【作者】 袁中伟；

【导师】 尹立子； 曹雨辰；

【作者基本信息】 四川农业大学 ， 兽医硕士（专业学位）， 2020， 硕士

【摘要】 耐甲氧西林金黄色葡萄球菌(Methicillin resistant Staphylococcus aureus,MRSA)是一种可以引起生物被膜介导感染的致病菌。本研究从体外结晶紫染色实验中筛选出具有抑制生物被膜形成和清除成熟生物被膜作用的百里香酚,并探究百里香酚联合万古霉素治疗小鼠生物被膜感染模型的效果。本研究分为以下三个部分:(1)百里香酚对TCH1516的体外抑菌机制初步探究采用微量肉汤稀释法和琼脂平板菌落记数法测定百里香酚对MRSA(TCH1516)的最低抑菌浓度(minimal inhibit concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC);通过测定菌液电导率、DNA外渗量、SDS-PAGE和电子显微镜确定百里香酚对TCH1516细胞膜、细胞壁、可溶性蛋白质代谢和菌体细胞的超微结构。结果显示百里香酚对TCH1516的MIC和MBC均为256μg/m L。与对照组相比,512μg/m L百里香酚作用菌体1 h后,菌液电导率增加18.08±1.80%,DNA外渗量增加123.40±8.06 ng/μL,作用24 h后,菌体可溶性蛋白质含量比对照组降低68.20±0.15%。百里香酚对TCH1516细胞壁和细胞膜具有破坏作用并干扰其正常的二分裂。结果表明百里香酚可以通过改变菌体细胞膜通透性,干扰蛋白质代谢和正常的二分裂来抑制细菌的生长。(2)百里香酚对TCH1516生物被膜的作用采用结晶紫染色法、扫描电镜、刚果红平板法、分光光度法和RT-PCR技术分别探究百里香酚对TCH1516生物被膜形成的抑制、清除成熟生物被膜的能力,对黏附多糖(polysaccharide intracellular adhesion,PIA)合成、eDNA(extracellular DNA)释放量、sar A、ica A和cid A基因转录水平的影响。结果显示百里香酚对细菌生物被膜的形成具有抑制作用,对成熟的生物被膜也具有清除作用。百里香酚能显著抑制PIA的合成。百里香酚能抑制细菌eDNA的释放量,其中32μg/m L的百里香酚作用细菌24 h后,与对照组相比,eDNA的释放量降低49.43±12.55%。百里香酚可以抑制TCH1516生物被膜相关基因的转录水平,其中64μg/m L百里香酚作用TCH1516 24 h后,与对照组相比,sar A、cid A和ica A转录水平分别降低94.01±2.84%、45.01±4.31%和30.85±17.83%。结果表明百里香酚对TCH1516生物被膜的抑制和清除作用显著。其作用机制可能是通过降低ica A、sar A和cid A基因转录水平,抑制PIA的合成,抑制eDNA的释放。(3)百里香酚提高小鼠腹腔植入性生物被膜感染模型中MRSA生物被膜的清除率30只雌性小鼠随机分为对照组、百里香酚组、万古霉素组、联合给药低剂量组、联合给药中剂量组和联合给药高剂量组。以输液管为载体,构建小鼠腹腔生物被膜感染模型后按上述分组注射相应药物。24 h后对载体表面活菌计数;扫描电子显微镜(SEM)观察载体表面生物被膜形态变化;对血液白细胞计数和测定炎症因子(TNF-α和IL-6)水平;显微镜下观察组织病理学改变。结果显示给药24 h后,药物处理组的载体表面菌落数均明显少于对照组,其中联合给药高剂量组几乎无细菌生长;SEM结果表明联合给药能有效清除生物被膜;病理切片结果显示对照组有大量炎症细胞,而联合给药高剂量组基本观察不到炎症细胞。药物单独作用组或联合组血白细胞计数均少于对照组,其中联合给药高剂量组的小鼠白细胞数恢复正常范围;该组TNF-α含量分别比对照组降低22.57±8.81%,IL-6含量分别比对照组升高26.96±9.95%。结果表明百里香酚对小鼠腹腔MRSA生物被膜具有一定的清除作用,与万古霉素联合应用可显著增强其对生物被膜的杀菌作用,且具有一定的治疗效果。综上所述,本研究表明:(1)百里香酚的抑菌机制为:通过改变细胞膜的通透性,破坏菌体细胞膜和细胞壁的完整性、干扰菌体内蛋白质的代谢等途径来抑制TCH1516的生长繁殖。(2)百里香酚可能是通过抑制ica A,cid A和sar A的转录水平降低PIA的合成和减少eDNA的释放来抑制生物被膜的形成和清除成熟的生物被膜。(3)百里香酚可以增强万古霉素对MRSA生物被膜感染的治疗效果。更多还原

【Abstract】 Methicillin-resistant Staphylococcus aureus(MRSA)is a common human pathogen,resulting biofilm-associated infections.In this study,we selected thymol which can inhibit the formation of biofilm and eliminate mature biofilms by crystal violet staining method in vitro,and explored the effect of thymol which enhanced the efficiency of vancomycin in a mouse infection model.This study is divided into the following three parts:1.Antibacterial mechanism of thymol on methicillin-resistant Staphylococcus aureus(MRSA)The inhibitory effect of thymol on MRSA(TCH1516)was determined via the micro-broth dilution method and the colony-counting method.Solution conductivity,DNA exosmosis amount,SDS-PAGE and transmission electron microscope(TEM)were detected to investigate the effect of thymol on cell membrane,cell wall,soluble protein synthesis and microscopic structures.Both MIC and MBC values of thymol against TCH1516 were 256 μg/m L.After treatment with 2 MIC thymol for 1 h,compared with control group,the conductivity improved 18.08 ± 1.80%.The content of DNA was increased by 123.40 ± 8.06 ng/μL than that in the control group.Compared with control group,after treatment with 2 MIC thymol for 24 h,the protein content decreased by 68.20±0.15%.According to TEM,thymol may have played antibacterial role by destroying cell membrane and cell wall,interfering binary fission.Thymol could damage the cell membrane,interfering the metabolism of protein,and binary fission.2.Effect of thymol on TCH1516 biofilms The abilities of thymol to inhibit biofilm formation,to eliminate mature biofilms,PIA(polysaccharide intracellular adhesion)synthesis,the release of extracellular DNA(eDNA)and the change of sar A,ica A and cid A transcriptional level were assessed by CV staining,SEM,congo red plate method,spectrophotometric and RT-PCR.The results showed that thymol could inhibit bacterial biofilm formation and remove the mature biofilms.As the thymol concentration increases,there is a gradual decrease in the synthesis of PIA,which indicates that thymol inhibits the synthesis of PIA in MRSA biofilms.Thymol could inhibit the release of eDNA from MRSA biofilms.After treatment with 32 μg/m L thymol for 24 h,release of eDNA decreased by 49.43± 12.55%,compared with control group.Afer treatment for 24 h,32 μg/m L thymol decreased the transcript levels of sar A,cid A and ica A by 94.01 ± 2.84%,45.01 ±4.31%,and 30.85 ± 17.83%,respectively,when compared with those in the untreated group.The results showed that thymol had significant inhibitory and removal effect on bacterial biofilms.The mechanism may be that it inhibits the synthesis of PIA and the release of eDNA by reducing the transcriptional levels of ica A,sar A and cid A genes.3.Thymol enhances clearance of MRSA biofilms in a mouse peritoneal implant infection model.30 female mice were randomly divided into control group,thymol group,vancomycin group,low-dose combination treatment group,middle-dose combination treatment group and high-dose combination treatment group.Using the infusion tube as the carrier,the mice biofilm infection models were established,and then the corresponding drugs were injected into models according to the above groups.After24 hours,the number of viable bacteria on the carrier surface were counted,the morphological changes of biofilms on the carrier surface were observed by scanning electron microscope(SEM).The white blood cell count and the levels of inflammatory factors(TNF-α and IL-6)were measured,and the histopathological changes were observed.The results showed that after 24 h of treatment,the colony count on the carrier surface of drug treatment group was significantly less than that of the control group,and there was almost no bacterial growth in the high dose group;under SEM,biofilms decreased significantly in combination treatment group.The results of pathological sections showed that there were many inflammatory cells in control group,but no inflammatory cells were observed in high-dose combination treatment group.The white blood cell count in the drug alone groups or combined groups decreased,compared with control group.In high-dose combination treatment group the white blood cell count returned to the normal range and the content of TNF-αdecreased by 22.57 ± 8.81%,and the content of IL-6 increased by 26.96 ± 9.95%,compared with control group.The results showed that thymol had a removal effect on MRSA biofilms in mice,and the combined application of thymol and vancomycin could significantly enhance its bactericidal effect on biofilms,and it had certain therapeutic effects.In summary,this study shows that:(1)the antibacterial mechanism of thymol is to inhibit the growth of TCH1516 by changing the permeability of cell membrane,destroying the integrity of cell wall and interfering the metabolism of protein,and binary fission in bacteria.(2)thymol may inhibit the formation of biofilms and eliminate mature biofilms by inhibiting the transcriptional levels of ica A,cid A and sar A,reducing the synthesis of PIA and the release of eDNA.(3)thymol can enhance the effect of vancomycin on MRSA biofilm infection.更多还原