彎曲菌抗生素敏感性及遺傳特征分析
發(fā)布時間：2019-05-20 04:18
【摘要】：彎曲菌主要導致食源性疾病,彎曲菌病的常見癥狀為腹瀉性腸炎,通常為自限性無需抗生素的治療,但對于免疫力低下、病程遷延的患者以及重癥患者而言,抗生素的治療是必要的。喹諾酮類和大環(huán)內酯類為臨床治療彎曲菌病的常用藥物,然而彎曲菌對于重要的臨床常用抗生素的耐藥性逐漸增加,尤其是對喹諾酮類的耐藥率已幾乎達到100%,嚴重影響了臨床治療的有效性,加重了疾病負擔。彎曲菌為人獸共患病原菌,其耐藥的產(chǎn)生和傳播機制相對復雜,畜禽養(yǎng)殖中抗生素作為飼料添加劑的使用以及臨床不合理用藥均會在一定程度上增加彎曲菌的耐藥性。為獲得我國菌株的耐藥特征及變化趨勢,本研究對實驗室收集保存的858株我國不同來源的空、結腸彎曲菌進行了抗生素敏感性分析,結果顯示我國菌株對于喹諾酮類為接近95%的高耐藥率。對空、結腸彎曲菌分別比較12種抗生素的耐藥率,發(fā)現(xiàn)結腸彎曲菌對紅霉素、鏈霉素等的耐藥率顯著高于空腸彎曲菌。對1994～2013年分離的空腸彎曲菌分四個時期的耐藥率結果表明,隨著時間的推進,我國空腸彎曲菌的整體耐藥率逐漸升高,耐藥趨勢嚴峻。本研究為我國耐藥彎曲菌的監(jiān)測及指導臨床用藥提供了數(shù)據(jù)支持。目前已經(jīng)從分子水平明確了彎曲菌對多種抗生素的耐藥機制,基因水平的研究對彎曲菌疾病的預防控制具有重要意義。目前已發(fā)現(xiàn)的大環(huán)內酯類耐藥機制有：靶向突變(23S rRNA及核糖體蛋白L4和L22)、CmeABC主動外排系統(tǒng)以及2013年最新報道的核糖體RNA甲基化酶基因ermB介導的水平轉移機制。臨床上除大環(huán)內酯類和喹諾酮類之外,在菌血癥及全身感染、重度感染中氨基糖苷類常作為備選藥物,因此對氨基糖苷類的耐藥機制研究也是必要的。本研究對158株紅霉素耐藥菌株進行大環(huán)內酯類耐藥相關23S rRNA點突變、核糖體RNA甲基化酶基因ermB篩查,82.91%(131/158)篩查到23S rRNA基因上A2075G位點突變,18.99%(30/158)篩查到ermB基因,30株ermB陽性菌株中9株已知ermB相關耐藥基因島的型別,3株已完成全基因組測序及耐藥島分析,8株分析進行中,10株測序進行中。ermB相關耐藥基因島及耐藥基因的出現(xiàn)表明彎曲菌可能是腸道中耐藥基因及基團傳播的儲存庫。菌株耐藥基因篩查結果顯示,23 S rRNA基因A2075G突變以及ermB基因的檢測可獲得我國近100%菌株的耐藥特征,據(jù)此可設計Real-time PCR對臨床病人糞便標本進行檢驗,在鑒定菌株的同時確定菌株的耐藥性,從而迅速有效的指導臨床用藥。對221株鏈霉素耐藥菌株進行了氨基糖苷類耐藥相關aadE-sat4-aphA-3耐藥基因簇的分布分析,陽性率為10.86%(24/221)。本研究為我國空、結腸彎曲菌耐藥機理的研究提供了理論依據(jù),同時為我國重要耐藥彎曲菌菌株的監(jiān)測提供依據(jù)�；蚍中途哂懈叻直媛�、重復性好、易于標準化及自動化等優(yōu)點,近年來分子生物學不斷發(fā)展,細菌的分型技術已不再局限于傳統(tǒng)的表型分型,PFGE、 MLST等分子分型技術廣泛應用于食源性病原微生物的暴發(fā)識別以及溯源分析等。彎曲菌的MLST分析技術具有高分辨率、較好的重復性、數(shù)據(jù)標準化、可與國際數(shù)據(jù)庫比較及共享且操作過程快速簡便等優(yōu)點,本研究的目的在于了解我國不同來源的耐藥彎曲菌有無顯著的克隆群,耐藥菌株間是否具有遺傳相關性以及家禽、家畜等儲存宿主來源的菌株有無與人感染菌株密切相關的特殊ST型別,因此使用該技術對158株不同來源紅霉素耐藥彎曲菌、100株鏈霉素耐藥彎曲菌進行分子分型,分型結果顯示,大部分腹瀉患者來源的結腸彎曲菌與雞來源的彎曲菌有著相似的基因型及耐藥性,耐藥菌株在彎曲菌中沒有形成顯著的克隆群分布。158株紅霉素耐藥彎曲菌中ST6322與ST1145均含有ermB基因,提示ermB基因可能與這兩種ST型別存在一定關聯(lián),需要增加菌株數(shù)量分析。本研究得到的結果將完善我國的彎曲菌監(jiān)測并對我國的食品安全有著重要意義。
[Abstract]:Campylobacter is the main cause of food-borne diseases. The common symptoms of Campylobacter are diarrhotic enteritis, usually self-care without the treatment of antibiotics, but the treatment of antibiotics is necessary for patients with low immunity, long course of disease and severe patients. The noonone and macrolides are commonly used drugs for clinical treatment of the bending diseases, however, the resistance of the bending bacteria to the important clinical common antibiotics is gradually increased, especially the drug resistance rate of the noonone is almost 100%, and the effectiveness of the clinical treatment is seriously affected, And the burden of the disease is increased. Campylobacter is a common pathogen of human, and its drug resistance is relatively complex. The use of antibiotics as feed additive in livestock and poultry culture and the irrational use of antibiotics will increase the drug resistance of the Campylobacter to some extent. In order to obtain the drug-resistant character and the changing trend of the strains in China, this study conducted the antibiotic sensitivity analysis of 858 strains of the Chinese strain which were stored in the laboratory, and the results showed that the strains of the Chinese strains were close to 95% of the high-resistance rate. The resistance rate of 12 kinds of antibiotics was compared with that of Campylobacter jejuni, and the resistance rate of Campylobacter coli to erythromycin and streptomycin was significantly higher than that of Campylobacter jejuni. The drug resistance rate of Campylobacter jejuni isolated from 1994 to 2013 showed that the resistance rate of Campylobacter jejuni in China increased with time, and the drug resistance trend was serious. This study provides data support for monitoring and guiding the clinical application of drug-resistant bending bacteria in China. At present, the resistance mechanism and gene level of Campylobacter jejuni have been identified from the molecular level, which is of great significance to the prevention and control of Campylobacter disease. The macrolide-resistant mechanisms that have been found are: targeted mutations (23S rRNA and ribosomal proteins L4 and L22), CmeC active efflux systems, and the latest reported ribosomal RNA methylase gene erB-mediated level transfer mechanism in 2013. In addition to macrolides and other types of macrolides, in the case of bacteremia and systemic infection, the amino-glycosides are often used as alternative drugs in severe infections, and therefore, the study of the drug-resistant mechanism of the amino-glycosides is also necessary. In this study, the 23S rRNA point mutation, the ribosomal RNA methylase gene erB screen and the 82.91% (131/158) of the 158 erythromycin resistant strains were screened to the A2075G site mutation of the 23S rRNA gene. 18.99% (30/158) were screened to the mB gene,9 of the 30 mB positive strains were known to be the type of the mB-related drug-resistant gene island,3 of the 3 strains had completed the whole genome sequencing and the drug-resistant island analysis, and the 8 strains were analyzed and the 10 strains were sequenced. The emergence of mB-associated drug-resistant gene and drug-resistant genes indicates that the Campylobacter may be a repository of drug-resistant genes and groups in the gut. The screening results of the drug-resistant gene of the strain show that the 23S rRNA gene A2075G mutation and the detection of the mB gene can obtain the drug resistance characteristic of the nearly 100% strain in China, so that the real-time PCR can be designed to test the stool sample of the clinical patient, and the drug resistance of the strain can be determined at the same time of the identification strain, So as to rapidly and effectively guide the clinical medication. The distribution of resistance-related aadE-sat4-apA-3-resistant gene cluster of 221 streptomycin-resistant strains was analyzed, and the positive rate was 10.86% (24/221). This study provides a theoretical basis for the study of the mechanism of drug resistance in the air and colon of our country, and provides the basis for the monitoring of the strain of the major drug-resistant strains in China. The gene typing has the advantages of high resolution, good repeatability, easy standardization and automation, and the like, MLST and other molecular typing techniques are widely used in the outbreak identification and trace analysis of food-borne pathogenic microorganisms. the MLST analysis technology of the bending bacteria has the advantages of high resolution, good repeatability, standardization of data, comparison and sharing with an international database, rapid and convenient operation, and the like, and the purpose of the research is to understand the presence or absence of a significant clone group of drug-resistant bending bacteria of different sources in China, if there is a genetic correlation between the resistant strains and the presence or absence of a special ST-type strain which is closely related to the human infection strain, The results showed that most of the diarrhea patients had similar genotype and drug resistance to the Campylobacter from the source of the chicken, and the drug-resistant strains did not form a significant colony distribution in the Campylobacter, and both ST6322 and ST1145 in the 158 strains of erythromycin-resistant strains contain the mB gene. It is suggested that the mB gene may be associated with the two ST types, and it is necessary to increase the number of the strains. The results obtained in this study will improve the monitoring of Campylobacter in China and have important significance for food safety in China.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5
本文編號：2481345
[Abstract]:Campylobacter is the main cause of food-borne diseases. The common symptoms of Campylobacter are diarrhotic enteritis, usually self-care without the treatment of antibiotics, but the treatment of antibiotics is necessary for patients with low immunity, long course of disease and severe patients. The noonone and macrolides are commonly used drugs for clinical treatment of the bending diseases, however, the resistance of the bending bacteria to the important clinical common antibiotics is gradually increased, especially the drug resistance rate of the noonone is almost 100%, and the effectiveness of the clinical treatment is seriously affected, And the burden of the disease is increased. Campylobacter is a common pathogen of human, and its drug resistance is relatively complex. The use of antibiotics as feed additive in livestock and poultry culture and the irrational use of antibiotics will increase the drug resistance of the Campylobacter to some extent. In order to obtain the drug-resistant character and the changing trend of the strains in China, this study conducted the antibiotic sensitivity analysis of 858 strains of the Chinese strain which were stored in the laboratory, and the results showed that the strains of the Chinese strains were close to 95% of the high-resistance rate. The resistance rate of 12 kinds of antibiotics was compared with that of Campylobacter jejuni, and the resistance rate of Campylobacter coli to erythromycin and streptomycin was significantly higher than that of Campylobacter jejuni. The drug resistance rate of Campylobacter jejuni isolated from 1994 to 2013 showed that the resistance rate of Campylobacter jejuni in China increased with time, and the drug resistance trend was serious. This study provides data support for monitoring and guiding the clinical application of drug-resistant bending bacteria in China. At present, the resistance mechanism and gene level of Campylobacter jejuni have been identified from the molecular level, which is of great significance to the prevention and control of Campylobacter disease. The macrolide-resistant mechanisms that have been found are: targeted mutations (23S rRNA and ribosomal proteins L4 and L22), CmeC active efflux systems, and the latest reported ribosomal RNA methylase gene erB-mediated level transfer mechanism in 2013. In addition to macrolides and other types of macrolides, in the case of bacteremia and systemic infection, the amino-glycosides are often used as alternative drugs in severe infections, and therefore, the study of the drug-resistant mechanism of the amino-glycosides is also necessary. In this study, the 23S rRNA point mutation, the ribosomal RNA methylase gene erB screen and the 82.91% (131/158) of the 158 erythromycin resistant strains were screened to the A2075G site mutation of the 23S rRNA gene. 18.99% (30/158) were screened to the mB gene,9 of the 30 mB positive strains were known to be the type of the mB-related drug-resistant gene island,3 of the 3 strains had completed the whole genome sequencing and the drug-resistant island analysis, and the 8 strains were analyzed and the 10 strains were sequenced. The emergence of mB-associated drug-resistant gene and drug-resistant genes indicates that the Campylobacter may be a repository of drug-resistant genes and groups in the gut. The screening results of the drug-resistant gene of the strain show that the 23S rRNA gene A2075G mutation and the detection of the mB gene can obtain the drug resistance characteristic of the nearly 100% strain in China, so that the real-time PCR can be designed to test the stool sample of the clinical patient, and the drug resistance of the strain can be determined at the same time of the identification strain, So as to rapidly and effectively guide the clinical medication. The distribution of resistance-related aadE-sat4-apA-3-resistant gene cluster of 221 streptomycin-resistant strains was analyzed, and the positive rate was 10.86% (24/221). This study provides a theoretical basis for the study of the mechanism of drug resistance in the air and colon of our country, and provides the basis for the monitoring of the strain of the major drug-resistant strains in China. The gene typing has the advantages of high resolution, good repeatability, easy standardization and automation, and the like, MLST and other molecular typing techniques are widely used in the outbreak identification and trace analysis of food-borne pathogenic microorganisms. the MLST analysis technology of the bending bacteria has the advantages of high resolution, good repeatability, standardization of data, comparison and sharing with an international database, rapid and convenient operation, and the like, and the purpose of the research is to understand the presence or absence of a significant clone group of drug-resistant bending bacteria of different sources in China, if there is a genetic correlation between the resistant strains and the presence or absence of a special ST-type strain which is closely related to the human infection strain, The results showed that most of the diarrhea patients had similar genotype and drug resistance to the Campylobacter from the source of the chicken, and the drug-resistant strains did not form a significant colony distribution in the Campylobacter, and both ST6322 and ST1145 in the 158 strains of erythromycin-resistant strains contain the mB gene. It is suggested that the mB gene may be associated with the two ST types, and it is necessary to increase the number of the strains. The results obtained in this study will improve the monitoring of Campylobacter in China and have important significance for food safety in China.
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5
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