【作者】 王中强；

【导师】 孙岩松； 宋宏彬；

【作者基本信息】 中国人民解放军军事医学科学院 ， 预防兽医学， 2011， 硕士

【摘要】 本研究通过军队传染病病原体网络化监测平台,建立标准化的病原体监测技术,对我国志贺菌、猪链球菌和鲍曼不动杆菌等重要病原菌进行分子分型、基因变异及菌群变迁特征分析,对及时发现传染病新的流行趋势和制定针对性的防控策略具有重要意义。在我国,志贺菌是一种重要的传染性致病菌,因其血清型众多,且传播过程中极易发生变异,对志贺菌感染导致的急性肠道传染病的防控带来很大困难。通过对165株志贺菌的区域性差异进行分析,发现昆明和新疆两个地区的福氏1b血清型菌株分别处于两个聚类中,表现出明显的地区差异。而宋内氏志贺菌的遗传较为稳定,不同年份分离的菌株基因型差异不大,相同基因型菌株可在不同地区间传播,没有明显的区域性差异;与之相反,福氏2型志贺菌作为我国主要流行血清型菌株则频繁的发生遗传变异,部分血清型不同的菌株基因型一致,可能与福氏志贺菌容易发生血清型的转换有关。同时,在对志贺菌进行血清分型过程中首次发现在我国存在福氏1c血清型菌株,并发现了两种新的福氏志贺菌血清型,暂时分别命名为福氏2c和福氏4y。其中福氏4y血清型菌株与其他血清型菌株在吲哚试验、VP试验、甘露醇发酵、山梨醇发酵和密二糖发酵5种生化反应上存在明显差异,生化鉴定结果显示:福氏4y血清型菌株52.9%可能性属于大肠杆菌属,44.5%可能性属于志贺菌属。针对上述发现的特殊血清型菌株,进一步通过PFGE和MLST技术对其遗传变异进行分析。PFGE分析结果显示处于不同聚类的福氏1c血清型菌株分别与福氏1a和1b血清型菌株相似性较高,MLST分析鉴定出福氏1c血清型菌株的3个新序列型:ST100、ST104和ST105,其中,分离自上海与分离自新疆的福氏1c志贺菌存在较大遗传差异,说明福氏1c志贺菌在我国可能存在多个流行克隆,而这些流行克隆可能起源于不同祖先克隆。而福氏2c血清型菌株PFGE分析结果显示,多数福氏2c血清型菌株与福氏2a或2b血清型遗传相似性较高,但是有2株福氏2c血清型菌株与其他福氏2型菌株亲缘关系较远,MLST结果显示全部福氏2c血清型菌株都属于新发现的ST100,菌株的看家基因并未发生变异。由此推测,福氏2c血清型菌株不仅可通过福氏2a和2b血清型菌株O抗原变异而来,还存在由非2型菌株变异或者其他地区传入的可能。其中,福氏4y血清型菌株最为特殊,与其他福氏志贺菌作遗传相似性比较,结果显示福氏4y血清型菌株单独处于一个福氏志贺菌之外的聚类中,相似性仅为44.0%,这与生化鉴定的结果相符。MLST分型结果中福氏4y血清型菌株属于新发现的ST99,出现了7个新的等位基因编号,表现出较大的差异,属于进化过程中的一个单独分支。福氏4y血清型菌株全基因组图谱初步的分析结果显示,大约有50%的基因属于大肠杆菌属,剩余部分则属于福氏志贺菌属。综合表型和基因型研究结果,我们推测该型菌株为大肠杆菌和志贺菌基因结合的突变体或处于遗传过程中的过渡状态。5株2007年分离自上海同一家医院的菌株表现出相对高的相似性,而另外1株2005年分离自上海的菌株则与2005年分离自北京的1株菌相似性较高。据此推测,在2005年福氏4y菌株便已在北京和上海两地间传播,或者上海的菌株发生了变异,并造成了2007年的一次小范围流行。这一极特殊血清型菌株的发现和流行引起了我们的高度重视,更为深入的研究仍在有序进行中。所有上述特殊血清型菌株耐药性研究表明特殊血清型菌株的多重耐药现象较为严重。全部福氏1c血清型菌株对氨苄西林、萘啶酸、四环素、氨苄西林/舒巴坦、阿莫西林/克拉维酸和氯霉素均耐药,对磺胺甲噁唑/甲氧苄啶的耐药率也高达85.7%;福氏2c血清型菌株对萘啶酸、氨苄西林、氯霉素、四环素、氨苄西林/舒巴坦、阿莫西林/克拉维酸、左氧氟沙星和磺胺甲噁唑/甲氧苄啶的耐药率依次为100%、94.1%、88.2%、82.4%、76.4%、76.4%、70.6%和64.7%;而福氏4y血清型菌株主要对氨苄西林、萘啶酸、阿莫西林/克拉维酸和庆大霉素4种抗生素耐药,耐药率依次为100%、100%、85.7%和85.7%,而国内外此前研究表明多数福氏志贺菌对庆大霉素是敏感的。同时,还对病原监测范围中另一种重要的人兽共患病原菌猪链球菌进行了分子分型分析。对32株猪源分离株及12株人源分离株的MLST分子分型显示:引起人类感染的是ST1和ST7高致病性猪链球菌,这两种序列型菌株在江苏和四川两地患者和病猪体内均可分离到,所占比例分别为43.2%和22.7%,为我国主要流行型别。除此之外,在国内还首次监测到ST19、ST20和ST91猪链球菌,所占比例依次为18.2%、6.8%和9.0%,均是从猪体内分离,其中部分ST19菌株无致病力,可以在猪体内携带而不引起疾病。猪链球菌具有遗传多样性,而且高致病性的猪链球菌近年来不断被发现,能够感染人的型别也在不断增加,故对猪链球菌的监测工作愈发重要,以防止猪链球菌引发的传染病再次大面积传播。另外,在病原监测过程中还检测出国内首株产1型新德里金属β-内酰胺酶(New Delhi metallo-β- lactamase 1,NDM-1)鲍曼不动杆菌,并及时上报,为NDM-1泛耐药菌感染的防控起到了重大作用。为研究该菌株在医院的传播规律,应用PFGE分型技术对该哨点医院同期分离的72株鲍曼不动杆菌进行了遗传相似性分析,发现该院鲍曼不动杆菌基因型众多,并未暴发某一特定型别菌株的大面积流行,仅存在少数几个型别菌株的小范围传播;产NDM-1菌株不属于院内主要的流行克隆群,且在监测的菌株中不含有与产NDM-1菌株基因型一致的菌株。结果表明,该菌株的感染并未在医院内传播流行,其为输入性病原菌还是鲍曼不动杆菌菌群变异产生有待进一步的调查研究。本研究开展了志贺菌、猪链球菌和鲍曼不动杆菌三种病原菌的监测、分子分型及遗传变异分析工作,取得了一系列有意义的研究成果:在国内发现了福氏志贺菌1c血清型菌株并检测到部分福氏2c和4y新血清型菌株,其中福氏4y血清型菌株两种菌属基因并存的现象具有重大研究价值;猪链球菌除已知的引起两次疫情的ST7菌株外,还普遍存在ST1菌株,并造成部分人的感染;此外,还监测到国内首株产NDM-1鲍曼不动杆菌。本研究所建立的分型技术和分析方法为今后传染病及感染性疾病的网络化监测、预警、暴发溯源奠定了技术基础,所监测到的新病原及其耐药性也为传染病防治和其它应用性研究提供了基础数据。更多还原

【Abstract】 Based on the army network monitoring platformfor infectious disease, this study was aimed to develop the standardized monitoring technique and to subtype the important bacterial pathogens including Shigella, Streptococcus suisand Acinetobacterbaumanniin order to analyse the genetic variation and changing trends of these pathogens. The results obtained in this study will play an important role inmaking and adjusting the prevention and control strategies.Shigella spp.,an important infectious agents with a number of serotypes which can cause acute diarrhea, easily experiences genetic variation in the process of transmission. So it will pose a major challenge for the prevention and control of shigellosis. In this study 165 Shigella strains were selected to analyse the regional variation. The Shigellaflexneriserotype 1b isolates from Kunming and Xinjiang formed different clusters respectively, revealing obvious regional variation. The S. sonnei isolates showed no clear regional difference as they had high genetic relatedness and the strains from different regions had the same genotypes. Frequent serotype switching was observed among the S. flexneriserotype2strains.The serotypes of strains were confirmed by using the commercially available antisera kit (Denka Seiken, Tokyo, Japan) and a panel of monoclonal antibodies against Shigellaflexneri (MASF) (Reagensia AB, Stockholm, Sweden). During the routine surveillance of shigellosis in our laboratory,S.flexneriserotype1cwas detectedin our country for the first time, and we also identifiedseveral newS.flexneriserotypes, provisionally named as S.flexneriserotype2c and serotype 4y. Api20E test kits were used to observe the biochemical reactions of the serologically atypical strains of S.flexneri. Surprisingly, serotype 4yhaddifferent biochemical characteristics from otherserotypes, and it was identified as a suspected biochemical profile with 52.9% probability of Escherichia coil and 44.5% probability of Shigellaby using Apilabplus software analysis.To explore the genetic diversity, regional variation and understand the epidemiology of Shigella, Pulsed field gel electrophoresis (PFGE) was used in this study to characterize the clinical isolates of Shigella. There are significant genotypicdifferences between the isolates of Shigella and strains of the same pulsetypespreadindifferent regions by using PFGEanalysis. The S.flexneri serotype 1c isolates in different genetic clusters showed high genetic similarity with the S.flexneri serotype 1a and 1b strains respectively. MLST analysis of S.flexneri serotype 1c showed that they belonged to ST100, ST104 and ST105 respectively. PFGE and MLST analysis had similar results that the serotype 1c strains isolated in Shanghai revealed different levels of genetic similarity for each other, and had higher genetic differences with the strains isolated in Xinjiang. So, S.flexneri serotype 1c strains in China may have several epidemic clones which may be derived from different ancestors.PFGE analysis of S.flexneri serotype 2c strains indicated that most strains had a high genetic similarity with S.flexneri serotype 2a and 2b strains, but two additional strains had low genetic relatedness with other S.flexneri serotype 2 strains. MLST analysis showed that all of the S.flexneri serotype 2c strains belonged to ST100.PFGE cluster analysis describing the genetic similarity among the serotype 4y and other serotypes of S.flexneri showed that different serotypes formed diverse clusters. Five S.flexneri serotype 4y strains isolated in 2007 from a same hospital in Shanghai showed high genetic similarity, and a strain isolated in 2005 inShanghai was closely related to a strain isolated in Beijing in the same year. This result suggested that S.flexneri serotype 4y strains may have spread between Beijing and Shanghai in 2005, and they experienced genetic variation during the dissemination process and then caused a small epidemic in Shanghai. S.flexneri serotype 4y strains had 44.0% similarity with other serotypes of S.flexneri, which was identical with the result of biochemical identification. MLST analysis of S.flexneri serotype 4y strains showed that they all belonged to another new ST (ST99) with 7 new alleles, which had great difference with other STs of S.flexneri.The antimicrobial susceptibilities of serologically atypical S.flexneri strains were determined by the Kirby-Bauer method with commercial antimicrobial discs (Oxoid, United Kingdom). The antibiotic discs used in this study were cefotaxime, ceftazidime, amoxicillin/clavulanic acid, ampicillin/sulbactam, ciprofloxacin, norfloxacin, levofloxacin, ampicillin, tetracycline, sulfamethoxazole/trimethoprim, chloramphenicol,imipenem, gentamicin and nalidixic acid. All of the serologically atypical strains showed serious multidrug resistance, among which . were resistant to imipenem. But S.flexneri serotype 1c strains were completely resistant to ampicillin,nalidixic acid,tetracycline,ampicillin/sulbactam,amoxicillin/clavulanic acid and chloramphenicol, and of them 85.7% were resistant to sulfamethoxazole/trimethoprim. Among the serotype 2c strains, 100%, 94.1%, 88.2%, 82.4%, 76.4%, 76.4%, 70.6% and 64.7% were resistant to nalidixic acid, ampicillin, chloramphenicol, tetracycline, ampicillin/sulbactam, amoxicillin/clavulanic, levofloxacin and sulfamethoxazole/trimethoprim respectively. All serotype 4y strains were resistant to nalidixic acid and ampicillin, and also 85.7% were resistant toamoxicillin/clavulanic and gentamicin. The emergence of multiple resistance among S.flexneri isolates will pose a major public health problem. In this study, 54.8% of the atypical strains were resistant to three commonly used antibiotics, including ampicillin, tetracycline, and sulfamethoxazole/trimethoprim.Furthermore, whole genome sequencing was conducted to explore the genetic difference and evolution of the serotype 4y, and the whole genome analysis is going on.In our country, most strains isolated from human were chosen in MLST analysis of Streptococcus suis. In order to understand the population structure and genetic relationship of S. suis, 32 strains isolated from pigs and 12 strains isolated from human were analysed by MLST. Five STs were identified, among which ST1andST7 were the two most common STs and mainly responsible for humanhighly pathogenicS.suis infection, and other three STs, ST19, ST20andST91, were detected for the first time in our country. Comparative analysis from the data of S.suis MLST database showed that three majorclonal groups (ST1, ST27 andST87) had existedin China. Strains belonging to ST1clonalgrouphadcaused2 large outbreaksin Jiangsu and SichuanProvince. It is reproted that some of the ST27 clone group strains can also result in humanhighly pathogenicS.suis infection in Thailand, and the strains belonging to the ST87clonalgroup can seldom infect human. However, in recent years, more and more STs of S. suis which can infect human have been found. So, the monitoring of S. suis is very important to prevent the large scale dissemination of S. suis .Carbapenem-resistant Acinetobacterbaumanni strains with New Delhimetallo-beta-lactamase–1 (NDM-1) were screened from the samples collected from a hospital, and a blaNDM-1-positive strain was detected. To the best of our knowledge, this is the first report of A. baumannii with NDM-1 inChina. PFGEanalysis showed thatthe blaNDM-1-positive strain was genetically distinct and had different PFGE banding pattern with other A. baumannii strains from this hospital. It was suggested that the blaNDM-1-positive strain did not spread in the hospital.更多还原