我国宋内志贺菌流行特征、耐药性及变异研究

发布时间：2018-05-15 03:06

本文选题：宋内志贺菌 + ONPG阴性　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：志贺菌(Shigella spp.)属于革兰氏阴性兼性厌氧肠杆菌,是引起细菌性痢疾的一种重要的肠道致病菌。据WHO统计数据显示,全球每年由志贺菌引起的细菌性痢疾的病例约为1.65亿,其中1.63亿发生在卫生条件较差的发展中国家,并且志贺菌感染导致的死亡人数高达110万,其中61%的死亡病例为5岁以下的儿童。近年来,尽管由细菌性痢疾导致的儿童死亡率已经下降,但是细菌性痢疾仍然是导致儿童死亡的主要原因之一。根据O抗原不同的结构,志贺菌属分4个血清群,分别是福氏志贺菌(Shigella flexneri)、痢疾志贺菌(Shigella dysenteriae)、鲍氏志贺菌(Shigella boydii)和宋内志贺菌(Shigella sonnei)。就志贺菌流行状况而言,发达国家的细菌性痢疾主要是宋内志贺菌引起,而在发展中国家长期以来以福氏志贺菌流行为主。但近年来,这一流行趋势正在逐渐改变,在亚洲的一些国家,宋内志贺菌正逐渐取代福氏志贺菌成为引起细菌性痢疾的主要病原菌。在我国,多年来志贺菌导致的细菌性痢疾亦主要由福氏志贺菌引起,尤以F2a亚型为主。但值得关注的是,近年来宋内志贺菌的流行比例逐年上升,同时在我国东部、北部等一些发达地区,宋内志贺菌甚至已超越福氏志贺菌成为主要的流行菌群。基于我国志贺菌流行变化的新趋势,本研究开展了对志贺菌的病原监测及相关的研究。本研究收集了全国14个省市不同年代的志贺菌3758株,并对其血清型进行了鉴定,其中福氏志贺菌2084株,宋内志贺菌1630株。结果发现宋内志贺菌的感染率呈逐年上升的趋势,尤其是近十年来,宋内志贺菌流行快速上升,并且正在逐渐超越福氏志贺菌成为主要的流行菌群。这一趋势在东南部、北部和中部一些经济发达的地区尤为明显。此外,我们首次发现不产β-半乳糖苷酶的宋内志贺菌即ONPG阴性宋内志贺菌在我国的流行,其在部分地区已超过ONPG阳性宋内志贺菌成为优势菌型。宋内志贺菌这一表型的改变使目前常用的商品化的鉴定产品梅里埃API 20E和VITEK2的鉴定率从99%分别降至50%和47%,临床上极易导致误诊。因此,宋内志贺菌这种新的流行变异趋势及其对公共健康的威胁不容忽视。在全部1630株宋内志贺菌中,ONPG阴性宋内志贺菌451株,占全部宋内志贺菌的27.7%。ONPG阴性宋内志贺菌在2005年首次监测到,其在2009年开始大量出现,在之后的时间呈流行状态,并在2011年首次超越ONPG阳性宋内志贺菌成为主要的流行菌型。为了进一步了解我国宋内志贺菌的遗传多态性及亲缘关系,本研究利用PFGE方法对不同地区宋内志贺菌,尤其是ONPG阴性宋内志贺菌进行了聚类分析。结果显示,onpg阴性与阳性宋内志贺菌的pfge图谱存在明显的差异,并且在聚类分析时,两种表型处在不同的流行簇中。在对上海、北京、甘肃、河南地区宋内志贺菌pfge的聚类分析中发现,上述各地区均存在具有相同带型onpg阴性宋内志贺菌的流行。对宋内志贺菌的遗传相关性分析发现,阴性菌形成了3个主要的克隆群,在3个克隆群中,主要以上海、甘肃和北京地区的阴性菌为主,还包括来自其他地区onpg阴性菌。研究结果显示,onpg阴性宋内志贺菌已经在我国很多地区出现并流行。为了解我国志贺菌的耐药特点,本研究对福氏和宋内志贺菌进行了抗生素敏感性检测。结果显示,这两种志贺菌对传统的抗生素具有很强的耐药性,耐药率从高到低依次为氨苄西林88.1%、替卡西林87.8%、四环素87.5%、复方新诺明73.2%和氯霉素49.2%。值得关注的是,福氏与宋内志贺菌对一些头孢类抗生素具有较强的耐药性,其中对头孢曲松、头孢唑林和头孢哌酮的耐药率分别达到了31.1%,32.4%和27.5%。对比宋内志贺菌与福氏志贺菌的抗生素耐药情况,结果发现,宋内志贺菌对传统抗生素也均具有很高耐药性,并且其对头孢类抗生素的耐药性强于福氏志贺菌,其中对头孢曲松、头孢唑林和头孢哌酮的耐药率分别达到了或超过35%。但宋内志贺菌对喹诺酮药物耐药性一直维持在较低水平。就临床常用的头孢类及喹诺酮类抗生素,本研究对不同地区和时间分离的菌株的耐药性进行了比较。结果发现,不同地区间抗生素耐药性存在差别:在东南、西北、中部和北部地区,宋内志贺菌对头孢类抗生素耐药性较高。从耐药性随时间的变化来看,宋内志贺菌对头孢曲松、头孢唑林和头孢哌酮的耐药性从2004年之后开始迅速上升,到2007-08年达到最大值,耐药率分别是62.2%、61.7%和59.6%,之后耐药性均下降,但仍然都维持在30%到40%之间。同时,本研究对两种不同onpg表型的宋内志贺菌的耐药性进行了比较,结果发现,onpg阳性菌的耐药性高于阴性菌,但onpg阴性宋内志贺菌耐药性随时间总体呈上升趋势,并且在2012年间出现过耐药性的一个短期的高点,这一情况的出现应该引起重视。上述研究结果对于宋内志贺菌临床诊断及抗生素的合理使用具有重要的参考价值。为了研究耐药性产生的分子机制,本研究对所有耐头孢类抗生素的宋内志贺菌中常见的耐药基因进行了pcr检测。结果发现,在所有耐头孢类抗生素是宋内志贺菌中普遍存在β-内酰胺酶(esbls)相关的耐药基因,其中blactx-m和blatem的阳性率分别为82.8%和70.5%。对blactx-m基因测序比对发现,blactx-m-14、blactx-m-79和blactx-m-15是最主要的型别,并且还发现两种不同型别的blactx-m基因同时存在于一个耐药菌中情况。在整合子基因的检测过程中发现,2类整合酶及其基因盒的检出率较高,分别为93.1%和86.0%。研究结果显示,在所有耐头孢类药物的宋内志贺菌中,耐药基因的比例较高,这些耐药基因在细菌间水平转移会加剧耐药菌的蔓延。基于之前的分析结果,本研究选取了67株宋内志贺菌,其中包括58株ONPG阴性宋内志贺菌,进行了全基因组测序。分析发现,ONPG阴性宋内志贺菌与阳性菌相比,在lac operon区域存在较大差异。对此区域设计引物进行PCR扩增后发现,ONPG阴性菌与阳性菌相比主要缺失了mhp operon的mhpB、mhpA和mhpR以及lac operon中的lacI、lacZ和lac基因,同时一个hypothetical protein(假定蛋白)基因,两个插入序列基因(IS1,IS600)也同时缺失。结合Holt等2012年发表文章的基因组数据及方法,本研究对宋内志贺菌的SNP位点进行了检测,并绘制了系统发育树。结果显示,我国的宋内志贺菌与部分国外菌株同处在一个大的分支上。同时,我国全部的67株宋内志贺菌处于都独立在一个分支上,菌株内部又有几个明显的小分支。推测我国的宋内志贺菌可能是来源于国外,并且在国内的进化上有着自己独立的特点。为了探究lac operon缺失对宋内志贺菌的影响,本研究采用λ-RED同源重组方法,成功构建了lac operon缺失的宋内志贺菌。对敲除株各种生化表型进行验证的结果发现,敲除株除ONPG表型变为阴性外还有6种表型发生变化,其中5种物质的代谢加强,分别是松二糖、龙胆二糖、D-鼠李糖、D-密二糖,D-丝氨酸。还有1种物质代谢变弱,为甲酸。对比不同温度下敲除株与野生株的生长情况,结果显示敲除株与野生株在不同温度下生长无差异。死亡率比较分析的结果显示,注射敲除株小鼠的死亡率没有升高。以上结果说明,lac operon缺失是导致ONPG阴性宋内志贺菌出现的原因,但本部分未能明确是何种优势导致ONPG阴性宋内志贺菌出现和流行,这部分内容需要之后更深入的研究。综上所述,本研究开展了我国宋内志贺菌特别是ONPG阴性菌的流行与分布规律研究;对不同地区宋内志贺菌的抗生素耐药性及耐药机制进行了分析;通过代表性菌株的全基因组测序,揭示ONPG阴性菌基因组的特点及其出现的分子机制。这些结果可以为宋内志贺菌引起的细菌性痢疾的监测、抗生素的合理使用、传染源的追溯、传播关系的调查以及防控策略的制订提供科学依据。
[Abstract]:Shigella spp., a gram-negative and anaerobically anaerobic enterobacter, is an important enteric pathogenic bacteria causing bacterial dysentery. According to WHO statistics, about 165 million of the cases of bacterial dysentery caused by Shigella are caused every year in the world, and 163 million of them occur in developing countries with poor health conditions and Shigella feeling. The number of deaths caused by dyeing is as high as 1 million 100 thousand, of which 61% of the deaths are children under 5 years of age. In recent years, although the mortality of children caused by bacterial dysentery has fallen, bacterial dysentery is still one of the main causes of death in children. According to the structure of O antigen, Shigella is divided into 4 serogroups, respectively. Shigella (Shigella flexneri), Shigella dysenterias (Shigella dysenteriae), Shigella Shigella (Shigella boydii) and Shigella Shigella (Shigella sonnei). In terms of the prevalence of Shigella, bacterial dysentery in developed countries is mainly caused by Shigella song, but it has been dominated by Shigella flexigella in developing countries for a long time. In recent years, this trend is changing gradually. In some Asian countries, Shigella Shigella is gradually replacing Shigella flexneri bacteria to become the main pathogenic bacteria causing bacillary dysentery. In our country, the bacterial dysentery caused by Shigella in many years is mainly caused by Shigella flexneri, especially F2a subtype. However, it is worth paying attention to in recent years The prevalence rate of Shigella in the Song Dynasty is increasing year by year. At the same time, in some developed regions such as eastern China and Northern China, Songchi Shika bacteria have even surpassed Shigella flexneri bacteria and become the main epidemic groups. Based on the new trend of the change of Shigella, the pathogen monitoring and related Research of Shigella are carried out in this study. 3758 strains of Shigella in 14 provinces and cities of China were identified, including 2084 Shigella flexneri strains and 1630 Shigella Shigella in Song Dynasty. The results showed that the infection rate of Shigella Shigella was increasing year by year, especially in the last ten years, the prevalence of Shigella Shigella in Song Dynasty was rising rapidly, and it was gradually surpassing Shigella flexneri bacteria. The main epidemic flora. This trend is particularly obvious in the southeast, North and central some economically developed areas. In addition, we first found that the ONPG negative Shigella Shigella, which does not produce beta galactosidase, is popular in China, and in some areas it has exceeded ONPG positive Shigella to become the dominant bacteria. Shiga Shiga in Song Dynasty The phenotypic changes of bacteria make the identification rates of API 20E and VITEK2 from 99% to 50% and 47% respectively, which are easily misdiagnosed in clinic. Therefore, the new trend of Shigella song and its threat to public health can not be ignored. In all 1630 strains of Shigella song, ONPG negative 451 Shigella song inner Shigella strains, which account for the 27.7%.ONPG negative Shigella Shigella in all Shigella Shigella, were first monitored in 2005. It began to appear in 2005 in a large amount. In the later period, the Shigella Shigella became popular. In 2011, the first ONPG positive Shigella Shigella became the main epidemic type. In order to further understand the remains of Shigella Shigella in China PFGE method was used to cluster analysis of Shigella Shigella in different areas, especially ONPG negative Shigella. The results showed that the PFGE map of ONPG negative and positive Shigella Shigella was distinctly different, and the two phenotypes were in different epidemic clusters in the cluster analysis. In Shanghai In the cluster analysis of Shigella Shigella PFGE in Beijing, Gansu and Henan, it was found that there were the same type of Shigella with the same type of ONPG negative Shigella. The genetic correlation analysis of Shigella song found that the negative bacteria formed 3 main clones in the 3 clones, mainly in Shanghai, Gansu and Beijing region. The negative bacteria were dominant, including the ONPG negative bacteria from other regions. The results showed that the ONPG negative Shigella Shigella had appeared in many areas in China. In order to understand the characteristics of the resistance of Shigella, the antimicrobial susceptibility test of Shigella flexneri and Shigella Shigella was carried out in this study. The results showed that the two Shigella strains were traditional to the traditional Shigella. The antibiotic resistance was very strong, and the resistance rate from high to low was ampicillin 88.1%, teicycline 87.8%, tetracycline 87.5%, compound Novamin 73.2% and chloramphenicol 49.2%., which were highly resistant to some cephalosporins, including ceftriaxone, cefzolin and cefoperazone. The drug resistance of Shigella Shigella and Shigella flexneri was 31.1%, 32.4% and 27.5%. respectively. The results showed that Shigella song had high resistance to traditional antibiotics, and its resistance to ceftriaxone, cefazolin and cefoperazone was better than that of Shigella flexneri. The resistance rate reached or exceeded 35%., respectively, but the drug resistance of Shigella song to quinolone has been maintained at a low level. The drug resistance of cephalosporins and quinolones, commonly used in clinic, was compared in different regions and time separated strains. The results showed that the antibiotic resistance existed in different regions. The resistance of Shigella song to ceftriaxone, cefazolin and cefoperazone began to rise rapidly from 2004 to 2007-08, and the drug resistance rate was 62.2%, 61.7% and 59.6 respectively. %, and then the resistance decreased, but still maintained between 30% and 40%. At the same time, this study compared the resistance of Shigella Shigella with two different ONPG phenotypes. The results showed that the resistance of ONPG positive bacteria was higher than that of negative bacteria, but the resistance of ONPG negative Shigella Shigella was on the rise with time and appeared in 2012 years. The results of this study have important reference value for the clinical diagnosis of Shigella song and the rational use of antibiotics. In order to study the molecular mechanism of the drug resistance, the common resistance of all cephalosporins to Shigella Shigella is in this study. The drug gene was detected by PCR. The results showed that all cephalosporins were resistant genes related to beta lactamase (ESBLs) in Shigella song. The positive rates of blactx-m and blatem were 82.8% and 70.5%. respectively, and blactx-m-14, blactx-m-79 and blactx-m-15 were the most important types for blactx-m gene sequencing. Two different types of blactx-m genes were found in a drug resistant strain. The detection rates of the integron genes were found to be higher in the 2 types of integrase and their gene boxes. The results of the 93.1% and 86.0%. studies showed that the proportion of the resistance genes in all the cephalosporin resistant Shigella, Shigella Shigella, was compared. On the basis of previous analysis, 67 strains of Shigella Shigella, including 58 ONPG negative Shigella Shigella, were sequenced. The results showed that the ONPG negative Shigella Shigella was compared with the positive bacteria, and there was a comparison between the lac operon region and the positive bacteria. After PCR amplification of the primers in this area, it was found that the ONPG negative bacteria mainly lost the mhpB of MHP operon, mhpA and mhpR, lacI, lacZ and lac genes in lac operon, at the same time, the two inserted sequence genes were also missing at the same time. 2012 The genomic data and methods of the article were published in this study. The SNP site of Shigella song was detected and the phylogenetic tree was plotted. The results showed that Shigella song in China and some foreign strains were in a large branch. At the same time, all 67 strains of Shigella Shigella in China were isolated on one branch. There are several obvious small branches in the interior. It is presumed that Shigella Shigella may originate from foreign countries and have its own independent characteristics in the domestic evolution. In order to explore the effect of lac operon deletion on Shigella song, this study successfully constructed the Shigella Shigella with the deletion of lac operon by the method of the homologous recombination of lac operon. The results of various biochemical phenotypes showed that there were 6 phenotypic changes in the knockout plant except for the negative ONPG phenotype, and the metabolism of 5 substances was strengthened, respectively: Pine two sugar, gentian two sugar, D- rat rhamnose, D- dense two sugar, D- serine, and 1 kinds of substances weakened, as formic acid. The results showed that there was no difference in the growth of the knockout plant and the wild plant at different temperatures. The mortality comparison analysis showed that the mortality of the injecting knockout strain mice did not increase. The above results indicated that the lac operon deletion was the cause of the emergence of the ONPG negative Shigella song, but this part failed to define the advantages of the ONP. G negative Shigella song internal Shigella and epidemic, this part of the content needs more in-depth study. In summary, this study carried out the study of the prevalence and distribution of Shigella Shigella especially ONPG negative bacteria in our country; the antibiotic resistance and resistance mechanism of Shigella Shigella in different regions were analyzed. Genome sequencing to reveal the characteristics of the genome of ONPG negative bacteria and their molecular mechanisms. These results can provide scientific basis for the monitoring of bacillary dysentery caused by Shigella song, the rational use of antibiotics, the traceability of the source of infection, the investigation of the transmission relationship and the formulation of control strategies.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R446.5

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