發(fā)布時(shí)間：2018-04-22 15:29

  本文選題：碳青霉烯酶 + 弗氏檸檬酸桿菌��； 參考：《吉林大學(xué)》2015年碩士論文

【摘要】：碳青霉烯類(lèi)抗生素是一類(lèi)抗菌譜廣、抗菌活性強(qiáng)的β-內(nèi)酰胺類(lèi)抗生素，對(duì)大多數(shù)革蘭氏陽(yáng)性菌、革蘭氏陰性菌和厭氧菌都有抗菌作用，且其活性不受β-內(nèi)酰胺酶的影響，對(duì)AmpC酶（AmpC-type enzymes）和超廣譜β-內(nèi)酰胺酶（extended-spectrum β-lactamases,ESBLs）高度穩(wěn)定。但近幾年，對(duì)碳青霉烯類(lèi)抗生素耐藥的菌株在全球范圍內(nèi)廣泛增多，尤其是革蘭氏陰性菌，且呈逐年增多形式。因此，日益嚴(yán)重的細(xì)菌耐藥性問(wèn)題引起了全球性的高度關(guān)注。碳青霉烯酶是一類(lèi)能水解碳青霉烯類(lèi)抗生素的β-內(nèi)酰胺酶，包括Ambler分類(lèi)中的A類(lèi)、B類(lèi)和D類(lèi)。A類(lèi)和D類(lèi)酶為絲氨酸酶，B類(lèi)酶為金屬酶。碳青霉烯酶被認(rèn)為是菌株對(duì)碳青霉烯酶類(lèi)抗生素產(chǎn)生耐藥性的最主要原因。 本研究的目的主要為探索分離自我國(guó)珠江醫(yī)院的多重耐藥弗氏檸檬酸桿菌112298耐藥的分子機(jī)制。共收集來(lái)自該醫(yī)院的70株耐碳青霉烯菌株，經(jīng)篩查選定同時(shí)表達(dá)碳青霉烯酶KPC和NDM的菌株112298進(jìn)行深入研究。112298分離自感染性休克患者的尿液標(biāo)本，通過(guò)16S rRNA鑒定112298的菌種，碳青霉烯酶基因和ESBLs基因篩查112298攜帶的基因，改良Carba NP法檢測(cè)碳青霉烯酶的類(lèi)型，通過(guò)最小抑菌濃度（minimum inhibitory concentration）測(cè)定菌株的抗生素敏感性，通過(guò)接合轉(zhuǎn)移實(shí)驗(yàn)驗(yàn)證攜帶碳青霉稀酶基因的質(zhì)粒是否有可轉(zhuǎn)移性。 結(jié)果顯示，弗氏檸檬酸桿菌112298同時(shí)產(chǎn)KPC型和NDM型碳青霉烯酶，blaKPC位于質(zhì)粒p112298-KPC（注冊(cè)號(hào)：KP987215），blaNDM位于質(zhì)粒p112298-NDM（注冊(cè)號(hào)：KP987216）。其中質(zhì)粒p112298-NDM為可接合型質(zhì)粒，通過(guò)接合轉(zhuǎn)移實(shí)驗(yàn)得到接合子112298-NDM-EC600，，而p112298-KPC不可通過(guò)接合轉(zhuǎn)移的方式進(jìn)行水平轉(zhuǎn)移。質(zhì)粒p112298-KPC和p112298-NDM都可通過(guò)電轉(zhuǎn)化的方式進(jìn)行水平轉(zhuǎn)移，得到轉(zhuǎn)化子112298-KPC-TOP10和112298-NDM-TOP10。112298攜帶有耐藥基因blaKPC、blaNDM、blaOXA-1group、blaCTX-M-9group和blaSHV，112298-NDM-EC600和112298-NDM-TOP10攜帶blaNDM和blaSHV，112298-KPC-TOP10攜帶blaKPC、blaOXA-1group和blaCTX-M-9group。改良Carba NP試驗(yàn)得出，112298產(chǎn)A類(lèi)和B類(lèi)碳青霉烯酶，112298-KPC-TOP10產(chǎn)A類(lèi)碳青霉烯酶，112298-NDM-EC600和112298-NDM-TOP10產(chǎn)B類(lèi)碳青霉烯酶。MIC值提示112298對(duì)青霉素類(lèi)、β-內(nèi)酰胺酶抑制劑、頭孢菌素類(lèi)、單酰胺菌素類(lèi)、碳青霉烯類(lèi)抗生素的耐藥性均可水平轉(zhuǎn)移，而對(duì)喹諾酮類(lèi)、呋喃類(lèi)抗生素的耐藥性則無(wú)法通過(guò)接合轉(zhuǎn)移或電轉(zhuǎn)化的方式轉(zhuǎn)移。對(duì)質(zhì)粒p112298-NDM和p112298-KPC的全序測(cè)定及分析得出，質(zhì)粒p112298-NDM為53.4kb的IncX3型質(zhì)粒，而p112298-KPC是大小為117kb的新型質(zhì)粒。質(zhì)粒p112298-KPC中攜帶blaKPC-2的Tn1721-based轉(zhuǎn)座子和其它抗性基因及可移動(dòng)元件共同組成一個(gè)多重耐藥（multidrug resistance，MDR）區(qū)。p112298-NDM與pNDM-HN380高度相似，其中blaNDM基因位于Tn125可變區(qū)， p112298-NDM中Tn125與其原型的不同之處在于上游的ISAba125被IS5替換及下游缺失1拷貝ISAba125. 弗氏檸檬酸桿菌112298同時(shí)產(chǎn)KPC-2型和NDM-1型碳青霉烯酶，使其對(duì)碳青霉烯類(lèi)抗生素的耐藥性增強(qiáng)。值得引起注意的是p112298-KPC除攜帶blaKPC-2外還有多種耐藥基因，包括氨基糖苷類(lèi)抗性基因、頭孢菌素類(lèi)抗性基因、氯霉素抗性基因、利福平抗性基因、季銨鹽類(lèi)抗性基因、磺胺類(lèi)抗性基因、喹諾酮抗性基因、氨芐西林抗性基因、鉻酸鹽抗性基因、磷霉素抗性基因等，且還有多個(gè)移動(dòng)元件，如插入序列、轉(zhuǎn)座子和1類(lèi)整合子。大量耐藥基因的共存勢(shì)必會(huì)加速菌株的進(jìn)化，使菌株快速獲得并保留不同的抗性基因，在不同的抗生素選擇壓力下，這將加速耐藥菌株的擴(kuò)散和持續(xù)，政府衛(wèi)生部門(mén)和醫(yī)務(wù)工作者應(yīng)加強(qiáng)監(jiān)控，以防微杜漸。
[Abstract]:Carbapenems, a class of antibiotics with broad antimicrobial spectrum and strong antibacterial activity, have antibacterial effects on most Gram-positive bacteria, Gram-negative and anaerobic bacteria, and their activity is not affected by beta lactamase, and AmpC enzyme (AmpC-type enzymes) and extended-spectrum beta lactamase (extended-spectrum beta -lactamase) S, ESBLs) are highly stable. However, in recent years, the strains resistant to carbapenems have increased worldwide, especially Gram-negative bacteria, and are increasing year by year. Therefore, the increasingly serious problem of bacterial resistance has attracted global attention. - lactamases, including class A, B and D classes in the Ambler classification. A and D enzymes are serine enzymes and B enzymes are metal enzymes. Carbapenem is considered to be the main cause of the strain's resistance to carbapenems.
The purpose of this study was to explore the molecular mechanism of multidrug-resistant citrate 112298 resistance in Zhujiang Hospital of China. A total of 70 strains of carbapenem resistant strains from the hospital were collected, and 112298 strains of carbapenem KPC and NDM were screened and selected to study.112298 isolated from patients with septic shock. The urine samples were identified by 16S rRNA, 112298 genes were screened by carbapenem gene and ESBLs gene, the Carba NP method was used to detect the type of carbapenem, and the antimicrobial susceptibility of the strain was measured by the minimum inhibitory concentration (minimum inhibitory concentration), and the carbon green was tested by the joint transfer experiment. Whether the plasmids of mycophenase gene are transferable.
The results showed that citric acid bacilli 112298 produced both KPC and NDM carbapenem, blaKPC was located in plasmid p112298-KPC (Registration Number: KP987215), blaNDM was located in plasmid p112298-NDM (Registration Number: KP987216). Plasmid p112298-NDM was a conjugable plasmid, and the zygote 112298-NDM-EC600 was obtained by grafting and transfer experiments, and p112298-KPC Horizontal transfer can not be carried out by conjugation. Both plasmids p112298-KPC and p112298-NDM can be transferred horizontally through electrical transformation. Transformants, 112298-KPC-TOP10 and 112298-NDM-TOP10.112298, carry resistant genes, blaKPC, blaNDM, blaOXA-1group, blaCTX-M-9group and blaSHV, 112298-NDM-EC600, and 112298-NDM-TOP10. Carrying blaNDM and blaSHV, 112298-KPC-TOP10 carrying blaKPC, blaOXA-1group and blaCTX-M-9group. improved Carba NP test, 112298 class A and B class carbapenenems, 112298-KPC-TOP10 producing carbapenenenase, 112298-NDM-EC600 and 112298-NDM-TOP10 producing carbapenenolase suggested 112298 against penicillins, beta lactamases The drug resistance of the agents, cephalosporins, monamamidins and carbapenems can be transferred horizontally, while the resistance to quinolones and furan antibiotics can not be transferred through joint transfer or electrical transformation. The full sequence determination and analysis of plasmid p112298-NDM and p112298-KPC shows that plasmid p112298-NDM is IncX3 of 53.4kb P112298-KPC is a new plasmid with the size of 117KB. The plasmid p112298-KPC carries the Tn1721-based transposon of blaKPC-2 and the other resistant genes and removable components together to make up a multidrug resistance (multidrug resistance, MDR) region.P112298-NDM and pNDM-HN380 highly similar, in which blaNDM gene is located in the Tn125 variable region. The difference between Tn125 and its prototype in NDM is that the upstream ISAba125 is replaced by IS5 and the downstream is missing 1 copies of ISAba125..
Citric acid bacilli 112298 produced both KPC-2 and NDM-1 carbapenem enzymes to enhance their resistance to carbapenems. It is worth noting that there are a variety of resistant genes, including aminoglycoside resistance genes, cephalosporins resistance genes, chloramphenicol resistance genes, rifampicin resistance, and the resistance of p112298-KPC to blaKPC-2. Sex gene, quaternary ammonium salt resistance gene, sulfonamide resistance gene, quinolone resistance gene, ampicillin resistance gene, chromate resistance gene, and fosfomycin resistance gene, as well as multiple mobile elements, such as insertion sequence, transposon and 1 integrons. Different resistance genes are retained and under the pressure of different antibiotic selection, this will accelerate the spread and persistence of the resistant strains, and the government health department and medical workers should strengthen monitoring to prevent the disease.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.61

【共引文獻(xiàn)】

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7 徐小紅;碳青霉烯類(lèi)耐藥腸桿菌科細(xì)菌耐藥機(jī)制及危險(xiǎn)因素分析[D];福建醫(yī)科大學(xué);2014年

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