亚胺培南耐药铜绿假单胞菌及金属β-内酰胺酶的分子流行病学研究

发布时间：2018-03-05 07:28

本文选题：铜绿假单胞菌　切入点：耐药性　出处：《华中科技大学》2006年硕士论文　论文类型：学位论文

【摘要】： 目的和意义铜绿假单胞菌是引起医院感染的重要病原体,多重耐药的铜绿假单胞菌感染可导致临床治疗困难,病死率增加。而亚胺培南是治疗多重耐药铜绿假单胞菌的有效药物,随着它的广泛使用,对其耐药的铜绿假单胞菌日益增多。本文分析亚胺培南耐药铜绿假单胞菌对常用抗菌药物的耐药性,研究对亚胺培南的耐药机制及所产金属β-内酰胺酶的类型,阐明同济医院铜绿假单胞菌对常用抗菌药物的耐药性及同源性,为指导临床合理使用抗菌药物特别是碳青霉烯类,新药研究及开发,加强感染控制措施,及时切断多重耐药株的克隆传播和水平传播,密切监测耐药株的变迁及其发展提供科学依据。方法收集同济医院2004年1月～9月亚胺培南耐药铜绿假单胞菌存活株29株,琼脂稀释法测定29株铜绿假单胞菌存活株对常用的10种抗菌药物的最低抑菌浓度(minimal inhibitory concentrations,MIC)。通过双纸片协同试验(double-disk synergy testing,DDST)、聚合酶链反应(polymerase chain reaction ,PCR)、序列分析(sequencing)等方法检测金属β-内酰胺酶并分析其类型。紫外分光光度法测定金属β-内酰胺酶的活性。脉冲场凝胶电泳(pulsed field gelelectrophoresis,PFGE)分析耐药菌株的同源性。结果经过WHONET5.3软件分析,同济医院1998年至2004年铜绿假单胞菌对亚胺培南的耐药性呈逐年上升趋势,由1998年7.3%上升至2004年14.3%。同济医院2004年1～9月共分离铜绿假单胞菌193株,对头孢噻肟、庆大霉素和四环素的耐药率分别为38.5%、42%和93.7%,其余抗菌药物的耐药率均低于20%。对亚胺培南和美罗培南或头孢他啶交叉耐药的铜绿假单胞菌分别为15株(11.0%)和14株(7.3%)。 193株铜绿假单胞菌中,亚胺培南耐药的铜绿假单胞菌(IMP-R-Pae)共39株。IMP-R-Pae和亚胺培南敏感的铜绿假单胞菌(IMP-S-Pae)对四环素的耐药率均在90%以上;对哌拉西林/他唑巴坦的耐药率分别为28.6%和17.9%,差异无显著性;对于其他抗菌药物的耐药率,IMP-R-Pae组明显高于IMP-S-Pae组,且差异呈极显著性。药物敏感性试验结果显示,存活的29株亚胺培南耐药铜绿假单胞菌均为多重耐药株。头孢噻肟和头孢他啶的耐药率分别为69%和27.6%。氨曲南和阿米卡星的耐药率均为44.8%,其MIC50和MIC90均为16μg/ml和256μg/ml。亚胺培南和美罗培南的耐药率分别为93.1%和69%, MIC50和MIC90均为32μg/ml和256μg/ml。29株铜绿假单胞菌中有34.5%(10/29)的菌株对亚胺培南的MIC32μg/ml,属于高水平耐药[11][12]。庆大霉素和阿米卡星的耐药率分别为82.8%和44.8%,MIC50分别是256μg/ml和16μg/ml, MIC90均为256μg/ml。环丙沙星、哌拉西林/他唑巴坦和四环素的耐药率分别为41.4%、48.3%和100%。双纸片协同试验检测29株铜绿假单胞菌中有11株金属β-内酰胺酶阳性,阳性率为37.9%。金属β-内酰胺酶基因检测显示有2株细菌产VIM-2型金属β-内酰胺酶(6.9%)。紫外分光光度法检测两株产酶株的酶活性分别为27.35±3.25U,26.58±3.18U。采用脉冲场凝胶电泳技术对29株铜绿假单胞菌进行分子分型,PFGE图谱共分为16个基因型,A、B、C、D、E、F、G型各有两株以上,其中D型3株,分别来自器官移植病房(2株)和神经内科病房(1株)的不同患者,标本采集时间相近,且均使用过呼吸机辅助呼吸,其余类型各1株,两株产金属β-内酰胺酶菌株不属于同一基因型,。结论同济医院2004年1～9月分离的铜绿假单胞菌,除对头孢噻肟、庆大霉素和四环素耐药率较高,分别为38.5%、42%和93.7%,对其他抗菌药物的耐药率均低于20%。亚胺培南耐药的铜绿假单胞菌对于抗菌药物的耐药率均高于亚胺培南敏感组,除四环素和哌拉西林/他唑巴坦外,差异均呈极显著性(P0.01),且亚胺培南耐药的铜绿假单胞菌通常表现为多重耐药。 29株亚胺培南耐药铜绿假单胞菌,对头孢噻肟、庆大霉素、美罗培南和四环素的耐药率大于60%;对环丙沙星、氨曲南、阿米卡星和哌拉西林/他唑巴坦耐药率相近,为41%～48%;对头孢他啶耐药率最低,为27.6%。双纸片协同试验产金属β-内酰胺酶菌株阳性率为37.9%。基因检测只发现2株产VIM-2型金属β-内酰胺酶。 PFGE分型结果提示同济医院亚胺培南耐药铜绿假单胞菌可能存在小范围的流行,即可能有耐药菌株的克隆传播。同济医院亚胺培南耐药铜绿假单胞菌之间尚未发现灭活酶基因的水平传播。
[Abstract]:Purpose and significance
Pseudomonas aeruginosa is an important pathogen of nosocomial infection, can lead to difficulties in the clinical treatment of multi drug resistant Pseudomonas aeruginosa infection, the mortality rate increased. Imipenem is an effective drug for the treatment of multi drug resistant Pseudomonas aeruginosa, with its widely used, increasing the drug resistance of Pseudomonas aeruginosa. The commonly used antimicrobial drug resistance of imipenem resistant Pseudomonas aeruginosa strains of this type, the mechanism of imipenem resistance in research and production of metallo beta lactamase, and homology to clarify the drug resistance of Pseudomonas aeruginosa in Tongji Hospital to commonly used antimicrobial drugs, to guide the rational use of clinical antibiotics especially carbapenem class, research and development of new drugs and strengthen infection control measures, cut off the spread of multi drug resistant strains of clonal spread and level in a timely manner, closely monitoring the changes and development of resistant strains and provide the scientific basis According to.
Method
From January 2004 to September in Tongji Hospital of imipenem resistant Pseudomonas strains 29 strains were survival, 29 strains of Pseudomonas aeruginosa strains survived the minimum inhibitory concentration of 10 kinds of commonly used antibiotics by agar dilution method (minimal inhibitory concentrations, MIC). The double disk synergy test (double-disk synergy, testing, DDST), polymerase chain the reaction (polymerase chain reaction, PCR), sequence analysis (sequencing) method lactamases and analysis of its type. The determination of metal beta UV spectrophotometry lactamase activity. Pulsed field gel electrophoresis (pulsed field gelelectrophoresis, PFGE) to analyze the homology of resistant strains.
Through the analysis of WHONET5.3 software, from 1998 to 2004 in Tongji Hospital of Pseudomonas aeruginosa to imipenem increased year by year, up from 7.3% in 1998 to 2004 14.3%. Tongji Hospital in 2004 1~9 months were isolated 193 strains of Pseudomonas aeruginosa, cefotaxime, gentamicin and tetracycline resistance rates were 38.5%, 42% and 93.7%, the drug resistance rates were lower than the 20%. of imipenem and meropenem and ceftazidime interresistant Pseudomonas aeruginosa were 15 strains (11%) and 14 strains (7.3%).
193 strains of Pseudomonas aeruginosa, Pseudomonas aeruginosa resistant to imipenem (IMP-R-Pae and.IMP-R-Pae) a total of 39 strains of imipenem susceptible Pseudomonas aeruginosa (IMP-S-Pae) of tetracycline resistance rate in 90% above; resistant to piperacillin / tazobactam rate were 28.6% and 17.9%, no difference significant; for other antimicrobial drug resistance rate of IMP-R-Pae group was significantly higher than that in IMP-S-Pae group, and the difference was significant.
Drug sensitivity test showed that 29 strains of imipenem resistant Pseudomonas aeruginosa survival were multidrug-resistant. Cefotaxime and ceftazidime resistance rates were 69% and 27.6%. resistant to aztreonam and Amikacin rate were 44.8%, MIC50 and MIC90 were resistant to 16 g/ml and 256 g/ml. imipenem and meropenem were 93.1% and 69%, MIC50 and MIC90 were 34.5% 32 g/ml and 256 g/ml.29 strains of Pseudomonas aeruginosa (10/29) MIC32 g/ml strains to imipenem, resistant to high level resistance to gentamicin and Amikacin [11][12]. respectively 82.8% and 44.8%, respectively, MIC50 256 g/ml and 16 g/ml, 256 g/ml. MIC90 were ciprofloxacin, piperacillin / tazobactam and tetracycline resistance rates were 41.4%, 48.3% and 100%.
Double disk synergy test of 29 strains of Pseudomonas aeruginosa in 11 strains of metallo beta lactamase positive, the positive rate of 37.9%. for the detection of metallo beta lactamase gene showed that 2 strains of VIM-2 producing bacteria of metallo beta lactamase (6.9%). UV spectrophotometry for the determination of enzyme producing strains the activity of the two strains were 27.35 + 3.25U, 26.58 + 3.18U.
The pulsed field gel electrophoresis of 29 strains of Pseudomonas aeruginosa by molecular typing, PFGE map is divided into 16 genotypes, A, B, C, D, E, F, G each have more than two strains, including 3 strains of type D, respectively, from the transplant ward (2 strains) and the Department of Neurology (1 strains) of different patients, the time of sample collection and are similar, use of ventilator assisted breathing, the rest of the type 1 strains, two strains of metallo beta lactamase producing strains do not belong to the same genotype.
conclusion
Pseudomonas aeruginosa isolated from Tongji Hospital in 2004 1~9 months, in addition to cefotaxime, gentamicin and tetracycline resistance rates were 38.5%, 42% and 93.7%, the resistance rates to other antimicrobial agents were lower than 20%. in Pseudomonas aeruginosa imipenem resistant to antibiotics were higher than imipenem sensitive group. In addition to tetracycline and piperacillin / tazobactam, the difference was significantly (P0.01), and Pseudomonas aeruginosa resistant to imipenem usually show multiple drug resistance.
29 strains of imipenem resistant Pseudomonas aeruginosa to gentamicin, cefotaxime, meropenem and tetracycline resistance rate of more than 60%; Ciprofloxacin, aztreonam, Amikacin and piperacillin / tazobactam resistant rate was 41% ~ 48%; similar to ceftazidime resistant rate of the low 27.6%.
The positive rate of metallo beta lactamase producing strain was detected by 37.9%. gene and only 2 strains of VIM-2 type metallo beta lactamase were found in the double paper synergistic test.
The results of PFGE typing suggested that there may be a small epidemic of imipenem resistant Pseudomonas aeruginosa in Tongji Hospital, that is, there may be a clone propagation of drug-resistant strains. No level of inactivated enzyme gene has been found between imipenem resistant Pseudomonas aeruginosa in Tongji Hospital.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R181.3

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