本文選題：鮑曼不動桿菌 + 生物被膜��； 參考：《河北北方學院》2017年碩士論文

【摘要】：鮑曼不動桿菌是造成呼吸系統(tǒng)感染的常見條件致病菌,隨著多重耐藥甚至泛耐藥鮑曼不動桿菌的出現(xiàn),臨床鮑曼不動桿菌感染的治療面臨極大挑戰(zhàn)。大部分鮑曼不動桿菌菌株能夠形成生物被膜,在增強細菌存活能力的同時也進一步提高了細菌對抗生素的耐受能力,因此,抑制或清除生物被膜是應對細菌耐藥的重要策略之一。裂解性噬菌體能夠特異感染并裂解宿主細菌,其作用機制與抗生素不同,在抗生素耐藥日益嚴重的今天又重新受到科學界的廣泛關注,被認為是治療耐藥菌感染的有效補充或替代手段。本研究以呼吸監(jiān)護室病人痰標本中分離的1株泛耐藥鮑曼不動桿菌為指示菌,從解放軍第307醫(yī)院污水中分離出1株裂解性噬菌體。在完成基本生物學鑒定和全基因組序列分析后,采用體外實驗系統(tǒng)觀察了噬菌體對鮑曼不動桿菌生物被膜的作用效果。為了研究該噬菌體的基本生物學特性,用雙層瓊脂法觀察噬菌斑大小和形態(tài),透射電子顯微鏡觀察噬菌體的形態(tài)結(jié)構,測定噬菌體的裂解譜及其一步生長曲線。為了進一步了解噬菌體的遺傳信息,用高通量測序的方法得到噬菌體的全基因組序列并進行基因注釋和同源性分析。實驗結(jié)果表明,噬菌體感染宿主菌后,可在雙層瓊脂平板上形成圓形且透亮的噬菌斑,噬菌斑周圍可見明顯的暈環(huán),說明噬菌體在感染過程中可產(chǎn)生類似解聚酶的物質(zhì);透射電子顯微鏡觀察可見噬菌體呈直徑約為48~50 nm的正六面體頭部和長度約為140~150 nm的可收縮長尾,形態(tài)類似肌尾噬菌體科(Myoviridae);該噬菌體可裂解包括宿主菌在內(nèi)的9株(9/31)耐碳青酶烯類鮑曼不動桿菌且具有較短的潛伏期(10 min)和較大的爆發(fā)量(229 pfu/cell);噬菌體的全基因組為線性雙鏈DNA分子,長度為41 243 bp,G+C含量為39.3%,編碼52個ORFs,16個ORFs編碼已知功能蛋白,不含整合酶、毒力因子或類轉(zhuǎn)座子等成分,與鮑曼不動桿菌噬菌體AB3,Fri1,Abp1,phiAB1和vB_AbaP_PD-6A3的全基因組序列高度同源。為了觀察噬菌體對細菌生物被膜的作用效果,體外培養(yǎng)鮑曼不動桿菌形成生物被膜后,利用掃描電子顯微鏡、共聚焦激光掃描顯微鏡、結(jié)晶紫染色半定量、生物被膜細菌計數(shù)以及生物被膜細菌ATP測定等方法評價噬菌體的作用效果。顯微鏡觀察結(jié)果表明,裂解性噬菌體能夠顯著破壞耐藥鮑曼不動桿菌生物被膜。細菌定量結(jié)果表明噬菌體可以有效抑制88.5%的耐藥鮑曼不動桿菌形成生物被膜,能夠清除52%~90%的生物被膜。ATP釋放實驗也進一步證明了裂解性噬菌體可以感染并破壞細菌生物被膜,進入生物被膜細菌內(nèi)部并使之裂解。除此以外,我們發(fā)現(xiàn)無論是對數(shù)生長期的鮑曼不動桿菌,還是鮑曼不動桿菌生物被膜,同樣會對噬菌體的感染產(chǎn)生耐受,對數(shù)生長期的鮑曼不動桿菌對裂解性噬菌體的不敏感突變(BIM)率為3×10-6 cfu。綜上所述,本研究成功分離到耐藥鮑曼不動桿菌裂解性噬菌體,鑒定了該噬菌體的基本生物學特性和基因組成分,證明了噬菌體不僅能夠有效殺滅宿主菌,而且能夠抑制并破壞宿主菌形成生物被膜,為耐藥鮑曼不動桿菌感染的治療提供了新思路,具有一定的應用前景。然而,在本研究中也發(fā)現(xiàn)了一些亟待解決的問題,如噬菌體解聚酶的分離、純化以及與其它抗菌物質(zhì)的聯(lián)合效應,如何克服噬菌體的耐受性等,相關研究仍在進行中。
[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.
【學位授予單位】：河北北方學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R446.5

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相關期刊論文 前1條

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本文編號：2052157