本文選題：宋內(nèi)志賀菌 + ONPG陰性�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年博士論文

【摘要】：志賀菌(Shigella spp.)屬于革蘭氏陰性兼性厭氧腸桿菌,是引起細(xì)菌性痢疾的一種重要的腸道致病菌。據(jù)WHO統(tǒng)計數(shù)據(jù)顯示,全球每年由志賀菌引起的細(xì)菌性痢疾的病例約為1.65億,其中1.63億發(fā)生在衛(wèi)生條件較差的發(fā)展中國家,并且志賀菌感染導(dǎo)致的死亡人數(shù)高達(dá)110萬,其中61%的死亡病例為5歲以下的兒童。近年來,盡管由細(xì)菌性痢疾導(dǎo)致的兒童死亡率已經(jīng)下降,但是細(xì)菌性痢疾仍然是導(dǎo)致兒童死亡的主要原因之一。根據(jù)O抗原不同的結(jié)構(gòu),志賀菌屬分4個血清群,分別是福氏志賀菌(Shigella flexneri)、痢疾志賀菌(Shigella dysenteriae)、鮑氏志賀菌(Shigella boydii)和宋內(nèi)志賀菌(Shigella sonnei)。就志賀菌流行狀況而言,發(fā)達(dá)國家的細(xì)菌性痢疾主要是宋內(nèi)志賀菌引起,而在發(fā)展中國家長期以來以福氏志賀菌流行為主。但近年來,這一流行趨勢正在逐漸改變,在亞洲的一些國家,宋內(nèi)志賀菌正逐漸取代福氏志賀菌成為引起細(xì)菌性痢疾的主要病原菌。在我國,多年來志賀菌導(dǎo)致的細(xì)菌性痢疾亦主要由福氏志賀菌引起,尤以F2a亞型為主。但值得關(guān)注的是,近年來宋內(nèi)志賀菌的流行比例逐年上升,同時在我國東部、北部等一些發(fā)達(dá)地區(qū),宋內(nèi)志賀菌甚至已超越福氏志賀菌成為主要的流行菌群�；谖覈举R菌流行變化的新趨勢,本研究開展了對志賀菌的病原監(jiān)測及相關(guān)的研究。本研究收集了全國14個省市不同年代的志賀菌3758株,并對其血清型進(jìn)行了鑒定,其中福氏志賀菌2084株,宋內(nèi)志賀菌1630株。結(jié)果發(fā)現(xiàn)宋內(nèi)志賀菌的感染率呈逐年上升的趨勢,尤其是近十年來,宋內(nèi)志賀菌流行快速上升,并且正在逐漸超越福氏志賀菌成為主要的流行菌群。這一趨勢在東南部、北部和中部一些經(jīng)濟(jì)發(fā)達(dá)的地區(qū)尤為明顯。此外,我們首次發(fā)現(xiàn)不產(chǎn)β-半乳糖苷酶的宋內(nèi)志賀菌即ONPG陰性宋內(nèi)志賀菌在我國的流行,其在部分地區(qū)已超過ONPG陽性宋內(nèi)志賀菌成為優(yōu)勢菌型。宋內(nèi)志賀菌這一表型的改變使目前常用的商品化的鑒定產(chǎn)品梅里埃API 20E和VITEK2的鑒定率從99%分別降至50%和47%,臨床上極易導(dǎo)致誤診。因此,宋內(nèi)志賀菌這種新的流行變異趨勢及其對公共健康的威脅不容忽視。在全部1630株宋內(nèi)志賀菌中,ONPG陰性宋內(nèi)志賀菌451株,占全部宋內(nèi)志賀菌的27.7%。ONPG陰性宋內(nèi)志賀菌在2005年首次監(jiān)測到,其在2009年開始大量出現(xiàn),在之后的時間呈流行狀態(tài),并在2011年首次超越ONPG陽性宋內(nèi)志賀菌成為主要的流行菌型。為了進(jìn)一步了解我國宋內(nèi)志賀菌的遺傳多態(tài)性及親緣關(guān)系,本研究利用PFGE方法對不同地區(qū)宋內(nèi)志賀菌,尤其是ONPG陰性宋內(nèi)志賀菌進(jìn)行了聚類分析。結(jié)果顯示,onpg陰性與陽性宋內(nèi)志賀菌的pfge圖譜存在明顯的差異,并且在聚類分析時,兩種表型處在不同的流行簇中。在對上海、北京、甘肅、河南地區(qū)宋內(nèi)志賀菌pfge的聚類分析中發(fā)現(xiàn),上述各地區(qū)均存在具有相同帶型onpg陰性宋內(nèi)志賀菌的流行。對宋內(nèi)志賀菌的遺傳相關(guān)性分析發(fā)現(xiàn),陰性菌形成了3個主要的克隆群,在3個克隆群中,主要以上海、甘肅和北京地區(qū)的陰性菌為主,還包括來自其他地區(qū)onpg陰性菌。研究結(jié)果顯示,onpg陰性宋內(nèi)志賀菌已經(jīng)在我國很多地區(qū)出現(xiàn)并流行。為了解我國志賀菌的耐藥特點,本研究對福氏和宋內(nèi)志賀菌進(jìn)行了抗生素敏感性檢測。結(jié)果顯示,這兩種志賀菌對傳統(tǒng)的抗生素具有很強(qiáng)的耐藥性,耐藥率從高到低依次為氨芐西林88.1%、替卡西林87.8%、四環(huán)素87.5%、復(fù)方新諾明73.2%和氯霉素49.2%。值得關(guān)注的是,福氏與宋內(nèi)志賀菌對一些頭孢類抗生素具有較強(qiáng)的耐藥性,其中對頭孢曲松、頭孢唑林和頭孢哌酮的耐藥率分別達(dá)到了31.1%,32.4%和27.5%。對比宋內(nèi)志賀菌與福氏志賀菌的抗生素耐藥情況,結(jié)果發(fā)現(xiàn),宋內(nèi)志賀菌對傳統(tǒng)抗生素也均具有很高耐藥性,并且其對頭孢類抗生素的耐藥性強(qiáng)于福氏志賀菌,其中對頭孢曲松、頭孢唑林和頭孢哌酮的耐藥率分別達(dá)到了或超過35%。但宋內(nèi)志賀菌對喹諾酮藥物耐藥性一直維持在較低水平。就臨床常用的頭孢類及喹諾酮類抗生素,本研究對不同地區(qū)和時間分離的菌株的耐藥性進(jìn)行了比較。結(jié)果發(fā)現(xiàn),不同地區(qū)間抗生素耐藥性存在差別:在東南、西北、中部和北部地區(qū),宋內(nèi)志賀菌對頭孢類抗生素耐藥性較高。從耐藥性隨時間的變化來看,宋內(nèi)志賀菌對頭孢曲松、頭孢唑林和頭孢哌酮的耐藥性從2004年之后開始迅速上升,到2007-08年達(dá)到最大值,耐藥率分別是62.2%、61.7%和59.6%,之后耐藥性均下降,但仍然都維持在30%到40%之間。同時,本研究對兩種不同onpg表型的宋內(nèi)志賀菌的耐藥性進(jìn)行了比較,結(jié)果發(fā)現(xiàn),onpg陽性菌的耐藥性高于陰性菌,但onpg陰性宋內(nèi)志賀菌耐藥性隨時間總體呈上升趨勢,并且在2012年間出現(xiàn)過耐藥性的一個短期的高點,這一情況的出現(xiàn)應(yīng)該引起重視。上述研究結(jié)果對于宋內(nèi)志賀菌臨床診斷及抗生素的合理使用具有重要的參考價值。為了研究耐藥性產(chǎn)生的分子機(jī)制,本研究對所有耐頭孢類抗生素的宋內(nèi)志賀菌中常見的耐藥基因進(jìn)行了pcr檢測。結(jié)果發(fā)現(xiàn),在所有耐頭孢類抗生素是宋內(nèi)志賀菌中普遍存在β-內(nèi)酰胺酶(esbls)相關(guān)的耐藥基因,其中blactx-m和blatem的陽性率分別為82.8%和70.5%。對blactx-m基因測序比對發(fā)現(xiàn),blactx-m-14、blactx-m-79和blactx-m-15是最主要的型別,并且還發(fā)現(xiàn)兩種不同型別的blactx-m基因同時存在于一個耐藥菌中情況。在整合子基因的檢測過程中發(fā)現(xiàn),2類整合酶及其基因盒的檢出率較高,分別為93.1%和86.0%。研究結(jié)果顯示,在所有耐頭孢類藥物的宋內(nèi)志賀菌中,耐藥基因的比例較高,這些耐藥基因在細(xì)菌間水平轉(zhuǎn)移會加劇耐藥菌的蔓延�；谥暗姆治鼋Y(jié)果,本研究選取了67株宋內(nèi)志賀菌,其中包括58株ONPG陰性宋內(nèi)志賀菌,進(jìn)行了全基因組測序。分析發(fā)現(xiàn),ONPG陰性宋內(nèi)志賀菌與陽性菌相比,在lac operon區(qū)域存在較大差異。對此區(qū)域設(shè)計引物進(jìn)行PCR擴(kuò)增后發(fā)現(xiàn),ONPG陰性菌與陽性菌相比主要缺失了mhp operon的mhpB、mhpA和mhpR以及l(fā)ac operon中的lacI、lacZ和lac基因,同時一個hypothetical protein(假定蛋白)基因,兩個插入序列基因(IS1,IS600)也同時缺失。結(jié)合Holt等2012年發(fā)表文章的基因組數(shù)據(jù)及方法,本研究對宋內(nèi)志賀菌的SNP位點進(jìn)行了檢測,并繪制了系統(tǒng)發(fā)育樹。結(jié)果顯示,我國的宋內(nèi)志賀菌與部分國外菌株同處在一個大的分支上。同時,我國全部的67株宋內(nèi)志賀菌處于都獨立在一個分支上,菌株內(nèi)部又有幾個明顯的小分支。推測我國的宋內(nèi)志賀菌可能是來源于國外,并且在國內(nèi)的進(jìn)化上有著自己獨立的特點。為了探究lac operon缺失對宋內(nèi)志賀菌的影響,本研究采用λ-RED同源重組方法,成功構(gòu)建了lac operon缺失的宋內(nèi)志賀菌。對敲除株各種生化表型進(jìn)行驗證的結(jié)果發(fā)現(xiàn),敲除株除ONPG表型變?yōu)殛幮酝膺�有6種表型發(fā)生變化,其中5種物質(zhì)的代謝加強(qiáng),分別是松二糖、龍膽二糖、D-鼠李糖、D-密二糖,D-絲氨酸。還有1種物質(zhì)代謝變?nèi)?為甲酸。對比不同溫度下敲除株與野生株的生長情況,結(jié)果顯示敲除株與野生株在不同溫度下生長無差異。死亡率比較分析的結(jié)果顯示,注射敲除株小鼠的死亡率沒有升高。以上結(jié)果說明,lac operon缺失是導(dǎo)致ONPG陰性宋內(nèi)志賀菌出現(xiàn)的原因,但本部分未能明確是何種優(yōu)勢導(dǎo)致ONPG陰性宋內(nèi)志賀菌出現(xiàn)和流行,這部分內(nèi)容需要之后更深入的研究。綜上所述,本研究開展了我國宋內(nèi)志賀菌特別是ONPG陰性菌的流行與分布規(guī)律研究;對不同地區(qū)宋內(nèi)志賀菌的抗生素耐藥性及耐藥機(jī)制進(jìn)行了分析;通過代表性菌株的全基因組測序,揭示ONPG陰性菌基因組的特點及其出現(xiàn)的分子機(jī)制。這些結(jié)果可以為宋內(nèi)志賀菌引起的細(xì)菌性痢疾的監(jiān)測、抗生素的合理使用、傳染源的追溯、傳播關(guān)系的調(diào)查以及防控策略的制訂提供科學(xué)依據(jù)。
[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.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R446.5

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