本文選題：噬菌體 + 多重耐藥�。� 參考：《大連理工大學(xué)》2014年碩士論文

【摘要】：肺炎克雷伯氏菌是醫(yī)院獲得性肺炎主要的條件致病菌。近年來抗菌藥物的不合理使用,導(dǎo)致肺炎克雷伯氏菌耐藥株增多、耐藥性增強(qiáng),給用藥及治療造成了極大的困擾,嚴(yán)重威脅人的生命健康。噬菌體是一類細(xì)菌性病毒,在自然環(huán)境中普遍存在,因其具有特異性、高效性、安全性、低成本、開發(fā)周期短等優(yōu)點(diǎn),被認(rèn)為是最具前景的抗生素替代品。本研究探索應(yīng)用裂解性噬菌體防治多重耐藥肺炎克雷伯氏菌引起的小鼠肺炎,為多重耐藥菌的治療提供一條新途徑。 從醫(yī)院患肺炎病人的痰液中分離純化病原菌,經(jīng)形態(tài)學(xué)觀察、生理生化實(shí)驗(yàn)、16SrRNA序列分析、藥敏實(shí)驗(yàn)分析、生物被膜形成實(shí)驗(yàn)對(duì)病原菌進(jìn)行分類鑒定,分離獲得一株產(chǎn)生物被膜的多重耐藥肺炎克雷伯氏菌(K. pneumoniae),命名為KP1513。將已分離的病原菌作為宿主菌,采用雙層平板法從醫(yī)院處理前污水中分離裂解性噬菌體。該噬菌體純化后,通過透射電鏡觀察,考察其氯仿敏感性、pH穩(wěn)定性、溫度穩(wěn)定性、宿主譜、潛伏期、裂解量等噬菌體生物學(xué)特性,將篩選獲得的裂解性噬菌體命名為phage1513。該噬菌體屬于有尾噬菌體目、長尾噬菌體科,宿主專一性強(qiáng),對(duì)氯仿不敏感,因而不含脂類物質(zhì),且在小于40℃,pH為5-10范圍內(nèi)較穩(wěn)定。感染宿主菌的潛伏期約30min,裂解量約264PFU/cell。phage1513基因組測(cè)序及功能基因分析表明,該噬菌體基因組大小為49462bp,編碼72個(gè)基因,含有4個(gè)功能模塊,與Tunalinkevirus噬菌體屬親緣關(guān)系最近,不含抗生素抗性基因及毒素基因,且其裂解機(jī)制與穿孔素及裂解酶相關(guān)。 通過測(cè)定細(xì)菌與噬菌體共培養(yǎng)物在650nm處的吸光值來考察噬菌體對(duì)病原菌的體外裂解效果。體外抑菌實(shí)驗(yàn)表明,phage1513具有快速感染并裂解宿主菌的能力,能夠抑制生物被膜的形成。體內(nèi)實(shí)驗(yàn)采用滴鼻的給藥方式。小鼠致死模型中,通過對(duì)比PBS組、噬菌體預(yù)防實(shí)驗(yàn)組(攻毒24h前給藥)和噬菌體治療實(shí)驗(yàn)組(攻毒2h后給藥)小鼠的存活率,評(píng)價(jià)噬菌體防治小鼠肺炎的效果。結(jié)果表明,治療組中不同感染復(fù)數(shù)的噬菌體均能一定程度上降低小鼠死亡率,且感染復(fù)數(shù)越高,死亡率越低,但噬菌體不能對(duì)小鼠肺炎起到預(yù)防作用。在小鼠亞致死模型中,通過考察小鼠肺部組織病理切片,小鼠體重變化,以及肺組織勻漿液中菌數(shù)、噬菌體數(shù)以及炎癥因子(IL-6,TNF-α)的變化,證明phage1513在體內(nèi)能高效裂解病原菌,改善肺炎癥狀,減輕炎癥反應(yīng)。 本研究篩選了針對(duì)多重耐藥肺炎克雷伯氏菌的裂解性噬菌體,研究其生物學(xué)性質(zhì),進(jìn)行基因組測(cè)序及功能基因分析,并對(duì)體外體內(nèi)裂解宿主的能力和效果進(jìn)行評(píng)價(jià),為噬菌體應(yīng)用于細(xì)菌性肺炎的防治提供了依據(jù)。
[Abstract]:Klebsiella pneumoniae is a major opportunistic pathogen of hospital acquired pneumonia. In recent years, the irrational use of antimicrobial agents has led to the increase of resistant strains of Klebsiella pneumoniae and the increase of drug resistance, which has caused great distress to drug use and treatment, and has seriously threatened the life and health of human beings. Bacteriophage is a kind of bacterial virus, which is widely used in natural environment. Because of its specificity, efficiency, safety, low cost and short development cycle, phage is considered as the most promising substitute for antibiotics. This study explored the application of lytic phage in the prevention and treatment of mouse pneumonia induced by multidrug resistant Klebsiella pneumoniae and provided a new approach for the treatment of multidrug resistant bacteria. The pathogenic bacteria were isolated and purified from the sputum of patients with pneumonia in hospital. The pathogens were classified and identified by morphological observation, physiological and biochemical experiments, 16s rRNA sequence analysis, drug sensitivity analysis and biofilm formation experiment. A strain of multidrug resistant Klebsiella pneumoniae was isolated and named KP1513. The lytic bacteriophage was isolated from pre-hospital sewage by double-layer plate method using isolated pathogenic bacteria as host bacteria. The bacteriophage was purified by transmission electron microscope. The pH stability, temperature stability, host spectrum, incubation period and cleavage amount of chloroform sensitive phage were investigated. The selected lytic phage was named phage1513. The bacteriophage belongs to the order Phage with tail. It has strong host specificity, is not sensitive to chloroform, so it does not contain lipids, and is stable in the range of pH 5-10 at less than 40 鈩，

本文編號(hào)：1901645