【摘要】：研究背景隨著各種抗生素的大量使用,多重耐藥甚至泛耐藥細菌不斷增多,給臨床診治帶來巨大的挑戰(zhàn),現(xiàn)已成為一個世界性難題。碳青霉烯類抗生素是治療多重耐藥革蘭陰性桿菌嚴重感染的最后一道防線,耐碳青霉烯革蘭陰性桿菌的快速傳播越來越引起關注。產(chǎn)碳青霉烯酶是這類細菌對碳青霉烯類抗生素耐藥的主要原因,碳青霉烯酶包括Ambler分子分類中的A、B、D三類酶,是指所有能明顯水解亞胺培南或美羅培南等碳青霉烯類抗生素的一類β-內酰胺酶。大多數(shù)碳青霉烯酶基因位于可轉移性基因元件上如質粒、整合子等,在同種屬和不同種屬的細菌可以通過接合、傳導、轉化、轉座等方式獲得耐藥基因,導致更多細菌產(chǎn)碳青霉烯酶。耐碳青霉烯革蘭陰性桿菌不僅對β-內酰胺類抗生素耐藥,同時多數(shù)也對喹諾酮、氨基糖苷等其他類抗生素耐藥,從而表現(xiàn)出多耐藥甚至泛耐藥的特性。因此,使用快速方法檢測細菌是否產(chǎn)生碳青霉烯酶,對控制這類細菌的傳播和感染具有重要意義。目前產(chǎn)碳青霉烯酶的檢測方法主要有改良的Hodge實驗(the modified Hodge test)這是一個表型實驗,這是美國臨床實驗室標準化協(xié)會(CLSI)推薦的表型確證試驗。另外還有亞胺培南-EDTA雙紙片協(xié)同試驗及Etest MBL商品化確認試條,專門針對B組碳青霉烯酶的檢驗[1]。但是,隨著耐藥菌株所含耐藥基因的組合和種類越趨復雜,上述方法的特異性和敏感性受到挑戰(zhàn),產(chǎn)碳青霉烯酶的檢測方法亟待革新。美國臨床與實驗室標準化協(xié)會(Clinical and Laboratory Standards Institute,CLSI)于2015年引入了Carba NP試驗[2],作為腸桿菌科、銅綠假單胞菌和不動菌屬細菌產(chǎn)生的碳青霉烯酶表型檢測的確證試驗,由法國南法醫(yī)學院Patrice Nordmann教授研究團隊于2012年首次報道[3],此后,陸續(xù)優(yōu)化該方法用于檢測產(chǎn)碳青霉烯酶假單胞菌及指示碳青霉烯酶類型的Carba NP試驗Ⅱ已有報道[4],該試驗能直接判定產(chǎn)碳青霉烯酶菌株的產(chǎn)酶類型,較改良的Hodge試驗更簡單、快速。隨著分子生物學和生物信息分析學的快速發(fā)展,使得全基因組測序技術得到廣泛的應用。全基因組測序是對物種未知基因組全序列進行測序。目前,全基因組測序技術應用比較多的主要是第二代測序技術(NGS)和第三代測序技術。第二代測序技術能夠快速、低成本的進行全基因組測序,其設備供應商主要是454(羅氏公司),SOLi D(AB公司)和Solexa。第三代測序技術于2011年開始推廣,其單分子實時測序技術(SMRT)與第二代測序完全不同,由Pacific Biosciences公司研發(fā),它的序列讀長高達3kb。高通量測序技術的出現(xiàn)使得人類獲得基因組方式出現(xiàn)重大變革,特別是在基因組信息較小的微生物領域,已成為病原微生物的鑒定分型、致病機理研究等一項不可取代的工具。本研究采用CLSI推薦的Carba NP試驗,對本院可疑產(chǎn)碳青霉烯酶多重耐藥革蘭陰性桿菌進行篩選,以多重PCR方法對篩選陽性的結果進行耐藥基因的檢測,以了解我院革蘭陰性桿菌產(chǎn)碳青霉烯酶流行情況,并探討Carba NP試驗檢測方法的優(yōu)劣。對來源同一病人的4株產(chǎn)碳青霉烯酶肺炎克雷伯菌進行接合/轉化試驗,比較接合/轉化菌和親本菌株的藥物敏感性差異,并分析4株菌的同源性。通過高通量測序技術對其中1株攜帶bla KPC-2的多重耐藥菌株作全基因組測序,通過生物信息學分析,了解肺炎克雷伯菌的耐藥機制和耐藥基因環(huán)境等特點。研究目的1.了解廣州醫(yī)科大學附屬第一醫(yī)院臨床分離產(chǎn)碳青霉烯酶多重耐藥革蘭陰性桿菌的流行及耐藥分子機制。2.了解Carba NP試驗檢測革蘭陰性桿菌產(chǎn)碳青霉烯酶的效果。3.來源同一病人4株肺炎克雷伯菌株耐藥性及同源性分析。4.用高通量測序方法分析1株KPC陽性肺炎克雷伯菌株的基因環(huán)境。研究方法1.收集廣州醫(yī)科大學附屬第一醫(yī)院微生物室2010年1月-2013年10月臨床分離的59株多重耐藥的革蘭陰性桿菌,所有菌株用VITEK2微生物全自動鑒定儀進行鑒定和藥敏實驗,篩選對三種以上抗菌藥物耐藥的革蘭陰性桿菌菌株,且對至少一種碳青霉烯類抗生素耐藥,即為可疑產(chǎn)碳青霉烯酶菌株,并對標本類型、碳青霉烯類藥物敏感性等資料進行統(tǒng)計,分析其流行病學特點。2.采用Carba NP試驗檢測產(chǎn)碳青霉烯酶的菌株,并用多重PCR技術對碳青霉烯酶表型試驗陽性菌株進行相關耐藥基因檢測,了解本院革蘭陰性桿菌碳青霉烯酶的主要基因型,并探討Carba NP試驗檢測革蘭陰性桿菌碳青霉烯酶的效果。3.通過接合試驗和電轉化試驗獲得4株肺炎克雷伯菌接合子或轉化子,受體菌攜帶耐藥基因bla NDM-1或bla KPC-2,提取質粒DNA,通過酶切技術分析質粒類型。4.分別提取多重耐藥菌株LJ1、LJ2、LJ3、LJ4的總DNA,ERIC-PCR方法分析同源性和質粒不相容群,通過PCR方法檢測7個肺炎克雷伯菌管家基因,利用MLST網(wǎng)上工具進行ST分型,進一步分析這些菌株的同源性。5.提取轉化子LJ4C總DNA,用Illumina Miseq和Pac Bio RS II平臺進行高通量測序。由Celera Assembler軟件進行組裝。在其PBc R pipeline中,用Illumina Mi Seq Reads(二代測序)的數(shù)據(jù)進行糾錯。RAST網(wǎng)上工具進行基因注釋,Res Finder和NCBI BLAST網(wǎng)上工具進行耐藥基因分析,NCBI BLAST網(wǎng)上工具進行耐藥基因遺傳環(huán)境分析,Plasmid Finder網(wǎng)上工具進行質粒序列分析。研究結果1.59株多重耐藥的革蘭陰性桿菌的流行病學和藥敏結果分析。59株多重耐藥革蘭陰性桿菌對亞胺培南、美洛培南、厄他培南耐藥率分別為62.71%,61.02%,64.41%。菌株主要來源于痰液35.6%(21/59)膽汁5.1%(3/59)、分泌物8.5%(5/59)、腹水5.1%((3/59)、尿液13.6%(8/59)、胸水3.4%(2/59)、血液23.7%(14/59)、引流液5.1%(3/59)。多重耐藥革蘭陰性桿菌中以攜帶NDM-1基因和攜帶KPC-2基因型菌株為主,而產(chǎn)生碳青霉烯酶的細菌以弗氏檸檬酸桿菌、銅綠假單胞菌和肺炎克雷伯菌等比較常見。2.Carba NP試驗和多重PCR檢測結果分析比較。Carba NP試驗確定33株產(chǎn)碳青霉烯酶菌株,分別為A類酶12株、B類酶21株,PCR法檢出多種碳青霉烯酶,包括KPC(12株)、IMP(7株)、NDM(12株)、VIM(3株)。與PCR法比較,Carba NP試驗敏感性和特異性分別為97.06%和100%。3.3株肺炎克雷伯菌LJ1-LJ3攜帶耐藥基因bla NDM-1,這些菌株可通過接合方式傳遞耐藥基因,攜帶耐藥基因bla KPC-2菌株LJ4接合不成功,需用電轉化獲得轉化子。S1核酸內切酶分析4株菌有三種質粒類型。4.用ERIC-PCR方法分析LJ1、LJ2、LJ3、LJ4菌株同源性,LJ1和LJ2高度同源,與LJ3、LJ4菌有不同的帶型。LJ3、LJ4不屬于18個已發(fā)現(xiàn)質粒不相容群的任何一種,LJ1、LJ2屬于不相容群Inc F II群。通過7個肺炎克雷伯菌管家基因檢測,MLST網(wǎng)上工具進行分析,在LJ1和LJ2為新發(fā)現(xiàn)ST型,均為ST1416,LJ3為ST20,LJ4為ST11。5.PCR法證實多重耐藥產(chǎn)碳青霉烯酶菌株LJ4攜帶bla KPC-2型碳青霉烯酶基因,對其進行高通量測序,獲得來自LJ4菌株的質粒p CT-KPC,是一個151466bp的環(huán)狀分子,包括120783bp的質粒骨架,有編碼,復制起始,轉移,維持和穩(wěn)定功能的編碼區(qū)以及包含兩段耐藥基因的編碼區(qū)(MRR)。p CT-KPC質粒含有53.81%的GC含量,總共有233個已鑒定的開放閱讀框ORFs,138個編碼基因與已知有功能的蛋白質有高度相似性。質粒LJ4C是由p HN7A8、p KPC-LK30的部分質粒結構及一個額外的tra區(qū)域構成一個嵌合體,接合轉移基因簇tra與細菌接合轉移相關。6.對質粒結構研究發(fā)現(xiàn),LJ4菌株的轉化子同時攜帶bla CTX-M-65、blafos A3、blarmt B、bla SHV、bla TEM1b及bla KPC-2多個耐藥基因。blafos A基因在家禽和寵物之間廣泛傳播,本研究結果顯示在病人身上分離的病原菌株質粒跟寵物來源的菌株質粒結構高度同源,不排除在病患寵物及家禽與人類來源細菌發(fā)生同源重組的可能。結論1.我院產(chǎn)生碳青霉烯酶的細菌以弗氏檸檬酸桿菌、銅綠假單胞菌和肺炎克雷伯菌等比較常見,提示臨床上如果檢出了此類多重耐藥菌株,應注意篩查其產(chǎn)生碳青霉烯酶的狀況,以便合理應用抗菌藥物。2.Carba NP試驗用于多重耐藥革蘭陰性桿菌產(chǎn)碳青霉烯酶的篩查簡單、易行、快速、準確,可在臨床實驗室中推廣使用。3.已出現(xiàn)同時攜帶ESBLs、fos A、rmt B、KPC基因質粒的菌株,質粒攜帶的bla NDM-1和bla KPC-2基因可通過接合或轉化的方式在同種細菌間傳播,預示這些多重耐藥基因在細菌間有水平播散的可能。4.高通量測序可快速、全面、準確地對耐藥基因、基因環(huán)境和細菌分型等開展詳細的分析,便于完成細菌耐藥機制的研究。
[Abstract]:Background with the extensive use of various antibiotics, multidrug-resistant and even pan resistant bacteria are increasing, which poses a great challenge to clinical diagnosis and treatment. It has become a worldwide problem. Carbapenems are the last line of defense for the severe infection of multiresistant gram-negative bacilli, carbapenem resistant gram-negative bacilli Carbon penicenase is the main cause of resistance to carbapenems. Carbapenenems include A, B, and D three enzymes in the classification of Ambler molecules, which are a class of beta lactamases, which can obviously hydrolyze imipenem or meropenem and other carbapenems. Most carbon green Mycophenase genes are located in transferable gene elements such as plasmids and integrons. Bacteria in the same genus and different species can obtain resistance genes through conjugation, conduction, transformation, transposing and so on, causing more bacteria to produce carbapenem. The carbapenems resistant bacteria are resistant to beta lactam antibiotics and most of them are also resistant to beta lactam antibiotics. The resistance to quinolone, aminoglycoside and other antibiotics, thus showing the characteristics of multidrug-resistant and even pan resistance. Therefore, the rapid detection of carbapenems by bacteria is of great significance in controlling the spread and infection of these bacteria. The main methods for the detection of carbapenems are the modified Hodge test (the m). Odified Hodge test) this is a phenotypic experiment. This is the phenotypic confirmatory test recommended by the American clinical laboratory standardization association (CLSI). There are also the imipenem -EDTA double paper synergistic test and the Etest MBL commercialization test, specifically for the test of carbopenicolenes in the B Group [1]. but with a group of resistant genes containing the resistant strain. The more complex and more complex, the specificity and sensitivity of the methods are challenged, and the detection methods for carbapenems are urgently needed to be innovated. The Clinical and Laboratory Standards Institute (CLSI) introduced the Carba NP test [2] in 2015 as the Enterobacteriaceae, Pseudomonas aeruginosa and the genus acomonas. The confirmatory test of the phenotypic phenotypic detection of carbapenenenes produced by bacteria was first reported in 2012 by the research team of Professor Patrice Nordmann of the French Academy of Southern forensic medicine (Prof Patrice Nordmann). Since then, the Carba NP test for the detection of carbapenem producing Pseudomonas sp. and the Carba NP test indicating the type of carbapenenenes has been reported. The test can be directly judged. The type of enzyme producing enzyme producing carbapenenenase is more simple and faster than the modified Hodge test. With the rapid development of molecular biology and bioinformatics, the whole genome sequencing technology is widely used. Whole genome sequencing is the whole sequence of unknown species of species. With more than second generation sequencing technology (NGS) and third generation sequencing technology, second generation sequencing technology can be quickly and low cost for whole genome sequencing. Its equipment suppliers are mainly 454 (Roche), SOLi D (AB) and Solexa. third generation sequencing technology began to be popularized in 2011, and their single molecule real time sequencing technology (SMRT) Different from the second generation sequencing, it was developed by Pacific Biosciences, and its sequence read long up to 3kb. high throughput sequencing technology has made human genome form a major change, especially in the field of microorganism with small genome information, which has become an identification of pathogenic microorganism, a study of pathogenic mechanism and so on. In this study, the Carba NP test recommended by CLSI was used to screen the multidrug-resistant gram-negative bacilli of suspected carbapenem production in our hospital. The multiple PCR method was used to detect the resistance genes of the screening positive results, so as to understand the prevalence of carbapenem production in Gram-negative bacilli in our hospital and to explore the Carba NP test. 4 strains of Klebsiella pneumoniae producing carbon penicylenzyme from the same patient were conjugation / conversion tests, and the differences in drug sensitivity of conjugation / transformed bacteria and parent strains were compared and the homology of 4 strains was analyzed. 1 strains of multiple resistant strains carrying bla KPC-2 were sequenced by high throughput sequencing. Through bioinformatics analysis, the drug resistance mechanism and drug resistance gene environment of Klebsiella pneumoniae were investigated. Objective 1. to understand the prevalence and molecular mechanism of multi resistant gram-negative bacilli of carbapenem production in the First Affiliated Hospital of Guangzhou Medical University and to understand the carbon production of gram-negative bacilli by Carba NP test. The effect of penicilenes on the resistance and homology of 4 strains of klebber strains from the same patient.3..4. high flux sequencing method was used to analyze the genetic environment of 1 strains of KPC positive klebber strain. Method 1. collected 59 strains of multiple tolerance of clinical isolation in the first hospital of Guangzhou Medical University, the first hospital microorganism room, October January 2010. Gram-negative bacilli of the drug, all strains were identified by VITEK2 microorganism automatic identification instrument and drug sensitivity test, screening the gram-negative bacilli resistant to more than three kinds of antimicrobial agents, and resistant to at least one carbapenene antibiotic, that is, suspected carbapenems, and the type of carbapenems, and the sensitivity of carbapenems. The epidemiological characteristics of.2. were analyzed by Carba NP test for the detection of carbapenem producing strains, and multiple PCR techniques were used to detect the related resistance genes of the positive strains of carbapenem test, to understand the main genotypes of carbapenems of gram-negative bacilli in our hospital and to explore the test of Carba NP test. The effect of gram-negative bacilli carbapenem.3. obtained 4 strains of Klebsiella pneumoniae zygote or transformant through conjugation test and electrical transformation test. The receptor bacteria carried the resistance gene bla NDM-1 or bla KPC-2 to extract plasmid DNA. The multiple resistant strain LJ1, LJ2, LJ3, LJ4 DNA were extracted by the enzyme cutting technique and analyzed the plasmid type.4. respectively. Methods the homologous and plasmid incompatible groups were analyzed, and 7 Klebsiella pneumoniae housekeeping genes were detected by PCR method. The ST typing was carried out by MLST online tools. The homologous.5. extracted transformants LJ4C total DNA was further analyzed. Illumina Miseq and Pac Bio RS II platform were used for high flux sequencing. In its PBc R pipeline, using the data of Illumina Mi Seq Reads (two generation sequencing) to carry on the error correction.RAST online tools to carry on the gene annotation, Res Finder and NCBI BLAST network tools to carry on the resistance gene analysis. Results the epidemiological and drug susceptibility results of 1.59 strains of gram-negative bacilli were analyzed. The resistance rates of.59 strains to imipenem, meropenem, and eopenem were 62.71% and 61.02% respectively. The strains of 64.41%. were mainly from 35.6% (21/59) bile 5.1% (3/59), 8.5% (5/59), ascites 5.1% (3/59), and 1 in urine. 3.6% (8/59), hydrothorax 3.4% (2/59), blood 23.7% (14/59), and drainage 5.1% (3/59). Multidrug-resistant gram-negative bacilli were mainly carried with NDM-1 gene and KPC-2 genotype strains, and carbapenem producing bacteria with citrate, Pseudomonas aeruginosa and Klebsiella pneumoniae were more common in.2.Carba NP test and multiple PCR. Analysis and comparison of the results of.Carba NP test to determine 33 strains of carbapenenenase producing carbapenenenase, respectively a class a enzyme 12, 21 B enzymes, and PCR method to detect a variety of carbapenems, including KPC (12 strains), IMP (7 strains), NDM (12 strains), VIM (3). The sensitivity and specificity of Carba NP test are 97.06% and 100%.3.3 strains Klebsiella pneumoniae respectively. Carrying resistance gene bla NDM-1, these strains can transfer drug-resistant genes through conjugation, carrying drug resistant gene bla KPC-2 strain LJ4 unsuccessfully, and use electrical transformation to obtain transformant.S1 nucleic acid endonuclease analysis of 4 strains of plasmid type.4. with ERIC-PCR method to analyze LJ1, LJ2, LJ3, LJ4 strain homology LJ4 bacteria have different band type.LJ3, LJ4 does not belong to any one of the 18 found plasmid incompatible groups, LJ1 and LJ2 belong to the incompatible group Inc F II group. The carbapenem strain LJ4 carries the BLA KPC-2 type carbapenem gene, which is sequenced high flux and obtained the plasmid P CT-KPC from the LJ4 strain. It is a 151466bp ring molecule, including the plasmid skeleton of 120783bp, which encodes, replicating, transferring, maintaining and stabilizing the coding region, as well as the coding region containing two segment resistant genes. (MRR) the.P CT-KPC plasmid contains 53.81% GC content, a total of 233 identified open reading frame ORFs, and 138 encoding genes are highly similar to the known functional proteins. Plasmid LJ4C is constructed from a partial plasmid structure of P HN7A8, P KPC-LK30 and an additional tra region to form a chimerism, joining the transfer gene cluster tra and bacterial connection. The plasmid structure study found that the transformants of LJ4 strain also carried bla CTX-M-65, blafos A3, blarmt B, BLA SHV, BLA TEM1b and multiple resistant genes to spread widely between poultry and pets. The results of this study showed that the plasmid isolated from the patient was with the strain of the pet source. The plasmid structure is highly homologous, which does not exclude the possibility of homologous recombination between the sick pets and the poultry and the human source bacteria. Conclusion 1. the carbapenem bacteria producing carbapenem in our hospital are more common, such as citric acid bacilli Freund, Pseudomonas aeruginosa and Klebsiella pneumoniae. It is suggested that the multiple drug resistant strains have been detected in the clinic and should be screened out. To find out the status of carbapenem production in order to apply the.2.Carba NP test to the screening of carbapenems of multi resistant gram-negative bacilli, it is easy, fast, accurate, and can be used in clinical laboratory to promote the use of.3. with ESBLs, Fos A, RMT B, KPC plasmid and Bla ND. M-1 and Bla KPC-2 genes can be transmitted in the same type of bacteria by conjugation or transformation. It indicates that the possible.4. high throughput sequencing of these multidrug resistant genes in bacteria is rapid, comprehensive and accurate in the analysis of drug resistance genes, genetic environment and bacterial genotyping, so as to facilitate the study of the mechanism of bacterial resistance.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R446.5

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