产KPC酶肺炎克雷伯菌对磷霉素耐药及传播机制研究

发布时间：2018-05-20 04:46

  本文选题：肺炎克雷伯菌 + KPC型碳青霉烯酶　； 参考：《浙江大学》2015年博士论文

【摘要】：KPC (Klebsiella pneumoniae carbapenemase)型碳青霉烯酶首次于2001年被报道,主要分布于肺炎克雷伯菌中。近年来,产KPC肺炎克雷伯菌(KPC-KP)菌株在全球多国家地区广泛流行,严重威胁人类健康。KPC-KP对几乎所有p-内酰胺类以及多种非p-内酰胺类抗菌药物耐药,使临床抗菌药物选择十分受限。近年来,磷霉素因对多重耐药菌株包括KPC-KP菌株仍有较好的抗菌活性而再度受到重视。我们前期研究发现浙医一院KPC-KP菌株磷霉素敏感率为43.4%,其中rmtB(质粒携带氨基糖甙类耐药基因,介导氨基糖甙类高水平耐药)阳性菌株仅为8.5%,然而他们对磷霉素的耐药机制尚不明确。 本研究第一部分对浙医一院2010.1-2013.2间收集的97株KPC-KP菌株,通过琼脂稀释法测定其对磷霉素及其他抗菌药物的MICs值,PCR和基因测序检测耐药基因携带情况,脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)方法分析其克隆相关性,并选取代表菌株行转化接合试验分析磷霉素灭活酶基因fosA3定位及其携带质粒在菌株间传播情况。最后fos基因阴性菌株通过PCR及测序检测其磷霉素靶酶基因murA和转运系统相关基因glpA、uhpT、uhpA、ptsI、cyaA是否存在突变；同时通过碳源生长实验检测转运系统的功能。 97株KPC-KP菌株中,共发现磷霉素耐药菌株57株(58.74%),其中44株为fosA3阳性,一株fosA阳性。其余12株不携带本研究中筛查的fos基因。这些菌株对所测抗菌药物包括碳青霉烯类、头孢菌素类、氨基糖甙类、喹诺酮类和四环素类皆呈高比例耐药,仅对多粘菌素E和替加环素表现出较高的敏感率,分别为100%和94.7%。57株磷霉素耐药菌株中50株携带rmtB基因,可以解释这些菌株对氨基糖甙类抗菌药物的高水平耐药。MLST共发现两个序列型(STs) ST11和ST494,前者包含56株,后者仅包含一株foS基因阴性菌株。PFGE共发现5个脉冲类型(PTS)。44株fosA3阳性菌株皆属于ST11-PTA,说明浙医一院fosA3阳性菌株为单克隆传播。而fos阴性菌株基因背景相对复杂,包括ST11-PTA(6株)、ST11-PTB(4株)、ST11-PTC(1株)和ST494-PTD(1株)afosA阳性菌株属于ST11-PTE.12株fos基因阴性菌株,靶酶基因未发现突变,且皆可在G-6-P为唯一碳源的M9培养基上生长,说明其磷酸己糖转运系统(UhpT)功能正常。4株磷霉素耐药菌株亦能在以G-3-P为唯一碳源的M9培养基上生长,说明其甘油-3-磷酸转运系统(G1pT)功能正常,其耐药机制需进一步研究。8株不能在以G-3一P为唯一碳源的M9培养基上生长,说明其G1pT系统存在功能异常,GlpT基因分析发现其中3株存在glpT基因及氨基酸序列改变,包含2株发生DNA的A477A突变,一株发生氨基酸Cys309Phe突变。其余5株未发现uhpT’基因序列改变,提示UhpT功能障碍可能与UhpT转录或表达水平下降有关。转运系统相关调节基因uhpA和cyaA未发现基因突变。而ptsl在12株磷霉素耐药菌株中普遍存在氨基酸改变Ile569Asn,结合碳源生长实验结果认为该突变为无义突变不影响ptsl编码蛋白功能。代表菌株KP1034磷霉素抗性不能通过接合实验传递到受体菌。其转化菌KP1034-T含有一大小约136kb的质粒,同时携带bldKPC-2、fosA3和rmtB基因,仅对替加环素、多粘菌素和喹诺酮类有足够的敏感性。 基于第一部分发现pKP1034同时携带有blaKPC-2、fosA3和rmtB多个重要耐药基因,本部分从转化菌KP1034-T中抽提质粒pKP1034送上海迈普科技有限公司进行全质粒测序。然后对pKP1034全序列进行注解分析,并同相关质粒比对,查看其质粒骨架基因及多重耐药区基因组成及结构特点,探讨其演化过程。 pKP1034为大小为136,848bp的闭合环形DNA分子,平均GC含量为54.5%。经过RAST注解共有191个阅读编码框,包含丰富的质粒稳定性相关基因和耐药基因(blaKPC、rmtB、fosA3、blaSHV-12、blaCTX-M-65、blaTEM-1和catA2),然而接合相关基因(tra基因)不完整,这与该质粒不能通过接合实验转移到受体菌大肠埃希菌J53相一致。该质粒为高度嵌合质粒,经质粒不相容群分析属于IncR-F33:A-:B-,可分为三个部分：携带catA2(氯霉素耐药基因)的复合转座子、tral和traB之间的片段(tral和traB皆为部分片段,被IS26截断)和余下的基因结构。第二部分traI和traB之间的片段跟国内流行fosA3阳性质粒pHN7A8高度同源,第三部分同台湾报道的肺炎克雷伯菌来源的blaKPC日性质粒pKPC-LK30高度同源。通过对PKP1034、pHN7A8和pKPC-LK30分析比较,可推测pKP1034的演化过程：pHN7A8中,携带catA2的复合转座子插入到tral基因中,同时另一份IS26插入到traB中,该IS26同catA2下游的IS26方向相同；②catA2下游的IS26同新插入的IS26发生同源重组,导致两份IS26中间的基因成分连同其中一份IS26从pHN7A8剪切下来,形成一个环形分子；③被切除的环形分子中的IS26同pKPC-LK30由blaSHV-11下游的IS26再次发生同源重组,从而使该环形分子插入到pKPC-LK30中；④pKPC-LK30中resD基因下游插入一个拷贝IS1,其与vagD基因下游的IS1(不完整基因)方向相同,二者之间发生同源重组导致中间基因结构连同完整的IS1基因被剪切下来,最终形成与pKP1034高度同源的质粒。其中,步骤③和④发生顺序可以改变。 pKP1034和相关质粒多重耐药区(MRRs)深入比较分析发现：①pKP1034中携带fosA3. blaCTX-M-65和blaTEM-1的MRR部分与pHN7A8的MRR基本相同,只是在pKP1034中,IS1294被新插入的IS26截断,然后新插入的IS26与fosA3上游的IS26发生同源重组导致中间结构包括fosA3方向发生逆转；②pKP1034中携带fosA3和blaSHV-12的MRR部分与pKPC-LK30的MRR基本相同,但是pKPC-LK30携带blaSHV-11而非blaSHV-12,前者为非超广谱β-内酰胺酶,而后者为超广谱p-内酰胺酶,提示超广谱p-内酰胺酶blaSHV-12可能由blaSHV-11发生突变演化而来。③pKP1034中blaKPC的基因环境来源于国内流行的携带blaKPC基因的Tn3-Tn4401复合转座子,只是在pKP1034中,Tn3部分可能因为IS26介导的同源重组而被部分切除掉。 结论：浙医一院KPC-KP菌株磷霉素耐药率较高(58.74%),其主要机制为携带(44株)灭活酶基因fosA3,另有一株携带fosA。这些菌株对所测抗菌药物皆高比例耐药,仅对多粘菌素E和替加环素表现出足够高的敏感率,分别为100%和94.7%。fosA3阳性KPC-KP菌株为单克隆传播,皆属于ST11-PTA型。代表菌株fosA3阳性质粒pKP1034为一高度嵌合的多重耐药质粒,由KPC阳性质粒pKPC-LK30和国内携带fosA3的流行质粒pHN7A8经经过IS26和IS1介导的多次同源重组演化而成。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R446.5

【参考文献】

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