本文選題：肺炎克雷伯菌 + KPC型碳青霉烯酶�。� 參考：《浙江大學(xué)》2015年博士論文

【摘要】：KPC (Klebsiella pneumoniae carbapenemase)型碳青霉烯酶首次于2001年被報(bào)道,主要分布于肺炎克雷伯菌中。近年來(lái),產(chǎn)KPC肺炎克雷伯菌(KPC-KP)菌株在全球多國(guó)家地區(qū)廣泛流行,嚴(yán)重威脅人類健康。KPC-KP對(duì)幾乎所有p-內(nèi)酰胺類以及多種非p-內(nèi)酰胺類抗菌藥物耐藥,使臨床抗菌藥物選擇十分受限。近年來(lái),磷霉素因?qū)Χ嘀啬退幘臧↘PC-KP菌株仍有較好的抗菌活性而再度受到重視。我們前期研究發(fā)現(xiàn)浙醫(yī)一院KPC-KP菌株磷霉素敏感率為43.4%,其中rmtB(質(zhì)粒攜帶氨基糖甙類耐藥基因,介導(dǎo)氨基糖甙類高水平耐藥)陽(yáng)性菌株僅為8.5%,然而他們對(duì)磷霉素的耐藥機(jī)制尚不明確。 本研究第一部分對(duì)浙醫(yī)一院2010.1-2013.2間收集的97株KPC-KP菌株,通過(guò)瓊脂稀釋法測(cè)定其對(duì)磷霉素及其他抗菌藥物的MICs值,PCR和基因測(cè)序檢測(cè)耐藥基因攜帶情況,脈沖場(chǎng)凝膠電泳(PFGE)和多位點(diǎn)序列分型(MLST)方法分析其克隆相關(guān)性,并選取代表菌株行轉(zhuǎn)化接合試驗(yàn)分析磷霉素滅活酶基因fosA3定位及其攜帶質(zhì)粒在菌株間傳播情況。最后fos基因陰性菌株通過(guò)PCR及測(cè)序檢測(cè)其磷霉素靶酶基因murA和轉(zhuǎn)運(yùn)系統(tǒng)相關(guān)基因glpA、uhpT、uhpA、ptsI、cyaA是否存在突變；同時(shí)通過(guò)碳源生長(zhǎng)實(shí)驗(yàn)檢測(cè)轉(zhuǎn)運(yùn)系統(tǒng)的功能。 97株KPC-KP菌株中,共發(fā)現(xiàn)磷霉素耐藥菌株57株(58.74%),其中44株為fosA3陽(yáng)性,一株fosA陽(yáng)性。其余12株不攜帶本研究中篩查的fos基因。這些菌株對(duì)所測(cè)抗菌藥物包括碳青霉烯類、頭孢菌素類、氨基糖甙類、喹諾酮類和四環(huán)素類皆呈高比例耐藥,僅對(duì)多粘菌素E和替加環(huán)素表現(xiàn)出較高的敏感率,分別為100%和94.7%。57株磷霉素耐藥菌株中50株攜帶rmtB基因,可以解釋這些菌株對(duì)氨基糖甙類抗菌藥物的高水平耐藥。MLST共發(fā)現(xiàn)兩個(gè)序列型(STs) ST11和ST494,前者包含56株,后者僅包含一株foS基因陰性菌株。PFGE共發(fā)現(xiàn)5個(gè)脈沖類型(PTS)。44株fosA3陽(yáng)性菌株皆屬于ST11-PTA,說(shuō)明浙醫(yī)一院fosA3陽(yáng)性菌株為單克隆傳播。而fos陰性菌株基因背景相對(duì)復(fù)雜,包括ST11-PTA(6株)、ST11-PTB(4株)、ST11-PTC(1株)和ST494-PTD(1株)afosA陽(yáng)性菌株屬于ST11-PTE.12株fos基因陰性菌株,靶酶基因未發(fā)現(xiàn)突變,且皆可在G-6-P為唯一碳源的M9培養(yǎng)基上生長(zhǎng),說(shuō)明其磷酸己糖轉(zhuǎn)運(yùn)系統(tǒng)(UhpT)功能正常。4株磷霉素耐藥菌株亦能在以G-3-P為唯一碳源的M9培養(yǎng)基上生長(zhǎng),說(shuō)明其甘油-3-磷酸轉(zhuǎn)運(yùn)系統(tǒng)(G1pT)功能正常,其耐藥機(jī)制需進(jìn)一步研究。8株不能在以G-3一P為唯一碳源的M9培養(yǎng)基上生長(zhǎng),說(shuō)明其G1pT系統(tǒng)存在功能異常,GlpT基因分析發(fā)現(xiàn)其中3株存在glpT基因及氨基酸序列改變,包含2株發(fā)生DNA的A477A突變,一株發(fā)生氨基酸Cys309Phe突變。其余5株未發(fā)現(xiàn)uhpT’基因序列改變,提示UhpT功能障礙可能與UhpT轉(zhuǎn)錄或表達(dá)水平下降有關(guān)。轉(zhuǎn)運(yùn)系統(tǒng)相關(guān)調(diào)節(jié)基因uhpA和cyaA未發(fā)現(xiàn)基因突變。而ptsl在12株磷霉素耐藥菌株中普遍存在氨基酸改變Ile569Asn,結(jié)合碳源生長(zhǎng)實(shí)驗(yàn)結(jié)果認(rèn)為該突變?yōu)闊o(wú)義突變不影響ptsl編碼蛋白功能。代表菌株KP1034磷霉素抗性不能通過(guò)接合實(shí)驗(yàn)傳遞到受體菌。其轉(zhuǎn)化菌KP1034-T含有一大小約136kb的質(zhì)粒,同時(shí)攜帶bldKPC-2、fosA3和rmtB基因,僅對(duì)替加環(huán)素、多粘菌素和喹諾酮類有足夠的敏感性。 基于第一部分發(fā)現(xiàn)pKP1034同時(shí)攜帶有blaKPC-2、fosA3和rmtB多個(gè)重要耐藥基因,本部分從轉(zhuǎn)化菌KP1034-T中抽提質(zhì)粒pKP1034送上海邁普科技有限公司進(jìn)行全質(zhì)粒測(cè)序。然后對(duì)pKP1034全序列進(jìn)行注解分析,并同相關(guān)質(zhì)粒比對(duì),查看其質(zhì)粒骨架基因及多重耐藥區(qū)基因組成及結(jié)構(gòu)特點(diǎn),探討其演化過(guò)程。 pKP1034為大小為136,848bp的閉合環(huán)形DNA分子,平均GC含量為54.5%。經(jīng)過(guò)RAST注解共有191個(gè)閱讀編碼框,包含豐富的質(zhì)粒穩(wěn)定性相關(guān)基因和耐藥基因(blaKPC、rmtB、fosA3、blaSHV-12、blaCTX-M-65、blaTEM-1和catA2),然而接合相關(guān)基因(tra基因)不完整,這與該質(zhì)粒不能通過(guò)接合實(shí)驗(yàn)轉(zhuǎn)移到受體菌大腸埃希菌J53相一致。該質(zhì)粒為高度嵌合質(zhì)粒,經(jīng)質(zhì)粒不相容群分析屬于IncR-F33:A-:B-,可分為三個(gè)部分：攜帶catA2(氯霉素耐藥基因)的復(fù)合轉(zhuǎn)座子、tral和traB之間的片段(tral和traB皆為部分片段,被IS26截?cái)?和余下的基因結(jié)構(gòu)。第二部分traI和traB之間的片段跟國(guó)內(nèi)流行fosA3陽(yáng)性質(zhì)粒pHN7A8高度同源,第三部分同臺(tái)灣報(bào)道的肺炎克雷伯菌來(lái)源的blaKPC日性質(zhì)粒pKPC-LK30高度同源。通過(guò)對(duì)PKP1034、pHN7A8和pKPC-LK30分析比較,可推測(cè)pKP1034的演化過(guò)程：pHN7A8中,攜帶catA2的復(fù)合轉(zhuǎn)座子插入到tral基因中,同時(shí)另一份IS26插入到traB中,該IS26同catA2下游的IS26方向相同；②catA2下游的IS26同新插入的IS26發(fā)生同源重組,導(dǎo)致兩份IS26中間的基因成分連同其中一份IS26從pHN7A8剪切下來(lái),形成一個(gè)環(huán)形分子；③被切除的環(huán)形分子中的IS26同pKPC-LK30由blaSHV-11下游的IS26再次發(fā)生同源重組,從而使該環(huán)形分子插入到pKPC-LK30中；④pKPC-LK30中resD基因下游插入一個(gè)拷貝IS1,其與vagD基因下游的IS1(不完整基因)方向相同,二者之間發(fā)生同源重組導(dǎo)致中間基因結(jié)構(gòu)連同完整的IS1基因被剪切下來(lái),最終形成與pKP1034高度同源的質(zhì)粒。其中,步驟③和④發(fā)生順序可以改變。 pKP1034和相關(guān)質(zhì)粒多重耐藥區(qū)(MRRs)深入比較分析發(fā)現(xiàn)：①pKP1034中攜帶fosA3. blaCTX-M-65和blaTEM-1的MRR部分與pHN7A8的MRR基本相同,只是在pKP1034中,IS1294被新插入的IS26截?cái)?然后新插入的IS26與fosA3上游的IS26發(fā)生同源重組導(dǎo)致中間結(jié)構(gòu)包括fosA3方向發(fā)生逆轉(zhuǎn)；②pKP1034中攜帶fosA3和blaSHV-12的MRR部分與pKPC-LK30的MRR基本相同,但是pKPC-LK30攜帶blaSHV-11而非blaSHV-12,前者為非超廣譜β-內(nèi)酰胺酶,而后者為超廣譜p-內(nèi)酰胺酶,提示超廣譜p-內(nèi)酰胺酶blaSHV-12可能由blaSHV-11發(fā)生突變演化而來(lái)。③pKP1034中blaKPC的基因環(huán)境來(lái)源于國(guó)內(nèi)流行的攜帶blaKPC基因的Tn3-Tn4401復(fù)合轉(zhuǎn)座子,只是在pKP1034中,Tn3部分可能因?yàn)镮S26介導(dǎo)的同源重組而被部分切除掉。 結(jié)論：浙醫(yī)一院KPC-KP菌株磷霉素耐藥率較高(58.74%),其主要機(jī)制為攜帶(44株)滅活酶基因fosA3,另有一株攜帶fosA。這些菌株對(duì)所測(cè)抗菌藥物皆高比例耐藥,僅對(duì)多粘菌素E和替加環(huán)素表現(xiàn)出足夠高的敏感率,分別為100%和94.7%。fosA3陽(yáng)性KPC-KP菌株為單克隆傳播,皆屬于ST11-PTA型。代表菌株fosA3陽(yáng)性質(zhì)粒pKP1034為一高度嵌合的多重耐藥質(zhì)粒,由KPC陽(yáng)性質(zhì)粒pKPC-LK30和國(guó)內(nèi)攜帶fosA3的流行質(zhì)粒pHN7A8經(jīng)經(jīng)過(guò)IS26和IS1介導(dǎo)的多次同源重組演化而成。
[Abstract]:KPC (Klebsiella pneumoniae carbapenemase) type carbapenem was first reported in 2001, mainly distributed in Klebsiella pneumoniae. In recent years, the strains of Klebsiella pneumoniae (KPC-KP) producing KPC are widely prevalent in many countries, which seriously threaten human health.KPC-KP to almost all p- lactam and a variety of non p- lactam. The drug resistance of bacteria is very limited. In recent years, fosfomycin has been paid more attention to the good antibacterial activity of multiple resistant strains, including KPC-KP strain. Our previous study found that the sensitivity rate of phosphamycin of KPC-KP strain of Zhejiang hospital was 43.4%, of which rmtB (plasmid carrying aminoglycoside resistance gene is mediated. The positive strains of aminoglycosides were only 8.5%, but the mechanism of their resistance to fosfomycin was not clear.
In the first part of this study, 97 strains of KPC-KP strains collected from the 2010.1-2013.2 of Zhejiang medical hospital were collected. The MICs values of fosfomycin and other antibiotics were measured by agar dilution, and the resistance genes were detected by PCR and gene sequencing. The cloning correlation was analyzed by pulse field gel electrophoresis (PFGE) and multiple point sequence typing (MLST). The fosA3 localization of phosphamycin inactivation enzyme gene and the transmission of plasmid in the strain were analyzed on behalf of the strain line transformation conjugation test. Finally, whether the fos gene negative strains were detected by PCR and sequencing to detect the mutation of murA and glpA, uhpT, uhpA, ptsI, cyaA, and the growth experiment of carbon source by carbon source. Check the function of the transport system.
Of 97 strains of KPC-KP, 57 strains of phosphamycin resistant strains (58.74%) were found, of which 44 were fosA3 positive and one was fosA positive. The other 12 strains did not carry the fos gene screened in this study. These strains were highly proportional to the antimicrobial agents, including carbapenems, cephalosporins, glucosinolides, quinolones and tetracycline. The high sensitivity of polymyxin E and tigocycline, 50 of the 100% and 94.7%.57 strains of fosfomycin resistant strains respectively carrying rmtB genes, can explain that these strains have two sequences (STs) ST11 and ST494 in the high level resistant.MLST of aminoglycosides, the former contains 56, and the latter contains only one foS base. The negative strain.PFGE found that 5 pulse types (PTS).44 strain fosA3 positive strains belonged to ST11-PTA, indicating that the fosA3 positive strain of Zhejiang hospital was McAb, while the genetic background of the FOS negative strains was relatively complex, including ST11-PTA (6 strains), ST11-PTB (4 strains), ST11-PTC (1 strains) and ST494-PTD (1 strains). Because of the negative strain, the target enzyme gene had no mutation and could grow on the M9 medium with G-6-P as the only carbon source. It indicated that the phosphate hexose transport system (UhpT), the normal.4 strain, was also able to grow on the M9 medium with G-3-P as the only carbon source, indicating that the function of the glycerol -3- phosphoric acid transport system (G1pT) was normal and its resistance to drug resistance was normal. The mechanism needs to further study that.8 strain can not grow on the M9 medium with G-3 1 P as the only carbon source, indicating that the G1pT system has abnormal function. The GlpT gene analysis found that 3 of them have the glpT gene and the amino acid sequence changes, including the A477A mutation of 2 DNA, a mutation of the Cys309Phe of the amino acid, and the other 5 did not find the uhpT 'base. The sequence changes suggest that UhpT dysfunction may be related to the decrease of UhpT transcriptional or expression levels. The gene mutations are not found in the regulatory gene uhpA and cyaA in the transport system, but the amino acid change Ile569Asn is commonly found in the 12 strains of phosphamycin resistant strains, and it is considered that the mutation is a nonsense mutation and does not affect the PTSL. KP1034 phosphamycin resistance, which represents the strain of the strain, can not be transmitted to the receptor bacteria by conjugation experiments. The transformant KP1034-T contains a plasmid with a size of about 136KB and carries the bldKPC-2, fosA3 and rmtB genes, and is only sensitive to tegicycline, polymyxin and quinolones.
Based on the first part, it was found that pKP1034 carried several important resistance genes of blaKPC-2, fosA3 and rmtB. The whole plasmid was sent to Shanghai MEP science and Technology Co., Ltd. from the transformation bacteria KP1034-T. The whole sequence of pKP1034 was annotated and compared with the related plasmids, and the plasmid skeleton gene was examined. The gene composition and structural characteristics of multidrug resistant regions were discussed.
PKP1034 is a closed loop DNA molecule with a size of 136848bp. The average GC content is 54.5%. after RAST annotation with 191 reading coding frames, which contain abundant plasmid stability related genes and resistant genes (blaKPC, rmtB, fosA3, blaSHV-12, blaCTX-M-65, blaTEM-1, etc.). However, the conjugation related genes are incomplete, which are not associated with the plasmid. The plasmid can be transferred to the J53 of Escherichia coli by conjugation. The plasmid is highly chimeric plasmids. The plasmid incompatible group analysis belongs to IncR-F33:A-: B-, which can be divided into three parts: a compound transposon carrying catA2 (chloramphenicol resistant gene), fragments between tral and traB (tral and traB are partial fragments, truncated by IS26). The remaining gene structure. The second part between traI and traB is highly homologous to the domestic fosA3 positive plasmid pHN7A8, and the third part is highly homologous to the blaKPC daily plasmid pKPC-LK30 derived from Klebsiella pneumoniae reported in Taiwan. By comparison of PKP1034, pHN7A8 and pKPC-LK30 analysis, the evolutionary process of pKP1034 is speculated: pHN7A8 The compound transposons carrying catA2 were inserted into the tral gene, while another IS26 was inserted into the traB. The IS26 was the same as the IS26 direction in the downstream of catA2; and the IS26 in the lower catA2 was homologous to the newly inserted IS26, causing the two IS26 intermediate genes to be cut down with one of them, forming a ring molecule. (3) the IS26 and pKPC-LK30 in the resected ring molecules are reorganized again by IS26 from the downstream of blaSHV-11, so that the ring molecule is inserted into the pKPC-LK30; (4) the resD gene is inserted into a IS1 in pKPC-LK30, which is the same as IS1 (incomplete gene) downstream of the vagD gene, and the homologous recombination between the two is caused by the cause. The gene structure and the complete IS1 gene were cut down to form a highly homologous plasmid with pKP1034. The sequence of steps 3 and 4 can be changed.
The comparative analysis of pKP1034 and multiple resistance regions (MRRs) of related plasmids found that: (1) the MRR part carrying fosA3. blaCTX-M-65 and blaTEM-1 in pKP1034 is basically the same as MRR in pHN7A8, but in pKP1034, IS1294 is truncated by newly inserted IS26. The 3 direction reverses, and the MRR part carrying fosA3 and blaSHV-12 in pKP1034 is basically the same as MRR of pKPC-LK30, but pKPC-LK30 carries blaSHV-11 rather than blaSHV-12, the former is a non super broad-spectrum beta lactamase, and the latter is a broad-spectrum p- lactamase, suggesting that the extended spectrum p- lactamase blaSHV-12 may be mutated by the mutation and evolution of the p- lactamase blaSHV-12. 3. (3) the genetic environment of blaKPC in pKP1034 is derived from the domestic Tn3-Tn4401 complex transposon carrying the blaKPC gene, only in pKP1034, part of Tn3 may be partially removed because of the homologous recombination mediated by IS26.
Conclusion: the resistance rate of KPC-KP strain of zhe hospital is high (58.74%), and its main mechanism is to carry (44 strains) inactivated enzyme gene fosA3, and another with fosA., which are highly proportional to the antimicrobial agents, and only have high sensitivity to polymyxin E and tigocycline, 100% and 94.7%.fosA3 positive KPC-KP, respectively. The strain is a monoclonal transmission, all of which belong to type ST11-PTA. The fosA3 positive plasmid pKP1034 of the representative strain is a highly chimeric multidrug resistant plasmid, which is evolved from KPC positive plasmid pKPC-LK30 and the popular plasmid pHN7A8 carrying fosA3 in domestic by multiple homologous recombination mediated by IS26 and IS1.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5

【參考文獻(xiàn)】

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