亚麻酸联合妥布霉素抗铜绿假单胞菌生物膜形成效应的群体感应系统机制研究

发布时间：2018-06-13 09:22

本文选题：铜绿假单胞菌 + 亚麻酸　；参考：《大连医科大学》2017年硕士论文

【摘要】：背景:自然界中的微生物为人类带来了有利同时又有害的影响。其有利的一面源于人类与微生物界的共生关系。然而人类一些疾病的发生使得看似“友善”的微生物成为了会导致人类许多微生物相关疾病的条件致病菌。许多细菌通过产生生物膜来抵御不利的生存环境,这是其中一种防御机制。生物膜的形成是一个主动过程,主要取决于当环境信号刺激细胞进入周期增长阶段的时间,并且生物膜的形成过程是由一系列由群体感应系统(QS)支配的基因调控的。另外,生物膜结构因多糖、蛋白质、脂类及胞外DNA等大分子物质的存在而被强化,这些物质使得微生物能抵抗杀菌药物。铜绿假单胞菌,一种广泛存在于自然界中的革兰阴性的条件致病菌,是医院内感染的主要病原菌之一。铜绿假单胞菌有三种相关的群体感应系统(QS),通过这些群体感应系统,调控一些毒力因子的产生,从而导致铜绿假单胞菌引起人类严重威胁生命的感染,这迫使临床医生大量使用抗生素来应对这些感染疾病,从而出现了多重耐药菌株。近年来,临床上常用妥布霉素治疗由铜绿假单胞菌引起的肺部感染,但是铜绿假单胞菌也产生了对妥布霉素的抗药性。目前通过研发和使用抗生素来治疗这些感染,尽管大量关于药物开发的研究都致力于减少微生物的耐药性,但是某些特定抗菌化合物的毒性和耐受性问题以及微生物的持续进化给研发带来很多困难,这些迫使研究者关注和筛选天然植物化学成分和膳食成分作为抗菌药物。脂肪酸是人体营养的重要组成部分,在人体生长过程中扮演十分重要的角色。脂肪酸作为活性物质和药物载体应用在制药行业,例如脂质体制剂以及ω-3脂肪酸在脂质异常、糖尿病、癌症以及抗炎和免疫调节活动中的作用,使得人们开始利用以及评估它在微生物相关传染病中的价值,尽管它有不利的一面,但它可能对人类健康有更多有益的价值。在细菌中,游离脂肪酸通过多种方式首先作用于细菌细胞膜,使细菌繁殖受阻或引起类细胞凋亡样的杀菌效应。这些游离脂肪酸有必需脂肪酸,例如亚油酸和亚麻酸,人体不能合成,但可以通过营养和膳食补充剂获得。亚麻酸连同其ω-3脂肪酸衍生物具有抗多种微生物的能力。所以我们推断亚麻酸也能具备抗铜绿假单胞菌生物膜,并能提高妥布霉素的药效。方法:为了证明这一猜想,本研究主要观察亚麻酸及妥布霉素对铜绿假单胞菌临床分离株、环境分离株和实验室标准株(ATCC 27853)的影响。采用结晶紫染色检测生物膜生物量厚度的差异来评估生物膜的形成。生物膜越厚,吸收的染液越多,与对照组相比,如果染色越轻,说明是由药物抑制作用引起的。由于染色法不能区分附着在生物膜上的活细胞和死细胞,我们采用Alarma Blue法来测定生物膜代谢活性,去更好地解释亚麻酸或妥布霉素的作用。此外,我们用荧光显微镜观察生物膜生物量的变化。由于生物膜的形成由群体感应系统控制,我们通过实时定量PCR检测了选择性群体感应、生物膜和毒力因子相关基因的表达水平。另一方面,铜绿假单胞菌产生的多种毒力因子会导致宿主组织损伤,因此我们也检测了QS相关毒力因子如群集运动、绿脓菌素、Las A活性以及偶氮酪蛋白酶的表达情况,从而确认亚麻酸或联合妥布霉素是否可以减少这些毒性表型特征的产生从而减轻了铜绿假单胞菌的毒性。结果:实验结果发现亚麻酸和妥布霉素对这些铜绿假单胞菌菌株的中位最小抑菌浓度分别是1.56mg/ml和0.3125mg/ml,其中一株临床分离株C2对最小抑菌亚浓度为0.078mg/ml的妥布霉素显示出抵抗,因此采用此株菌来研究在不受妥布霉素影响下,最小抑菌亚浓度的亚麻酸对细菌的作用效果。在单独使用亚麻酸以及联合使用妥布霉素会破坏铜绿假单胞菌生物膜的形成,减少生物膜细胞的代谢活动。在动力试验中,与未处理对照组相比,使用最大药物剂量能进一步阻止群集运动。亚麻酸也可以减少绿脓菌素的产生以及降低偶氮酪蛋白的活性,并具有剂量依赖性,而妥布霉素不然。同样也发现Las A活性受到抑制。基因表达分析结果也证实联合使用亚麻酸和妥布霉素会下调铜绿假单胞菌群体感应系统、生物膜和毒力因子相关基因的表达。药物相互作用分析结果也证实了这种协同作用。结论:因此,亚麻酸可以抑制铜绿假单胞菌生物膜的形成从而可以成为一种潜在的辅助药物。这种联合疗法能够降低妥布霉素的药用剂量从而减少氨基糖苷类抗生素对人体的负面影响,这为未来治疗和控制铜绿假单胞菌的感染奠定坚实基础。
[Abstract]:Background: the microbes in nature bring the beneficial and harmful effects of human beings. Their beneficial side is derived from the symbiotic relationship between human and microbial communities. However, the occurrence of some human diseases makes the seemingly "friendly" microbes become the conditional pathogenic bacteria that cause many human microbes to be related to diseases. The formation of the biofilm is an active process, mainly depending on the time when the environmental signal stimulates the cell to enter the period of growth, and the formation of the biofilm is regulated by a series of genes controlled by the QS. The membrane structure is strengthened by the presence of macromolecules such as polysaccharides, proteins, lipids and extracellular DNA, which make microbes resistant to bactericidal drugs. Pseudomonas aeruginosa, a gram-negative conditional pathogenic bacteria widely found in nature, is one of the main pathogens in hospital infection. There are three phases of Pseudomonas aeruginosa. The QS, which regulates the production of some virulence factors through these quorum sensing systems, leads to a serious threat to life by Pseudomonas aeruginosa, which compels clinicians to use antibiotics to cope with these infections, thus producing multidrug resistant strains. Mycophentin is a treatment for pulmonary infection caused by Pseudomonas aeruginosa, but Pseudomonas aeruginosa has also produced resistance to tobramycin. Currently, these infections are treated by development and use of antibiotics, although a large number of studies on drug development are committed to reducing microbial resistance, but the toxicity of certain specific antibacterial compounds The problem of tolerance and the continuous evolution of microbes have brought many difficulties to research and development, which compel researchers to focus on and screen the chemical constituents and dietary ingredients of natural plants as an antiseptic. Fatty acids are an important part of human nutrition and play an important role in the growth of human body. Fatty acids are used as active substances and as active substances. The use of drug carriers, such as liposomes, and omega -3 fatty acids, in lipid abnormalities, diabetes, cancer, and anti-inflammatory and immunomodulatory activities, makes people start to use and assess the value of it in microbiological related infectious diseases, although it has a negative side, but it may have more human health. The value of benefit. In bacteria, free fatty acids first act in the bacterial cell membrane in a variety of ways, causing bacterial resistance to obstruct or induce apoptosis like effects. These free fatty acids have essential fatty acids, such as linoleic acid and linolenic acid, which can not be synthesized by human body, but can be obtained by nutrition and dietary supplements. Linolenic acid can be obtained. The same Omega -3 fatty acid derivatives have the ability to resist a variety of microorganisms. Therefore, we infer that linolenic acid can also have anti Pseudomonas aeruginosa biofilm and can improve the efficacy of tobramycin. Methods: to prove this conjecture, this study mainly observed the clinical isolates of linolenic acid and tobramycin on Pseudomonas aeruginosa, environmental isolates and The influence of laboratory standard strain (ATCC 27853). The formation of biofilm is evaluated by the difference of the thickness of biomembrane biomass using the crystal violet staining. The thicker the biofilm is, the more the dye is absorbed. Compared with the control group, the lighter the staining is, it is caused by the inhibition of the drug. Because the staining method can not distinguish the life attached to the biofilm. Cells and dead cells, we use Alarma Blue method to determine biofilm metabolic activity, to better explain the role of linolenic acid or tobramycin. In addition, we use fluorescence microscopy to observe the changes in biofilm biomass. Because of the formation of biofilm by a quorum induction system, we detected a sense of selective population by real-time quantitative PCR. The expression level of genes related to biofilm and virulence. On the other hand, a variety of virulence factors produced by Pseudomonas aeruginosa can cause tissue damage. Therefore, we also detected the expression of QS related virulence factors such as cluster motion, Pseudomonas aeruginosa, Las A activity, and azo protein egg white enzyme, thus confirming linolenic acid or combination. Whether brycomycin can reduce the production of these toxic phenotypes and reduce the toxicity of Pseudomonas aeruginosa. Results: the results showed that the minimal inhibitory concentration of linolenic acid and tobramycin on these strains was 1.56mg/ml and 0.3125mg/ml respectively, and one of the clinical isolates C2 was the minimal inhibitory concentration of Pseudomonas aeruginosa. In order to show resistance to tobramycin 0.078mg/ml, this strain was used to study the effect of linolenic acid on the minimal inhibitory concentration of linolenic acid on bacteria without the effect of tobramycin. The use of linolenic acid alone and the combination of tobramycin could destroy the formation of Pseudomonas aeruginosa biofilm and reduce the metabolic activity of biofilm cells. In dynamic tests, the maximum drug dose could further prevent the cluster movement compared with the untreated control group. Linolenic acid could also reduce the production of Pseudomonas aeruginosa and reduce the activity of azo casein, with dose-dependent, and not in tobramycin. The Las A activity was also inhibited. Gene expression analysis also found that It was confirmed that the combination of linolenic acid and tobramycin reduced the expression of the quorum induction system, the biofilm and the virulence related genes in Pseudomonas aeruginosa. The results of the drug interaction analysis also confirmed this synergistic effect. Conclusion: therefore, linolenic acid can inhibit the formation of Pseudomonas aeruginosa biofilm and can be a potential one. The combined therapy can reduce the dose of tobramycin and reduce the negative effects of aminoglycoside antibiotics on the human body, which lays a solid foundation for the future treatment and control of Pseudomonas aeruginosa infection.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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