC35218中的聚集,这一现象可能与AcrAB-TolC被抑制导致抗生素外排减少相关。同时,当超过一定浓度时,抗生素的聚集量反出现降低,可能与细胞膜通透性的改变有关。更多还原

【Abstract】 Objective:Antibiotic plays a key role in the treatment of diseases caused by bacterial infection in clinic. However, after the long-term exposure to antibiotics, the bacteria alter their metabolic pathways, produce inactivated enzyme, and regulate the expressions of efflux pump proteins, which result in the bacterial resistance against some antibiotics in some degree. A study on gram-negative bacteria resistance have documented that the most common gram-negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli (E. coli) became resistant against many antibiotics including ceftazidime, cefoperazone/sulbactam, ciprofloxacin, gentamicin and etc. those were widely used in clinic.By now there was no such a successful strategy exceptβ-lactamase inhibitors to restore antibacterial activities of antibiotics. In the previous study, we found that 256μg/ml Artesunate (AS), a derivative of artemisinin (ART), which was widely used against falciparum malaria and cerebral malaria, could decrease the MIC of Escherichia coli ATCC35218 to gentamicin from 4μg/ml to 0.25μg/ml (16-fold). Expanding the combined range of AS, we found that AS also increased the susceptibility of Escherichia coli ATCC35218 to ampicillin sodium-sulbactam sodium and cefpiramide. Excitedly, the synergetic antibacterial effect was also observed in Escherichia coli isolates and methicillin resistant staphylococcus aureus (MRSA). Meanwhile, AS increased the accumulation of daunorubicin within ATCC35218. Importantly, ART could significantly decrease the mortality of mice challenged with live E. coli when it was used with ampicillin together.However, how AS potentiate the antibacterial effects of various antibiotics in vitro is still not clarified. In the present study we first measured the AS’s antibacterial potentiation on variousβ-lactam antibiotics against E. coli ATCC35218. Then we investigated the role of the main efflux pump related genes in the artesunate’s antibacterial potentiation effect against E. Coli. Based on these, we tried to illuminate the possible mechanism for AS’s antibacterial potentiation effect and provide an efficient approach to reduce the resistance of gram -negative bacteria in the treatment of infectious diseases in clinic.Methods:1.Two-fold diluted method was used to measure the susceptibility of E. Coli ATCC35218 and clinical isolates to variousβ-lactam antibiotics in the absence or presence of AS;2.Dynamic growth assay of effects of variousβ-lactam antibiotics alone or combined with AS on E. coli ATCC35218 and clinical isolates;3.RT-PCR analysis of the mRNA levels of the main efflux pump related gene(s) and upstream regulator(s) within E. coli ATCC35218 after the treatments of AS with different concentrations;4.qRT-PCR analysis of the mRNA level(s) of the differential gene(s) and and upstream regulator(s) within E. coli ATCC35218 after the treatment of 256μg/ml AMP alone or in combination with 256μg/ml AS;5.Two-fold diluted method was used to measure the susceptibility of E. Coli AG100A lacking AcrA and AcrB in the absence or presence of AS treatment.6.Antisense oligonucleotide technique was employed to knockdown the main differential genes to investigate its or their role of the differential gene(s) in the antibacterial potentiation effect produced by AS;7.Western blot analysis of protein(s) expression levels of the differential gene(s) within E. coli ATCC35218 after the treatment of AMP alone or in combination with AS;8.The accumulation of AMP in E. coli ATCC35218 by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS).Results:1. Although AS alone had no significant antibacterial effect, it almost increased the susceptibility of E. Coli to all testedβ-lactam antibiotics including ampicillin, ampicillin sodium-sulbactam sodium, penicilin G, oxacillin, cefpiramide, and ect.;2. Single AS had little inhibitory effect on E. coli ATCC35218, while AS in combination with variousβ-lactam antibiotics significantly suppressed the bacterial growth compared with single antibiotics treatments;3. RT-PCR results showed that different concentrations AS (512-32μg/ml) could not significantly reduce the expression of AcrA, MdtA, MdtC, and MdtF. In fact, 512μg/ml AS markedly down-regulated the expressions of AcrB and MdtE. In addition, AS (512-32μg/ml) had no obvious effects on the common outer membrane channel, TolC;4. qRT-PCR results demonstrated that 256μg/ml AS in combination with 256μg/ml AMP significantly reduced the mRNA levels of AcrB compared with single AS or AMP treatment. However, the expressions of AcrA, MdtA, MdtC, MdtE, and MdtF were not significantly changed in the presence or absence of AS. Meanwhile, the expression of TolC was not significantly changed;5. E. Coli AG100A lacking AcrA and AcrB genes became very sensitive to variousβ-lactam antibiotics. However, the additional AS treatment could not further increase the bacterial susceptibility;6. The transformation of as-ODNs targeting AcrA and AcrB significantly increased the the bacterial susceptibility to AMP. However, the bacterial resistance was not further decreased even after the additional AS adding when AcrB gene was knockdown.7. Except MarA gene, AS (512-32μg/ml) had no significant effect on the upstream regulators of AcrAB-TolC. However, AS (256μg/ml) in combination with AMP (256μg/ml) significantly down-regulated expressions of the positive regultors including SoxS, and MarA compared with single AS or AMP treatment. However, AS had no significant effects on the negative regulators such as AcrR and MppA;8. High concentration AS (512μg/ml) significantly down-regulted the expression of AcrB protein within ATCC35218. However, AS with lower concentration (32-256μg/ml) had no significant inhibitory effect on AcrB protein;9. AS could significantly increase the accumulation of AMP in the bacteria. The largest accumulation was found after the additional AS treatment at a concentration of 128μg/ml. Then the accumulation product was gradually decreased accompanied by increasing concentration of AS.10. Artesunate could not increase the accumulation of daunorubicin in E. coli AG100A, suggesting that artesunate lost its main effect when AcrB gene was knockout. Also, artesunate could not increase the the accumulation of daunorubicin when AcrB was overexpressed. We speculated it was due to the limited artesunate that could not play its antibacterial potentiation effect in front of the overexpression of AcrB. Conclusions:1. AS widely increased the susceptibility of E. coli toβ-lactam antibiotics;2. AS inhibited the expression of a major multi-drug efflux pump system AcrAB-TolC;3. AcrB played a major role in the antibacterial potentiation produced by AS, and AcrA played a minor role in this event;4. AS alone or in combination with AMP significantly down-regulated the positiveregulators and up-regultated the negative regulator on the upstream of AcrAB-TolC, thereby reducing the expression of AcrAB-TolC, which decreased the bacterial resistance against AMP;5. AS increased the accumulation of AMP within E. coli ATCC35218, which might be associated with the decreased the efflux output of antibiotics by the inhibition of the main efflux pump system AcrAB-TolC. While, the accumulation of the antibiotics was reduced when AS was over some certain concentration.6. AS could not increase the accumulation of DNR in E. coli AG100A (△AcrB), suggesting AS lost its main target when AcrB was knockout. And, AS could not increase the accumulation of DNR in E. coli AG100A transfected with pQE30-AcrB, indicating AS could not play its inhibitory effect on efflux pumps in the presence of the AcrB overexpression.更多还原