【摘要】：替加環(huán)素(tigecycline,Tgc)是FDA批準(zhǔn)臨床使用的第一個(gè)甘氨酰類(lèi)抗生素,主要用于復(fù)雜腹腔感染、皮膚感染和社區(qū)獲得性肺炎的治療,和黏菌素一起被稱為人類(lèi)抵抗細(xì)菌感染的最后"防線"。但是,近年來(lái),有關(guān)替加環(huán)素耐藥菌株發(fā)現(xiàn)的報(bào)道越來(lái)越多,給臨床治療效果和感染控制帶來(lái)了挑戰(zhàn)。替加環(huán)素2007年獲美國(guó)FDA批準(zhǔn),2012年底正式在國(guó)內(nèi)應(yīng)用于臨床,本課題對(duì)本三甲醫(yī)院2012年底使用替加環(huán)素以來(lái),臨床分離報(bào)告替加環(huán)素耐藥的鮑曼不動(dòng)桿菌進(jìn)行研究。鮑曼不動(dòng)桿菌具有很強(qiáng)的耐藥基因獲得能力,可以在干燥、輻射、消毒劑等極端條件下生存,是導(dǎo)致院內(nèi)感染的重要條件致病菌。根據(jù)衛(wèi)計(jì)委全國(guó)細(xì)菌耐藥監(jiān)測(cè)網(wǎng)Mohnarin數(shù)據(jù)顯示,臨床多重藥耐藥(MDR)鮑曼不動(dòng)桿菌近5年分離率均高于50%,而且泛耐藥(XDR)和全耐藥(PDR)的鮑曼不動(dòng)桿菌也逐年增多,并且已經(jīng)開(kāi)始對(duì)替加環(huán)素產(chǎn)生耐藥性。本研究利用高通量測(cè)序技術(shù)和分子生物學(xué)手段,從分子流行病學(xué)、比較基因組學(xué)和比較轉(zhuǎn)錄組學(xué)角度,系統(tǒng)研究了臨床分離的鮑曼不動(dòng)桿菌替加環(huán)素耐藥性發(fā)展的基因組學(xué)基礎(chǔ)和轉(zhuǎn)錄調(diào)控機(jī)制,并探討了在替加環(huán)素耐藥鮑曼不動(dòng)桿菌中毒力因子和耐藥基因的分布和關(guān)系。通過(guò)本研究,系統(tǒng)闡明了鮑曼不動(dòng)桿菌對(duì)替加環(huán)素耐藥的形成機(jī)制,為耐藥控制和院感防控提供了一定的理論基礎(chǔ)。在第一部分研究中,對(duì)本醫(yī)院替加環(huán)素使用一年半以來(lái)的臨床實(shí)驗(yàn)室分離報(bào)告替加環(huán)素耐藥的鮑曼不動(dòng)桿菌,使用微量肉湯稀釋法測(cè)定替加環(huán)素的體外最低抑菌濃度(MIC),確定了 56株替加環(huán)素不敏感株,進(jìn)行樣本類(lèi)型、科室分布、疾病、和抗生素使用等分析,揭示替加環(huán)素不敏感鮑曼不動(dòng)桿菌臨床分布特征;分子型別(MLST)分析揭示了其分子流行特征。第二部分研究以第一部分研究為基礎(chǔ),選擇替加環(huán)素不敏感鮑曼不動(dòng)桿菌3株以及分離自相同病人的不同替加環(huán)素耐藥程度菌株3株,共6株鮑曼不動(dòng)桿菌,進(jìn)行了高通量基因組測(cè)序和比較基因組學(xué)分析,構(gòu)建遺傳進(jìn)化樹(shù),比較基因組序列、結(jié)構(gòu)和功能基因的差異,研究發(fā)現(xiàn)了1株替加環(huán)素耐藥菌可能是由同病人分離的另外2株替加環(huán)素耐藥性較低的菌株之間同源重組形成的,并且約9kbp的基因組重組區(qū)域包含了 6個(gè)基因,其中4個(gè)是外排泵、轉(zhuǎn)錄調(diào)控因子或膜蛋白。對(duì)這3株菌在不同替加環(huán)素濃度(0μg/mL,0.5μg/mL和1μg/mL)條件下基因表達(dá)情況進(jìn)行鏈特異性轉(zhuǎn)錄組測(cè)序和比較轉(zhuǎn)錄組學(xué)分析,發(fā)現(xiàn)了在梯度增加的替加環(huán)素濃度下基因轉(zhuǎn)錄水平的變化,并確定了所有顯著差異表達(dá)的基因。這些基因的轉(zhuǎn)錄水平變化進(jìn)一步通過(guò)半定量qRT-PCR進(jìn)行了實(shí)驗(yàn)驗(yàn)證�；蚪M水平上包括6個(gè)基因的變異可能導(dǎo)致了相應(yīng)基因功能變化,結(jié)合轉(zhuǎn)錄水平的表達(dá)調(diào)控分析討論了其可能對(duì)替加環(huán)素耐藥產(chǎn)生的影響及途徑,差異轉(zhuǎn)錄組展示了基因調(diào)控可能對(duì)替加環(huán)素耐藥的較大影響并且確定了起顯著調(diào)控作用的基因。同時(shí),本研究對(duì)所有替加環(huán)素耐藥的鮑曼不動(dòng)桿菌19株進(jìn)行了比較基因組學(xué)研究,發(fā)現(xiàn)了19株鮑曼不動(dòng)桿耐藥菌在進(jìn)化關(guān)系上比較接近,耐藥基因和毒力因子均具有多態(tài)性,這些耐藥菌之間以及與GeneBank已有耐藥菌全基因組相比較,可以通過(guò)marker基因進(jìn)行不同耐藥基因或毒力因子型的區(qū)分。
[Abstract]:Tigecycline (Tgc) is the first glycyl antibiotic approved by the FDA for clinical use. It is mainly used in the treatment of complex abdominal infections, skin infections and community-acquired pneumonia. Together with mucin, tigecycline is known as the last line of defense against bacterial infections in humans. Tigacycline was approved by FDA in 2007 and formally used in clinical practice in China at the end of 2012. Since the end of 2012, Tigacycline-resistant Acinetobacter baumannii has been reported in clinical isolates from our tertiary hospital. Strong ability to acquire resistant genes that can survive in extreme conditions such as dryness, radiation, disinfectants and so on is an important conditional pathogen causing nosocomial infections. Acinetobacter baumannii resistant to tigacycline is also increasing year by year, and has begun to develop resistance to tigacycline. This study systematically studied the development of tigacycline resistance in clinical isolates of Acinetobacter baumannii from molecular epidemiology, comparative genomics and comparative transcriptome perspectives using high-throughput sequencing and molecular biology techniques. The genomics basis and transcriptional regulation mechanism of Tigacycline-resistant Acinetobacter baumannii were studied. The distribution and relationship of virulence factors and drug-resistant genes in Tigacycline-resistant Acinetobacter baumannii were discussed. Through this study, the mechanism of Tigacycline-resistant Acinetobacter baumannii was systematically elucidated. In part of the study, Acinetobacter baumannii, which was reported to be resistant to tigacycline, was isolated from the clinical laboratory of our hospital for one and a half years. Minimum inhibitory concentration (MIC) of tigacycline in vitro was determined by broth dilution method. Fifty-six tigacycline-insensitive strains were identified for sample type, Department distribution, disease, and resistance. Analysis of biotin use revealed the clinical distribution of tigacycline-insensitive Acinetobacter baumannii, and molecular typing (MLST) revealed its molecular epidemiological characteristics. Three strains and six strains of Acinetobacter baumannii were analyzed by high throughput genome sequencing and comparative genomics. A genetic evolutionary tree was constructed to compare the differences of genome sequence, structure and function genes. It was found that one strain of tigacycline-resistant bacteria might be the same as the other two strains with lower tigacycline resistance isolated from the same patient. The recombinant region consisted of six genes, four of which were efflux pumps, transcription regulators or membrane proteins. The gene expression profiles of the three strains under different tigacycline concentrations (0 ug/mL, 0.5 ug/mL and 1 ug/mL) were sequenced by chain-specific transcriptome analysis and comparative transcriptome analysis. Changes in gene transcription levels at gradient tigacycline concentrations were further validated by semi-quantitative qRT-PCR. Variations in six genes at the genome level may result in changes in gene function associated with transcriptional water Differential transcriptomes showed that gene regulation may have a greater effect on tigacycline resistance and identified genes that play a significant role in the regulation. In addition, 19 tigacycline-resistant Acinetobacter baumannii strains were compared. Genomic analysis revealed that 19 strains of Baumann fixed-rod resistant bacteria were similar in evolutionary relationship, and the drug-resistant genes and virulence factors were polymorphic. Compared with the genome of the existing drug-resistant bacteria in GeneBank, marker gene could be used to distinguish different drug-resistant genes or virulence factors.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R446.5

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