氨基糖苷类耐药基因armA实时荧光PCR检测方法的建立、应用及NDM-5阳性大肠埃希菌的特征研究

发布时间：2018-07-08 17:06

本文选题：实时荧光PCR + armA基因　；参考：《中国疾病预防控制中心》2015年硕士论文

【摘要】：16S rRNA甲基化酶armA是介导菌株对氨基糖苷类药物高水平耐药的重要机制,其快速检测方法的建立将有助于了解该酶的种属分布特征和流行传播规律。本研究根据armA基因的特征序列设计引物和探针,优化反应条件,建立armA基因的实时荧光PCR检测方法,并在多种常见致病菌和粪便模拟标本评价其灵敏度和特异性。结果显示所建立的实时荧光PCR检测方法对质粒标准品的灵敏度为4.10×101 copy/ml;对粪便模拟样品的检测下限为1.5×104 cfu/ml。而且该引物探针特异性高,对其他常见菌种DNA及氨基糖苷类耐药基因均无交叉反应。应用该实时荧光PCR检测方法,对收集的846株临床来源革兰氏阴性菌和107株动物来源克雷伯菌菌株中armA基因进行检测。结果显示,临床来源菌株armA基因携带率为22.0%,其中不动菌属armA基因携带率最高,达到66.5%,其他种属菌株携带率从高到低依次为：沙雷菌属51.9%,肠杆菌属27.1%,克雷伯菌属9.1%,埃希氏菌属3.3%,假单胞菌属3.0%。动物来源克雷伯菌属菌株的armA基因携带率为65.4%。应用Vitek 2 Compact微生物鉴定药敏系统对菌株阿米卡星和庆大霉素两种抗菌药物的最小抑菌浓度(MIC)进行测定,结果表明846株临床来源菌株对阿米卡星和庆大霉素的耐药率分别为31.3%和60.4%；其中不动菌属对阿米卡星和庆大霉素的耐药率最高,分别达到86.4%和90.9%,其他种属菌株对两种药物的耐药率依次为：沙雷菌属55.6%和55.6%；肠杆菌属35.4%和62.5%；克雷伯菌属12.7%和48.1%；埃希氏菌属7.7%和63.7%；假单胞菌属10.6%和53.0%。动物来源107株克雷伯菌株对阿米卡星和庆大霉素的耐药率分别为74.8%和79.4%。临床来源菌株携带armA基因与氨基糖苷类耐药表型的关系分析显示：携带armA基因的菌株对阿米卡星耐药率高达94.6%,对庆大霉素耐药率高达97.8%,armA基因的携带与氨基糖苷类药物耐药表型具有很高的一致性。此外,本实验也对收集的4株NDM-5阳性的大肠埃希菌的特征进行了研究。这四株菌来自国内四个不同城市的患者,分别导致了患者的尿路感染、伤口感染、细菌性阴道炎和菌血症。药敏检测结果显示四株菌株对除氨曲南以外几乎所有的β-内酰胺类药物均耐药。多位点序列分析(MLST)结果显示：其中两株属于ST167型,另外两株分别是ST2608型和ST2294型。脉冲场凝胶电泳(PFGE)-S1酶切和Southern杂交结果显示：尽管4株菌株携带的质粒数量和质粒大小不尽相同,但携带的blaNDM-5基因均位于大小相同的IncX3质粒上。上述结果提示我们,携带blaNDM-5基因的ST167型大肠埃希菌和IncX3质粒可能介导了blaNDM-5基因在我国的传播。
[Abstract]:16s rRNA methylase armA is an important mechanism of high level resistance to aminoglycoside drugs. The rapid detection of 16s rRNA methylase armA will be helpful to understand the characteristics of species distribution and epidemic spread of the enzyme. In this study, primers and probes were designed according to the characteristic sequence of armA gene, and the reaction conditions were optimized. A real-time fluorescence PCR method for detection of armA gene was established, and its sensitivity and specificity were evaluated in a variety of common pathogenic bacteria and stool samples. The results showed that the sensitivity of the established real-time PCR method for plasmid standard was 4.10 脳 101 copy / ml, and the detection limit for fecal simulated samples was 1.5 脳 10 ~ 4 cfur / ml. Moreover, the primer probe was highly specific and had no cross reaction to other common strains of DNA and aminoglycoside resistant genes. The armA gene of 846 strains of Gram-negative bacteria and 107 strains of Klebsiella fauna were detected by real-time fluorescence PCR. The results showed that the carrying rate of armA gene was 22. 0%, among which the armA gene carrying rate of Acinetobacter genus was the highest. The results showed that the carrying rate of other strains was 51.9% of Shareh, 27.1m of Enterobacter, 9.1% of Klebsiella, 3.33% of Ehisiella and 3.0% of Pseudomonas. The armA gene carrying rate of Klebsiella strain was 65.4%. The minimal inhibitory concentration (MIC) of amikacin and gentamicin was determined by Vitek 2 Compact microbiological identification system. The results showed that the resistance rates of 846 clinical strains to amikacin and gentamicin were 31.3% and 60.4%, respectively, and the resistant rates of acinetobacter to amikacin and gentamicin were the highest. The resistance rates of other strains to the two drugs were 55.6% and 55.6% for Shareh, 35.4% and 62.5% for Enterobacter, 12.7% and 48.1% for Klebsiella, 7.7% and 63.7% for Escherichia, 10.6% and 53.0% for Pseudomonas, respectively. The resistance rates of 107 Klebsiella strains to amikacin and gentamicin were 74.8% and 79.4%, respectively. Analysis of the relationship between armA gene and aminoglycoside resistance phenotype of clinical strains showed that the resistance rate of armA gene to amikacin was 94.6, and to gentamicin was 97.8% to aminoglycosides. Drug resistance phenotypes are highly consistent. In addition, the characteristics of four NDM-5 positive Escherichia coli strains were also studied. The four strains were isolated from four different cities in China, leading to urinary tract infection, wound infection, bacterial vaginitis and bacteremia respectively. The results of drug sensitivity test showed that the four strains were resistant to almost all 尾-lactams except aztreonam. The results of multilocus sequence analysis (MLST) showed that two of them belonged to ST167 and the other two were ST2608 and ST2294 respectively. The results of pulsed field gel electrophoresis (PFGE) -S1 digestion and Southern blotting showed that although the number of plasmids and plasmid size of the four strains were different, the blaNDM-5 gene was located on the same size of IncX3 plasmid. These results suggest that ST167 Escherichia coli and IncX3 plasmid carrying blaNDM-5 gene may mediate the transmission of blaNDM-5 gene in China.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R440

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