NDM-1泛耐药菌流行病学及分子特征研究

发布时间：2018-04-23 05:25

本文选题：NDM-1 + 流行病学特征　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：人类与各种感染性疾病的斗争中,抗生素起到关键作用,但由抗菌素应用产生的抗生素耐药问题逐渐显现,尤其是近几十年来,由于抗生素使用不当等问题加速了细菌耐药问题的恶化,给临床治疗带来沉重负担。尤其2009年新德里金属β-内酰胺酶(New Delhi metallo-β-lactamase-1,NDM-1)的出现和流行更新了人们对“超级细菌”的认识,引起了世界范围内广泛关注和恐慌,最初报道的产NDM-1的肺炎克雷伯菌和大肠杆菌对除了多粘菌素和替加环素以外的所有抗生素耐药,截止2016年,NDM-1耐药菌已经在全球范围内形成总体散发,局部地区高流行的流行趋势,越来越多的研究表明以NDM-1耐药基因为代表的细菌耐药问题已经成为健康和食品安全领域的最大威胁之一,能对人类健康产生巨大隐患。目前,我国大量报道了关于产NDM-1阳性菌的流行、blaNDM-1耐药谱及传播机制和遗传学特征等研究。报道的NDM-1阳性菌大部分为临床个案报道,病例之间无流行病学研究和联系,对于NDM-1阳性菌在全国的流行病学特征尚不明确,携带NDM-1菌株的特性及其播散的机制仍不清楚,给NDM-1的整体治疗、防控带来巨大挑战。因此,需系统研究NDM-1阳性菌株在各地的分布情况、病人类型、医疗环境、危险因素以及流行病学溯源和NDM-1分子特征和变异情况。基于以上现况,本研究在CNKI和Pub Med文献数据库检索了从2007年1月至2015年12月所有报道中国产NDM-1细菌研究的中英文文献,共检索中英文文献767篇,排除阴性结果文献、综述、NDM-1分子生物学基础研究、非人感染、中英文重复文献,共纳入研究文献50篇。对文献报道的NDM-1阳性菌株的流行病学特征,菌株来源、耐药谱及传播机制等方面信息进行汇总和分析,结果发现:截至2015年12月,我国共有25个省市、地区报道blaNDM-1阳性菌株,其中东南部沿海地区分布较多,广东地区报道的阳性病例为109例,占39.49%,明显多于其他地区(P0.05),NDM-1阳性菌主要分布在肺炎克雷伯菌和阴沟肠杆菌(P0.05);感染NDM-1阳性菌的男性患者明显多于女性患者(P0.01),主要集中在2个年龄段:10岁以下和60~80岁之间(P0.05);NDM-1阳性菌主要分离于痰液标本,为40株,占41.24%(P0.05),NDM-1阳性菌感染的病例主要分布在ICU、儿科和呼吸科(P0.05),肺部疾病患者更容易分离出阳性菌(P0.05),blaNDM-1阳性菌对阿米卡星、替加环素的总体耐药率最低,分别为7.69%和2.33%。文献分析显示我国共有13个菌属276株细菌携带NDM-1基因,blaNDM-1编码在大小为55kb~360kb范围的质粒上,表明编码blaNDM-1的质粒不仅可以实现高效水平传播,还具有较强的跨种传播能力;结果分析显示NDM-1阳性菌对34种抗生素的耐药性,只有对阿米卡星和多粘菌素E的耐药率低于10%,甚至还对9种抗生素出现100%耐药情况,证实了NDM-1阳性菌具有强耐药性。说明产NDM-1阳性菌感染已经在全国范围流行和传播,需要进一步开展主动监测,深入研究其发生发展规律。为进一步系统比较产NDM-1阳性菌的流行病学及生物学特性,本次研究选取全国5个地区8个临床哨点医院收集多重耐药菌株样本,使用梅里埃VITEK 2Compact全自动细菌鉴定及药敏分析系统进行菌种鉴定和药敏测定,对碳青霉烯类抗生素耐药的细菌标本使用普通PCR和实时定量PCR方法进行NDM-1阳性菌筛查,对筛选出的阳性菌株使用E-test法进行金属酶表型鉴定。在筛查的2367份样本中,一共鉴定出5份NDM-1阳性样本,阳性率为0.21%,通过菌种鉴定显示1株为肺炎克雷伯菌,其他4株均为不动杆菌属细菌,药敏结果显示:肺炎克雷伯菌只对多粘菌素和替加环素敏感,4株不动杆菌属细菌对庆大霉素、妥布霉素、阿米卡星、左氧氟沙星、多粘菌素和替加环素呈现敏感或中度耐药。E-test法检测5株NDM-1阳性菌金属酶表型验证试验均为阳性。通过上以上研究发现,虽然NDM-1耐药菌已经在我国部分医院中传播,但本次监测的哨点医院患者NDM-1耐药基因阳性率比文献报道的检出率较低。筛选的5株NDM-1阳性菌株有4株(80%)为不动杆菌属细菌和1株肺炎克雷伯菌与国内NDM-1阳性菌流行现况分析结果一致,显示我国NDM-1耐药基因主要在肠杆菌科细菌中不动杆菌属细菌中流行和传播。携带blaNDM-1不动杆菌属细菌不仅仅对多粘菌素和替加环素敏感。为了进一步研究NDM-1耐药基因所在质粒位置及周围序列结构特征,以期揭示耐药基因的分子特征。首先应用Southern blot法对blaNDM-1进行基因定位;通过PCR mapping进一步鉴定NDM-1所在质粒的结构;使用试剂盒提取质粒DNA并进行高通量测序。通过Southern blot法对5株NDM-1阳性菌进行基因定位发现:4株NDM-1阳性菌bla NDM-1均编码于质粒上,1株NDM-1阳性菌bla NDM-1丢失。其中3株NDM-1阳性菌blaNDM-1分别编码在大小约30-6kb的质粒,分别为Pm131-NDM-1、PNDM-BJ02、PAb NDM-1,1株NDM-1阳性菌blaNDM-1编码在大小约240-280kb质粒上。按照4个质粒扩增19对引物鉴定质粒结构,通过PCR mapping鉴定2个NDM-1阳性菌携带NDM-1耐药基因的质粒为p NDM-BJ01。4株细菌编码NDM的质粒测序结果显示:所有NDM-1阳性菌质粒均含有经典的Tn125转座子结构,但Tn125下游结构均发生丢失,NJ-18号和305-118号细菌质粒在Tn125上游有插入序列IS26和IS5,该插入序列可能介导NDM-1耐药基因进行跨种传播,而305-118号细菌NDM耐药基因亚型为NDM-5。综上所述,本研究结果显示:(1)我国blaNDM-1呈现整体散发局部流行的流行现状,具备独特流行病学特点,不动杆菌属细菌是bla NDM-1的主要优势菌,需要进一步开展主动监测,深入研究其发生发展规律;(2)产NDM-1阳性菌株在我国部分医院呈散发状态,病例之间无流行病学关联,阳性检出率低于文献报道平均水平;(3)blaNDM-1基因可能会在基础体质和免疫力较差人群或人群所在地点暴发流行,菌株主要集中在不动杆菌属细菌,还应重点关注肺炎克雷伯菌。(4)blaNDM-1位于质粒上,其全长30kb左右,1株NDM-1耐药菌质粒全长约240-280kb,质粒结构显示均含有Tn125转座子结构,且其下游结构部分缺失,部分Tn125结构上游含有插入序列IS26和IS5,Tn125转座子和插入序列共同介导耐药基因的水平转移。
[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.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;R516

【参考文献】