發(fā)布時(shí)間：2018-04-23 05:25

  本文選題：NDM-1 + 流行病學(xué)特征��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文

【摘要】：人類與各種感染性疾病的斗爭(zhēng)中,抗生素起到關(guān)鍵作用,但由抗菌素應(yīng)用產(chǎn)生的抗生素耐藥問(wèn)題逐漸顯現(xiàn),尤其是近幾十年來(lái),由于抗生素使用不當(dāng)?shù)葐?wèn)題加速了細(xì)菌耐藥問(wèn)題的惡化,給臨床治療帶來(lái)沉重負(fù)擔(dān)。尤其2009年新德里金屬β-內(nèi)酰胺酶(New Delhi metallo-β-lactamase-1,NDM-1)的出現(xiàn)和流行更新了人們對(duì)“超級(jí)細(xì)菌”的認(rèn)識(shí),引起了世界范圍內(nèi)廣泛關(guān)注和恐慌,最初報(bào)道的產(chǎn)NDM-1的肺炎克雷伯菌和大腸桿菌對(duì)除了多粘菌素和替加環(huán)素以外的所有抗生素耐藥,截止2016年,NDM-1耐藥菌已經(jīng)在全球范圍內(nèi)形成總體散發(fā),局部地區(qū)高流行的流行趨勢(shì),越來(lái)越多的研究表明以NDM-1耐藥基因?yàn)榇淼募?xì)菌耐藥問(wèn)題已經(jīng)成為健康和食品安全領(lǐng)域的最大威脅之一,能對(duì)人類健康產(chǎn)生巨大隱患。目前,我國(guó)大量報(bào)道了關(guān)于產(chǎn)NDM-1陽(yáng)性菌的流行、blaNDM-1耐藥譜及傳播機(jī)制和遺傳學(xué)特征等研究。報(bào)道的NDM-1陽(yáng)性菌大部分為臨床個(gè)案報(bào)道,病例之間無(wú)流行病學(xué)研究和聯(lián)系,對(duì)于NDM-1陽(yáng)性菌在全國(guó)的流行病學(xué)特征尚不明確,攜帶NDM-1菌株的特性及其播散的機(jī)制仍不清楚,給NDM-1的整體治療、防控帶來(lái)巨大挑戰(zhàn)。因此,需系統(tǒng)研究NDM-1陽(yáng)性菌株在各地的分布情況、病人類型、醫(yī)療環(huán)境、危險(xiǎn)因素以及流行病學(xué)溯源和NDM-1分子特征和變異情況�；谝陨犀F(xiàn)況,本研究在CNKI和Pub Med文獻(xiàn)數(shù)據(jù)庫(kù)檢索了從2007年1月至2015年12月所有報(bào)道中國(guó)產(chǎn)NDM-1細(xì)菌研究的中英文文獻(xiàn),共檢索中英文文獻(xiàn)767篇,排除陰性結(jié)果文獻(xiàn)、綜述、NDM-1分子生物學(xué)基礎(chǔ)研究、非人感染、中英文重復(fù)文獻(xiàn),共納入研究文獻(xiàn)50篇。對(duì)文獻(xiàn)報(bào)道的NDM-1陽(yáng)性菌株的流行病學(xué)特征,菌株來(lái)源、耐藥譜及傳播機(jī)制等方面信息進(jìn)行匯總和分析,結(jié)果發(fā)現(xiàn):截至2015年12月,我國(guó)共有25個(gè)省市、地區(qū)報(bào)道blaNDM-1陽(yáng)性菌株,其中東南部沿海地區(qū)分布較多,廣東地區(qū)報(bào)道的陽(yáng)性病例為109例,占39.49%,明顯多于其他地區(qū)(P0.05),NDM-1陽(yáng)性菌主要分布在肺炎克雷伯菌和陰溝腸桿菌(P0.05);感染NDM-1陽(yáng)性菌的男性患者明顯多于女性患者(P0.01),主要集中在2個(gè)年齡段:10歲以下和60~80歲之間(P0.05);NDM-1陽(yáng)性菌主要分離于痰液標(biāo)本,為40株,占41.24%(P0.05),NDM-1陽(yáng)性菌感染的病例主要分布在ICU、兒科和呼吸科(P0.05),肺部疾病患者更容易分離出陽(yáng)性菌(P0.05),blaNDM-1陽(yáng)性菌對(duì)阿米卡星、替加環(huán)素的總體耐藥率最低,分別為7.69%和2.33%。文獻(xiàn)分析顯示我國(guó)共有13個(gè)菌屬276株細(xì)菌攜帶NDM-1基因,blaNDM-1編碼在大小為55kb~360kb范圍的質(zhì)粒上,表明編碼blaNDM-1的質(zhì)粒不僅可以實(shí)現(xiàn)高效水平傳播,還具有較強(qiáng)的跨種傳播能力;結(jié)果分析顯示NDM-1陽(yáng)性菌對(duì)34種抗生素的耐藥性,只有對(duì)阿米卡星和多粘菌素E的耐藥率低于10%,甚至還對(duì)9種抗生素出現(xiàn)100%耐藥情況,證實(shí)了NDM-1陽(yáng)性菌具有強(qiáng)耐藥性。說(shuō)明產(chǎn)NDM-1陽(yáng)性菌感染已經(jīng)在全國(guó)范圍流行和傳播,需要進(jìn)一步開(kāi)展主動(dòng)監(jiān)測(cè),深入研究其發(fā)生發(fā)展規(guī)律。為進(jìn)一步系統(tǒng)比較產(chǎn)NDM-1陽(yáng)性菌的流行病學(xué)及生物學(xué)特性,本次研究選取全國(guó)5個(gè)地區(qū)8個(gè)臨床哨點(diǎn)醫(yī)院收集多重耐藥菌株樣本,使用梅里埃VITEK 2Compact全自動(dòng)細(xì)菌鑒定及藥敏分析系統(tǒng)進(jìn)行菌種鑒定和藥敏測(cè)定,對(duì)碳青霉烯類抗生素耐藥的細(xì)菌標(biāo)本使用普通PCR和實(shí)時(shí)定量PCR方法進(jìn)行NDM-1陽(yáng)性菌篩查,對(duì)篩選出的陽(yáng)性菌株使用E-test法進(jìn)行金屬酶表型鑒定。在篩查的2367份樣本中,一共鑒定出5份NDM-1陽(yáng)性樣本,陽(yáng)性率為0.21%,通過(guò)菌種鑒定顯示1株為肺炎克雷伯菌,其他4株均為不動(dòng)桿菌屬細(xì)菌,藥敏結(jié)果顯示:肺炎克雷伯菌只對(duì)多粘菌素和替加環(huán)素敏感,4株不動(dòng)桿菌屬細(xì)菌對(duì)慶大霉素、妥布霉素、阿米卡星、左氧氟沙星、多粘菌素和替加環(huán)素呈現(xiàn)敏感或中度耐藥。E-test法檢測(cè)5株NDM-1陽(yáng)性菌金屬酶表型驗(yàn)證試驗(yàn)均為陽(yáng)性。通過(guò)上以上研究發(fā)現(xiàn),雖然NDM-1耐藥菌已經(jīng)在我國(guó)部分醫(yī)院中傳播,但本次監(jiān)測(cè)的哨點(diǎn)醫(yī)院患者NDM-1耐藥基因陽(yáng)性率比文獻(xiàn)報(bào)道的檢出率較低。篩選的5株NDM-1陽(yáng)性菌株有4株(80%)為不動(dòng)桿菌屬細(xì)菌和1株肺炎克雷伯菌與國(guó)內(nèi)NDM-1陽(yáng)性菌流行現(xiàn)況分析結(jié)果一致,顯示我國(guó)NDM-1耐藥基因主要在腸桿菌科細(xì)菌中不動(dòng)桿菌屬細(xì)菌中流行和傳播。攜帶blaNDM-1不動(dòng)桿菌屬細(xì)菌不僅僅對(duì)多粘菌素和替加環(huán)素敏感。為了進(jìn)一步研究NDM-1耐藥基因所在質(zhì)粒位置及周?chē)蛄薪Y(jié)構(gòu)特征,以期揭示耐藥基因的分子特征。首先應(yīng)用Southern blot法對(duì)blaNDM-1進(jìn)行基因定位;通過(guò)PCR mapping進(jìn)一步鑒定NDM-1所在質(zhì)粒的結(jié)構(gòu);使用試劑盒提取質(zhì)粒DNA并進(jìn)行高通量測(cè)序。通過(guò)Southern blot法對(duì)5株NDM-1陽(yáng)性菌進(jìn)行基因定位發(fā)現(xiàn):4株NDM-1陽(yáng)性菌bla NDM-1均編碼于質(zhì)粒上,1株NDM-1陽(yáng)性菌bla NDM-1丟失。其中3株NDM-1陽(yáng)性菌blaNDM-1分別編碼在大小約30-6kb的質(zhì)粒,分別為Pm131-NDM-1、PNDM-BJ02、PAb NDM-1,1株NDM-1陽(yáng)性菌blaNDM-1編碼在大小約240-280kb質(zhì)粒上。按照4個(gè)質(zhì)粒擴(kuò)增19對(duì)引物鑒定質(zhì)粒結(jié)構(gòu),通過(guò)PCR mapping鑒定2個(gè)NDM-1陽(yáng)性菌攜帶NDM-1耐藥基因的質(zhì)粒為p NDM-BJ01。4株細(xì)菌編碼NDM的質(zhì)粒測(cè)序結(jié)果顯示:所有NDM-1陽(yáng)性菌質(zhì)粒均含有經(jīng)典的Tn125轉(zhuǎn)座子結(jié)構(gòu),但Tn125下游結(jié)構(gòu)均發(fā)生丟失,NJ-18號(hào)和305-118號(hào)細(xì)菌質(zhì)粒在Tn125上游有插入序列IS26和IS5,該插入序列可能介導(dǎo)NDM-1耐藥基因進(jìn)行跨種傳播,而305-118號(hào)細(xì)菌NDM耐藥基因亞型為NDM-5。綜上所述,本研究結(jié)果顯示:(1)我國(guó)blaNDM-1呈現(xiàn)整體散發(fā)局部流行的流行現(xiàn)狀,具備獨(dú)特流行病學(xué)特點(diǎn),不動(dòng)桿菌屬細(xì)菌是bla NDM-1的主要優(yōu)勢(shì)菌,需要進(jìn)一步開(kāi)展主動(dòng)監(jiān)測(cè),深入研究其發(fā)生發(fā)展規(guī)律;(2)產(chǎn)NDM-1陽(yáng)性菌株在我國(guó)部分醫(yī)院呈散發(fā)狀態(tài),病例之間無(wú)流行病學(xué)關(guān)聯(lián),陽(yáng)性檢出率低于文獻(xiàn)報(bào)道平均水平;(3)blaNDM-1基因可能會(huì)在基礎(chǔ)體質(zhì)和免疫力較差人群或人群所在地點(diǎn)暴發(fā)流行,菌株主要集中在不動(dòng)桿菌屬細(xì)菌,還應(yīng)重點(diǎn)關(guān)注肺炎克雷伯菌。(4)blaNDM-1位于質(zhì)粒上,其全長(zhǎng)30kb左右,1株NDM-1耐藥菌質(zhì)粒全長(zhǎng)約240-280kb,質(zhì)粒結(jié)構(gòu)顯示均含有Tn125轉(zhuǎn)座子結(jié)構(gòu),且其下游結(jié)構(gòu)部分缺失,部分Tn125結(jié)構(gòu)上游含有插入序列IS26和IS5,Tn125轉(zhuǎn)座子和插入序列共同介導(dǎo)耐藥基因的水平轉(zhuǎn)移。
[Abstract]:Antibiotics have played a key role in the struggle against all kinds of infectious diseases, but the antibiotic resistance caused by antibiotics has gradually emerged. Especially in recent decades, the problem of antibiotic resistance has been accelerated because of improper use of antibiotics, which brings a heavy burden to clinical treatment, especially in 2009 New Delhi metal beta. The emergence and epidemic of New Delhi metallo- beta -lactamase-1 (NDM-1) has renewed the awareness of "superbacteria", causing widespread concern and panic worldwide. The initial reports of NDM-1 producing Klebsiella pneumoniae and Escherichia coli were resistant to all antibiotics except polymyxin and tegafine, up to 2016. In the year, NDM-1 resistant bacteria have formed a global distribution and a high prevalence trend in local areas. More and more studies have shown that the problem of bacterial resistance represented by NDM-1 resistant genes has become one of the greatest threats in the field of health and food safety, and it can produce huge hidden dangers to human health. The prevalence of NDM-1 positive bacteria, the blaNDM-1 resistance spectrum, the transmission mechanism and the genetic characteristics were studied. Most of the reported NDM-1 positive bacteria were reported in clinical cases. There were no epidemiological studies and connections between cases. The epidemiological characteristics of NDM-1 positive bacteria were not clear in the country, and the characteristics and dissemination of the NDM-1 strains were carried out. The mechanism is still unclear, which brings great challenges to the overall treatment and prevention of NDM-1. Therefore, we need to systematically study the distribution of NDM-1 positive strains, patient type, medical environment, risk factors and epidemiological traceability and NDM-1 molecular characteristics and variation. Based on the above status, this study was retrieved in the CNKI and Pub Med literature database. From January 2007 to December 2015, all the Chinese and English literature reports on NDM-1 producing bacteria in China were reported, and 767 Chinese and English literature were retrieved, the negative results were excluded, the basic research of NDM-1 molecular biology, the non human infection, the Chinese and English literature were included, and the literature was included in the literature. The epidemiological characteristics of the NDM-1 positive strains reported in the literature were special. The data were collected and analyzed. The results were as follows: as of December 2015, there were 25 provinces and cities in China, and blaNDM-1 positive strains were reported in the region, among which the southeast coastal areas were more distributed, and the positive cases reported in Guangdong area were 109 cases, accounting for 39.49%, obviously more than other regions (P0.05), NDM-1 The positive bacteria were mainly distributed in Klebsiella pneumoniae and Enterobacter cloacae (P0.05); the male patients infected with NDM-1 positive bacteria were significantly more than those of women (P0.01), mainly in 2 age groups: under 10 years of age and between 60~80 years (P0.05); NDM-1 positive bacteria were mainly isolated from sputum specimens, 40, 41.24% (P0.05), NDM-1 positive bacteria infected cases. The main distribution in ICU, pediatrics and Department of respiration (P0.05), lung disease patients more easily isolate the positive bacteria (P0.05), blaNDM-1 positive bacteria to Amikacin, tegatrine's overall resistance rate is the lowest, respectively 7.69% and 2.33%. literature analysis showed that 13 bacteria in our country, 276 strains of bacteria carrying NDM-1 gene, blaNDM-1 coding in size 55kb~360kb On the scope of the plasmid, the plasmid encoding blaNDM-1 can not only achieve high level of transmission, but also has strong cross species transmission ability. Results analysis showed that NDM-1 positive bacteria were resistant to 34 antibiotics, only the resistance rate to Amikacin and polymyxin E was less than 10%, and even 100% resistance to 9 antibiotics was confirmed. The NDM-1 positive bacteria have strong resistance. It shows that the infection of NDM-1 positive bacteria has been popular and spread throughout the country. It is necessary to further develop the active monitoring and study its occurrence and development. In order to further compare the epidemiological and biological characteristics of NDM-1 positive bacteria, this study selected 8 clinical sentinel doctors in 5 regions of the country. A sample of multidrug resistant strains was collected in the hospital. The bacterial identification and drug sensitivity of the milier VITEK 2Compact automatic bacterial identification and drug sensitivity analysis system were used. The specimens of carbapenem resistant bacteria were screened by common PCR and real-time quantitative PCR method for NDM-1 positive bacteria, and E-test method was used for the screened positive strains. In the 2367 samples of the screening, 5 NDM-1 positive samples were identified, the positive rate was 0.21%. 1 strains were Klebsiella pneumoniae and the other 4 were Acinetobacter. The drug sensitivity showed that Klebsiella pneumoniae was only sensitive to polymyxin and tigocycline and 4 strains of Acinetobacter. Gentamicin, tobramycin, Amikacin, levofloxacin, polymyxin and tigocycline showed a sensitive or moderate resistance.E-test assay for 5 NDM-1 positive bacteria phenotypic validation tests. The above study found that although NDM-1 resistant bacteria had been transmitted in some hospitals in China, the sentinel medicine for this monitoring The positive rate of NDM-1 resistance gene in hospital patients was lower than that reported in the literature. 5 strains of NDM-1 positive strains screened were 4 (80%) of Acinetobacter and 1 strains of Klebsiella pneumoniae and domestic NDM-1 positive bacteria, which showed that the NDM-1 resistance genes in our country were mainly in the bacteria of the Enterobacteriaceae. Epidemic and spread. The bacteria carrying blaNDM-1 Acinetobacter are not only sensitive to polymyxin and tigacycline. In order to further study the location of plasmids and the characteristics of the surrounding sequence structure of the NDM-1 resistant genes, the molecular characteristics of the resistant genes are revealed. First, the Southern blot method is used to locate blaNDM-1 gene, and PCR mapping enters into the gene. The plasmid structure of NDM-1 was identified step by step. The plasmid DNA was extracted with the kit and the high flux sequencing was used. The gene location of 5 NDM-1 positive bacteria was detected by Southern blot method. The 4 NDM-1 positive bacteria bla NDM-1 were encoded on the plasmid, and the BLA NDM-1 was lost in 1 strains of NDM-1 positive bacteria. 3 strains of NDM-1 positive bacteria were encoded in the size respectively. The plasmids of about 30-6kb were encoded in Pm131-NDM-1, PNDM-BJ02, and PAb NDM-1,1 NDM-1 positive bacteria on the size 240-280kb plasmids. The plasmid structure was amplified by 19 pairs of primers according to 4 plasmids, and the plasmid sequencing of 2 NDM-1 positive bacteria carrying the NDM-1 resistant gene was identified by PCR mapping. The results showed that all NDM-1 positive bacteria plasmid contained the classical Tn125 transposon structure, but the downstream structure of Tn125 was lost, NJ-18 and No. 305-118 bacterial plasmids inserted sequence IS26 and IS5 upstream of Tn125, and the insertion sequence may mediate trans species transmission of NDM-1 resistant genes, and the NDM resistance gene subtype of No. 305-118 bacteria is NDM-5. synthesis. The results of this study show that: (1) the prevalence of local epidemic in China's blaNDM-1 is presented as a whole, with unique epidemiological characteristics. Acinetobacter is the main dominant bacteria of BLA NDM-1. It is necessary to carry out active monitoring and further study the law of its occurrence and development; (2) the positive strains producing NDM-1 are distributed in some hospitals in our country. There was no epidemiological association between the cases, the positive detection rate was lower than the average level of the literature reported. (3) the blaNDM-1 gene may be prevalent in the base constitution and the location of the poor immunity group or population. The strain mainly concentrated in the Acinetobacter, and should focus on Klebsiella pneumoniae. (4) blaNDM-1 is located on the plasmid. The total length of 1 strains of NDM-1 resistant bacteria was about 240-280kb. The plasmid structure showed that all of the plasmid structures contained Tn125 transposon structure, and the downstream structure of the plasmid was missing, the upstream of the Tn125 structure contained the insertion sequence IS26 and IS5, the Tn125 transposon and the insertion sequence mediate the horizontal transfer of the drug resistance gene together.

【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5;R516

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本文編號(hào)：1790658