【作者】 秦晓庆；

【导师】 侯振中；

【作者基本信息】 东北农业大学 ， 兽医产科学， 2013， 博士

【摘要】 空肠弯曲杆菌(Campylobacter Jejuni)是一种人畜共患病的病原菌,在世界范围内引起大规模的感染,该菌对生存条件的要求较为严苛。近年来有学者发现,该菌在一定条件下可以形成一种能够抵抗外界环境不良影响的生物膜,这也是该菌能够在严峻的环境中存活并产生耐药性的重要原因。本研究临床采取猪的盲肠部位作为分离空肠弯曲杆菌的试验样品,对空肠弯曲杆菌进行分离培养,分别采用常规生化鉴定以及PCR检测两种方法对分离到的菌株进行鉴定,比较两种方法的差异。经鉴定确认为空肠弯曲杆菌的菌株,通过结晶紫染色、扫描电镜等方法,确认空肠弯曲杆菌是否具有在体外形成生物膜的能力,建立其生物膜体外模型,并对其形成的过程进行了观察；正交实验法检测空气条件,环境温度以及培养基类型3个因素对空肠弯曲杆菌生物膜形成的影响。结果表明：用厌氧罐与焦性没食子酸结合的方法分离空肠弯曲杆菌是经济有效的；PCR法鉴定空肠弯曲杆菌,相对常规生化方法来说具有省时省力且灵敏准确的优点；分离所得到的空肠弯曲杆菌具有在体外形成生物膜的能力；空肠弯曲杆菌生物膜形成的的最佳条件为常规空气条件,即37℃以及MHB培养基。生物膜菌在其生存方式、胞外物质的合成以及对动物的致病性方面均与浮游菌存在有很大程度的差异,本试验采用蒽酮-硫酸法和考马斯亮蓝法,分别对不同时间生物膜状态和浮游状态下空肠弯曲杆菌合成的胞外多糖和菌体总蛋白量进行测定。结果发现生物膜型细菌在各时间点合成的水溶性胞外多糖的量均低于浮游型细菌,两者之间差异性显著(P<0.05)；而各时间点细菌合成水不溶性多糖的量则是生物膜型细菌高于浮游型细菌,二者在48h时差异不显著(P>0.05),其它时间点差异均显著(P<0.05)。两种状态下菌体总蛋白的量随时间的延长有增加趋势,两者在12h差异不显著(P>0.05),其它时间点差异均显著(P<0.05)。将普通清洁级小鼠用5倍剂量的抗生素处理,消除其肠道共生微生物菌群之后作为研究空肠弯曲杆菌与宿主之间相互作用的动物模型。分别用生物膜型细菌和浮游型的空肠弯曲杆菌感染试验小自鼠,观察小鼠临床症状及出现症状的时间,对攻毒小鼠的外周血进行细胞因子和抗氧化指标的检测,以发现两种状态下的空肠弯曲杆菌对小鼠的致病性,以及对感染小鼠的免疫机能的影响。结果表明,两种状态的细菌感染小鼠后,小鼠未出现明显的临床症状,仅有少数出现了腹泻或精神沉郁,且症状较轻或可以自愈。在经过空肠弯曲杆菌感染后的第1d,生物模型和浮游型的细菌处理的小鼠肝脏匀浆液中可发现细菌定植。两种状态的细菌在肝脏中的生长状态与清除时间明显不同。浮游型的细菌数量在6-7d达到峰值,且在肝脏中的清除时间长达15d。而生物膜型的细菌数量的峰值出现在感染后的第10d,且在肝脏中的清除时间比浮游菌处理组更长。空肠弯曲杆菌虽然是一种众所周知的肠道致病菌,但是却能够引起全身性或系统性的感染,因此空肠弯曲杆菌与宿主免疫之间的相互影响近年来不断受到关注。试验结果证实,对于两种状态的空肠弯曲杆菌感染小鼠的血浆中几种促炎性细胞因子以及抗炎性细胞因子的水平进行了检测,以此来研究该病原菌与宿主的免疫之间的联系。结果显示：感染后第1d起血浆的IFN-γ的水平就出现了一定程度的升高,但并不明显。浮游菌组IFN-γ的峰值出现在第4-7d,而生物膜菌组出现在第10d；浮游菌组TNF-α第4d达到峰值,之后迅速下降。而生物膜菌组峰值出现在第4-7d之间,直至第10d均处于较高水平：两实验组IL-2峰值均出现在感染后的7d左右,同一时间点血浆1L-2水平,生物膜菌组显著高于浮游菌组(P<0.05);IL-4在浮游菌组峰值出现在感染后的第4-7d,之后直至第10d菌居高不下,粘附菌组,感染前期并无显著升高,后期,该细胞因子持续升高且在监控的时间内并未达到峰值;IL-6的水平感染的早期阶段并没有显著增加,从第4d开始显著升高,峰值出现在第7d。归纳来讲,促炎性细胞因子出现在感染的前期阶段,且多在炎症的急性期达到峰值,而抗炎性细胞因子的升高是跟随在促炎性细胞因子升高之后,比较浮游细菌和生物膜型细菌对宿主的免疫防御的影响,可以看出,由粘附细菌引起的感染具有从炎症急性期向慢性化转变的趋势,而宿主的免疫也从先天免疫转向获得性免疫。两种型态的细菌对宿主机体抗氧化系统的影响的研究结果表明：SOD在浮游菌组显著降低(P<0.05):MDA在生物膜菌组显著降低(P<0.05); GSH在两实验组没有出现显著差异：LDH在两个实验组均显著升高(P<0.05),且浮游菌组显著高于生物膜菌组(P<0.05)。综上所述,本试验研究空肠弯曲杆菌生物膜的上述儿个方面取得了阶段性的进展,但是由于细菌生物膜的形成是一个极为复杂的过程,与此相关的影响因素涉及到各个方面,难以逐一模拟研究。另外由于空肠弯曲杆菌自身的特性,对于该菌生物膜的研究还非常有限,在其形成机制,基因水平的调控,以及耐药机制和致病机制等方面还存在很多不解之处,另外由于试验的局限性,体外实验并不能完全模仿体内环境,因此要想对该菌生物膜进行更深入了解还需要后期不断研究。更多还原

【Abstract】 Campylobacter Jejuni, as a worldwide prevalence of zoonotic disease pathogens, living conditions are more stringent. In recent years, some scholars found that under certain conditions the bacteria can form a biofilm which resistant to negative effects of the external environment, which is the bacteria to survive in harsh environments and major cause of resistance.The clinical specimen was selected as test samples for isolation of Campylobacter Jejuni, Campylobacter Jejuni was isolated and cultured respectively detected routine biochemical identification and PCR detections of isolated strains, to compare differences of the two methods in the identification of the pathogen.Confirmed by the above two methods of identification for strains of Campylobacter Jejuni, establishing biofilm models in vitro, its characteristics and influencing factors were studied by PCR. Crystal Violet staining, SEM and orthogonal experimental method etc., providing some rationale to study the formation of biofilm and remove of the bacteria.Biofilm bacteria, and called "block health bacteria", its survival way, synthesis of extracellular materials, and the pathogenicity on animal was different from floats bacteria in large degree, In this experiment, under the two states exopolysaccharides and total protein volume of bacteria were determine by using anthracene ketone-sulfate law and Coomassie brilliant blue law respectively, to compare the differences of the two bacteria in the synthesis of extracellular materials. Mice were infected by biofilm bacteria and planktonic bacteria infections respectively, observing symptoms and attack time in mice, our experiments detected antioxidant indexes and Cytokines in peripheral blood detections of toxicity attacked mice, to discover the differences on animal pathogenicity of the two bacteria, and to analyse their possible causes.Campylobacter Jejuni is an important pathogen of zoonotic diseases, its biofilm formation is one of the major cause of turning a chronic infection and develop resistance. In recent years, there are varieties of other bacteria biofilm research reports at home and abroad, but reports of Campylobacter Jejuni Biofilms are very limited. Our experiments established the bacteria biofilm model in vitro, and researched its possible effect factors, best conditions that formed the bacteria biofilm in vitro were discovered; the differences of bacteria synthesized extracellular materials in biofilm and floating state in different time, the possible causes of differences were discussed, and its significances in biofilm formed were analysised; the influence of the two bacteria in aspects total anti-oxidation capacity and immune function were obtained by detecting antioxidant indexes and Cytokines in peripheral blood for laboratory animals.5tmes idoses antibiotic treatment to ordinary clean mice to eliminate the intestinal microbial flora and then used them as animal models to stady the interaction between host and jejunum campylobacter. Infected test mice with C.jejuni of planktonic and adhesive states Separately, observed he symptoms and occurrence time, and tested plasma cytokines and antioxidant indicators of infected mouses, to find the differences in animal pathogenic and the influence to the body’s immune function of infected animals, of the two knids of campylobacter jejuni, and analyzed the possible reasons.. The results showed that the mouses did not appear obvious clinical symptoms, after infection with two states of bacteria only a few appeared diarrhea or spirit is depressed, and symptoms were very light or could recover by themselves. On the1st day after infections, the two knids of bacteria could be found colonize in the liver homogenate of the infected mouses. The two knids of bacteria were obviously different in the growth states and the removement in the live.The nmber of planktonic bacteria reached the peak during6-7d, and stayed in the liver for15days, while the number of adhesion bacteria peaked on the10th day after infection, and stayed longerin the liver.1d after infection on the plasma levels of IFN-gamma has been a certain degree of rise, but not obvious. Planktonic bacteria group of IFN-gamma peak appeared in4to7d after infection, bacteria adhesion of IFN-gamma peak appears in the10d; both the experimental group TNF alpha showed a trend of rising, plankionic bacteria group4DTNF alpha level reaches its peak, after falling rapidly. The adhesion of bacteria group peak between4to7d, until10d were significantly higher than the blank control group; Two experimental IL-2compared with the blank control group, to some extent, the rise of peak occurred at about7d after infection, compared the same time point of the two states of bacteria influence on mice serum IL-2, found that, unlike IFN-gamma and TNF alpha, IL-2levels of bacteria adhesion group were higher than in most of the time points of planktonic bacteria group, and significant difference (P<0.05); IL-4peak appeared after infection in the planktonic bacteria group of4to7d, until after10d bacteria high adhesion bacterium group, there is no marked increase in the early stage of the infection, later period, the cytokines did not continue to rise and the monitoring time to peak; Early stages of infection, the level of IL-6compared with the blank control group was not significantly increased, starting from4d after infection, the experimental group significantly increased the level of IL-6. Peak appears in the7d after infection.For two kinds of state of bacteria to the host organism oxidation system research has shown that the influence of SOD in the planktonic bacteria group is lower than the blank control group, and significant difference (P<0.05), adhesive bacteria group compared with blank control group no significant difference; MDA in adhesive bacteria group is lower than the blank control group, and significant difference (P<0.05), planktonic bacteria group and the blank control group difference was not significant; GSH in two states bacteria treatment group compared with blank control group, there were no significant differences; LDH in the group of planktonic bacteria and bacteria adhesion were significantly higher than that of blank control group (P<0.05), compared with two kinds of state of bacteria in mice LDH, the influence of planktonic bacteria group is higher than the bacteria adhesion, and significant difference (P<0.05).Campylobacter Jejuni is an important pathogen of zoonotic disease, its biofilm formation is one of the major cause of turning a chronic infection and develop resistance. In recent years, there are varieties of other bacteria biofilm research reports at home and abroad, but reports of Campylobacter Jejuni Biofilms are very limited. Our experiments established the bacteria biofilm model in vitro, and researched its possible effect factors, best conditions that formed the bacteria biofilm in vitro were discovered; the differences of bacteria synthesized extracellular materials in biofilm and floating state in different time, the possible causes of differences were discussed, and its significances in biofilm formed were analysised; the influence of the two bacteria in aspects total anti-oxidation capacity and immune function were obtained by detecting antioxidant indexes and Cytokines in peripheral blood for laboratory animals更多还原