质粒介导的多重耐药弗氏柠檬酸杆菌耐药机制研究

发布时间：2018-04-22 15:29

本文选题：碳青霉烯酶 + 弗氏柠檬酸杆菌　；参考：《吉林大学》2015年硕士论文

【摘要】：碳青霉烯类抗生素是一类抗菌谱广、抗菌活性强的β-内酰胺类抗生素，对大多数革兰氏阳性菌、革兰氏阴性菌和厌氧菌都有抗菌作用，且其活性不受β-内酰胺酶的影响，对AmpC酶（AmpC-type enzymes）和超广谱β-内酰胺酶（extended-spectrum β-lactamases,ESBLs）高度稳定。但近几年，对碳青霉烯类抗生素耐药的菌株在全球范围内广泛增多，尤其是革兰氏阴性菌，且呈逐年增多形式。因此，日益严重的细菌耐药性问题引起了全球性的高度关注。碳青霉烯酶是一类能水解碳青霉烯类抗生素的β-内酰胺酶，包括Ambler分类中的A类、B类和D类。A类和D类酶为丝氨酸酶，B类酶为金属酶。碳青霉烯酶被认为是菌株对碳青霉烯酶类抗生素产生耐药性的最主要原因。本研究的目的主要为探索分离自我国珠江医院的多重耐药弗氏柠檬酸杆菌112298耐药的分子机制。共收集来自该医院的70株耐碳青霉烯菌株，经筛查选定同时表达碳青霉烯酶KPC和NDM的菌株112298进行深入研究。112298分离自感染性休克患者的尿液标本，通过16S rRNA鉴定112298的菌种，碳青霉烯酶基因和ESBLs基因筛查112298携带的基因，改良Carba NP法检测碳青霉烯酶的类型，通过最小抑菌浓度（minimum inhibitory concentration）测定菌株的抗生素敏感性，通过接合转移实验验证携带碳青霉稀酶基因的质粒是否有可转移性。结果显示，弗氏柠檬酸杆菌112298同时产KPC型和NDM型碳青霉烯酶，blaKPC位于质粒p112298-KPC（注册号：KP987215），blaNDM位于质粒p112298-NDM（注册号：KP987216）。其中质粒p112298-NDM为可接合型质粒，通过接合转移实验得到接合子112298-NDM-EC600，，而p112298-KPC不可通过接合转移的方式进行水平转移。质粒p112298-KPC和p112298-NDM都可通过电转化的方式进行水平转移，得到转化子112298-KPC-TOP10和112298-NDM-TOP10。112298携带有耐药基因blaKPC、blaNDM、blaOXA-1group、blaCTX-M-9group和blaSHV，112298-NDM-EC600和112298-NDM-TOP10携带blaNDM和blaSHV，112298-KPC-TOP10携带blaKPC、blaOXA-1group和blaCTX-M-9group。改良Carba NP试验得出，112298产A类和B类碳青霉烯酶，112298-KPC-TOP10产A类碳青霉烯酶，112298-NDM-EC600和112298-NDM-TOP10产B类碳青霉烯酶。MIC值提示112298对青霉素类、β-内酰胺酶抑制剂、头孢菌素类、单酰胺菌素类、碳青霉烯类抗生素的耐药性均可水平转移，而对喹诺酮类、呋喃类抗生素的耐药性则无法通过接合转移或电转化的方式转移。对质粒p112298-NDM和p112298-KPC的全序测定及分析得出，质粒p112298-NDM为53.4kb的IncX3型质粒，而p112298-KPC是大小为117kb的新型质粒。质粒p112298-KPC中携带blaKPC-2的Tn1721-based转座子和其它抗性基因及可移动元件共同组成一个多重耐药（multidrug resistance，MDR）区。p112298-NDM与pNDM-HN380高度相似，其中blaNDM基因位于Tn125可变区， p112298-NDM中Tn125与其原型的不同之处在于上游的ISAba125被IS5替换及下游缺失1拷贝ISAba125. 弗氏柠檬酸杆菌112298同时产KPC-2型和NDM-1型碳青霉烯酶，使其对碳青霉烯类抗生素的耐药性增强。值得引起注意的是p112298-KPC除携带blaKPC-2外还有多种耐药基因，包括氨基糖苷类抗性基因、头孢菌素类抗性基因、氯霉素抗性基因、利福平抗性基因、季铵盐类抗性基因、磺胺类抗性基因、喹诺酮抗性基因、氨苄西林抗性基因、铬酸盐抗性基因、磷霉素抗性基因等，且还有多个移动元件，如插入序列、转座子和1类整合子。大量耐药基因的共存势必会加速菌株的进化，使菌株快速获得并保留不同的抗性基因，在不同的抗生素选择压力下，这将加速耐药菌株的扩散和持续，政府卫生部门和医务工作者应加强监控，以防微杜渐。
[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.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【共引文献】