裂解性噬菌体对耐药鲍曼不动杆菌生物被膜的作用研究

发布时间：2018-06-22 08:00

本文选题：鲍曼不动杆菌 + 生物被膜　；参考：《河北北方学院》2017年硕士论文

【摘要】：鲍曼不动杆菌是造成呼吸系统感染的常见条件致病菌,随着多重耐药甚至泛耐药鲍曼不动杆菌的出现,临床鲍曼不动杆菌感染的治疗面临极大挑战。大部分鲍曼不动杆菌菌株能够形成生物被膜,在增强细菌存活能力的同时也进一步提高了细菌对抗生素的耐受能力,因此,抑制或清除生物被膜是应对细菌耐药的重要策略之一。裂解性噬菌体能够特异感染并裂解宿主细菌,其作用机制与抗生素不同,在抗生素耐药日益严重的今天又重新受到科学界的广泛关注,被认为是治疗耐药菌感染的有效补充或替代手段。本研究以呼吸监护室病人痰标本中分离的1株泛耐药鲍曼不动杆菌为指示菌,从解放军第307医院污水中分离出1株裂解性噬菌体。在完成基本生物学鉴定和全基因组序列分析后,采用体外实验系统观察了噬菌体对鲍曼不动杆菌生物被膜的作用效果。为了研究该噬菌体的基本生物学特性,用双层琼脂法观察噬菌斑大小和形态,透射电子显微镜观察噬菌体的形态结构,测定噬菌体的裂解谱及其一步生长曲线。为了进一步了解噬菌体的遗传信息,用高通量测序的方法得到噬菌体的全基因组序列并进行基因注释和同源性分析。实验结果表明,噬菌体感染宿主菌后,可在双层琼脂平板上形成圆形且透亮的噬菌斑,噬菌斑周围可见明显的晕环,说明噬菌体在感染过程中可产生类似解聚酶的物质;透射电子显微镜观察可见噬菌体呈直径约为48~50 nm的正六面体头部和长度约为140~150 nm的可收缩长尾,形态类似肌尾噬菌体科(Myoviridae);该噬菌体可裂解包括宿主菌在内的9株(9/31)耐碳青酶烯类鲍曼不动杆菌且具有较短的潜伏期(10 min)和较大的爆发量(229 pfu/cell);噬菌体的全基因组为线性双链DNA分子,长度为41 243 bp,G+C含量为39.3%,编码52个ORFs,16个ORFs编码已知功能蛋白,不含整合酶、毒力因子或类转座子等成分,与鲍曼不动杆菌噬菌体AB3,Fri1,Abp1,phiAB1和vB_AbaP_PD-6A3的全基因组序列高度同源。为了观察噬菌体对细菌生物被膜的作用效果,体外培养鲍曼不动杆菌形成生物被膜后,利用扫描电子显微镜、共聚焦激光扫描显微镜、结晶紫染色半定量、生物被膜细菌计数以及生物被膜细菌ATP测定等方法评价噬菌体的作用效果。显微镜观察结果表明,裂解性噬菌体能够显著破坏耐药鲍曼不动杆菌生物被膜。细菌定量结果表明噬菌体可以有效抑制88.5%的耐药鲍曼不动杆菌形成生物被膜,能够清除52%~90%的生物被膜。ATP释放实验也进一步证明了裂解性噬菌体可以感染并破坏细菌生物被膜,进入生物被膜细菌内部并使之裂解。除此以外,我们发现无论是对数生长期的鲍曼不动杆菌,还是鲍曼不动杆菌生物被膜,同样会对噬菌体的感染产生耐受,对数生长期的鲍曼不动杆菌对裂解性噬菌体的不敏感突变(BIM)率为3×10-6 cfu。综上所述,本研究成功分离到耐药鲍曼不动杆菌裂解性噬菌体,鉴定了该噬菌体的基本生物学特性和基因组成分,证明了噬菌体不仅能够有效杀灭宿主菌,而且能够抑制并破坏宿主菌形成生物被膜,为耐药鲍曼不动杆菌感染的治疗提供了新思路,具有一定的应用前景。然而,在本研究中也发现了一些亟待解决的问题,如噬菌体解聚酶的分离、纯化以及与其它抗菌物质的联合效应,如何克服噬菌体的耐受性等,相关研究仍在进行中。
[Abstract]:Acinetobacter Bauman is a common pathogenic bacteria causing respiratory infection. With the emergence of multidrug-resistant and even pan resistant Acinetobacter, the clinical treatment of Acinetobacter Bauman is facing great challenges. Most of the strains of Acinetobacter Bauman can form biofilm, and further raise the viability of the bacteria. Therefore, the inhibition or removal of the biofilm is one of the important strategies to deal with bacterial resistance. The lysate phage can infect and cleavage the host bacteria specifically. The mechanism is different from the antibiotic. 1 strains of Pan resistant Acinetobacter Bauman isolated from the sputum specimens from patients with respiratory monitoring room were used as an indicator to separate 1 strains of lysate phage from the sewage of No.307 Hospital of PLA. In this study, after the basic biological identification and whole genome sequence analysis were completed, the experiment was carried out in vitro. The effect of phage on the biofilm of Acinetobacter Bauman was systematically observed. In order to study the basic biological characteristics of the phage, the size and morphology of phage plaque was observed by double agar method. The morphological structure of phage was observed by transmission electron microscope, and the lysis spectrum and one step growth curve of phage were measured. The whole genome sequence of the phage was obtained by high flux sequencing, and the whole genome sequence of the phage was obtained and the gene annotation and homology analysis were carried out. The experimental results showed that the phage formed a round and bright phage plaque on the double agar plate after the phage infected the host bacteria. The phage showed a clear halo in the circumference of the phage, indicating that the phage was infected. In the process, a substance similar to the depolymerization enzyme can be produced; transmission electron microscopy shows that the phage with a positive hexahedron head with a diameter of 48~50 nm and a long tailed long tail of about 140~150 nm is similar to the myocele phage (Myoviridae), and the phage can break 9 (9/31) 9 (9/31) carbenolenes, including the host bacteria. Bacilli have short incubation period (10 min) and large explosive amount (229 pfu/cell); the whole genome of phage is linear double stranded DNA molecule, length 41243 BP, G+C content 39.3%, encoding 52 ORFs, 16 ORFs coding known functional proteins, without integrase, toxic factor or class transposon, and phage AB3 of Acinetobacter Bauman The whole genome sequence of Fri1, Abp1, phiAB1 and vB_AbaP_PD-6A3 is highly homologous. In order to observe the effect of phage on bacterial biofilm, in vitro culture of Acinetobacter Bauman to form biofilm, scanning electron microscope, confocal laser scanning microscope, crystal violet staining semi quantitative, biological membrane bacteria count and biology are used. The effect of bacteriophage was evaluated by the method of membrane bacteria ATP. The microscope observation showed that the lysate phage could significantly destroy the biofilm of the resistant Acinetobacter Bauman. The bacterial quantitative results showed that the bacteriophage could effectively inhibit the formation of the biofilm of 88.5% resistant Acinetobacter Bauman, and could clear the biofilm of 52%~90%. The ATP release experiment further demonstrated that the Lysic phage can infect and destroy the bacterial biofilm and break into the biofilm bacteria. In addition, we find that the logarithmic growth period of Acinetobacter Bauman, or the biofilm of the Acinetobacter, will also tolerate the infection of the phage. The insensitive mutation (BIM) rate of Acinetobacter Bauman in the growing period was 3 x 10-6 cfu.. This study successfully isolated the lysate phage of Acinetobacter spp., and identified the basic biological and genomic components of the phage, which proved that phage not only effectively killed the host bacteria, but also proved that the bacteriophage could kill the host bacteria effectively. Enough inhibition and destruction of host bacteria to form biofilm, which provides new ideas for the treatment of Acinetobacter resistant Acinetobacter infection, and has a certain application prospect. However, some urgent problems have been found in this study, such as the separation of phage depolymerization enzymes, purification and combined effects with other antibacterial substances, and how to overcome phage. Tolerance and so on, the related research is still in progress.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5

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