耐氨基糖苷类铜绿假单胞菌的分子耐药机制研究

发布时间：2018-02-24 19:16

本文关键词： 铜绿假单胞菌琼脂稀释法氨基糖苷类修饰酶基因 16SrRNA甲基化酶基因聚类分析整合子多重耐药 PCR RFLP 耐药基因盒　出处：《广州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：铜绿假单胞菌( Pseudomonas aeruginosa, Pae)是院内感染的主要病原菌之一。在中国重症监护病房分离的革兰阴性菌中,铜绿假单胞菌位列第一。由于具有天然和获得性耐药,铜绿假单胞菌对很多抗菌药物耐药,给临床治疗带来很大困难。β-内酰胺类、氨基糖苷类、喹诺酮类抗菌药物是临床治疗Pae感染的常用药,近年来随着新的耐药基因的不断出现,Pae对这三类抗菌药物的耐药性普遍增加,甚至出现多药耐药(MDR)和泛耐药(PDR)菌株。因氨基糖苷类抗生素抗菌谱广、疗效卓越,在医学临床和畜牧兽医业应用广泛,但由于滥用和过度使用氨基糖苷类药物的耐药问题随之凸现,细菌对该类药物耐药是因为细菌产生氨基糖苷类修饰酶(AMEs)、16SrRNA甲基化酶和氨基糖苷类抗生素作用靶位16S rRNA基因突变而致。氨基糖苷类修饰酶按功能可分成乙酰转移酶(AAC)、磷酸转移酶(APH)、核苷转移酶(ANT)三类,分别修饰氨基糖苷药物相应位置上的- OH和- NH2 ,降低或丧失其对靶位核糖体的亲和力。编码AMEs的基因起源于抗生素产生菌,该类基因所表达的酶在抗生素产生菌中起到自我保护作用,许多研究表明抗药性产生菌所存在的自我保护机制与临床耐药菌的耐药机制相同。整合子是一个能够通过自身编码的整合酶来获取保外游离基因或基因片段并使之表达的遗传元件系统。整合子既可位于细菌染色体上,又可位于质粒上,还可位于转座子上。对细菌抗生素耐药机制的研究,导致了包括转座子和共轭质粒在内的许多移动元件的发现,而通过对这些元件序列进行比较分析后,最终发现了整合子的存在。研究目的铜绿假单胞菌是院内感染的主要条件致病菌之一,可引起严重的院内获得性感染。本研究试图了解临床常用的七种抗生素对60株铜绿假单胞菌(30株分离自门诊病人,30株分离自住院病人)的抗菌活性和多黏菌素B与其它六种抗菌药物(庆大霉素、奈替米星、阿米卡星、妥布霉素、环丙沙星、亚胺培南)体外联合抗菌效应,以及各种抗菌药物对不同标本来源菌株的抗菌效应有无差别;了解氨基糖苷类耐药相关基因和16SrRNA甲基化酶基因存在情况以及菌株之间的亲缘性;另外通过对临床分离的铜绿假单胞菌的耐药特征进行分析、检测相关耐药基因和整合子携带情况等,初步探讨铜绿假单胞菌对临床常用抗菌药物的分子耐药机制,从而了解不同的耐药机制间相互作用规律以及耐药基因在菌株间水平传播的特点,为临床用药提供实验室依据和帮助。研究方法 1、药敏试验和联合药敏试验 60株铜绿假单胞菌均来自广州医学院第一附属医院的临床标本,对这些菌株采用琼脂稀释法测定7种临床常用抗生素的最低抑菌浓度(Minimal inhibitory concentration,MIC)和药敏情况,分析铜绿假单胞菌对这些抗生素的药物敏感性;同时检测多粘菌素B与其它6种药物的联合药敏情况,分析它们之间的联合药敏情况,试图开辟治疗Pae感染新的途径,在用药选择、降低用药量和毒副作用方面起到一定作用。 2、多药耐药铜绿假单胞菌氨基糖苷类修饰酶和16SrRNA甲基化酶基因分析针对临床分离的Pae菌株,采用聚合酶链反应分析氨基糖苷类修饰酶、16SrRNA甲基化酶基因型及其它基因型,运用SPSS统计分析软件对菌株样本亲缘性做聚类分析。 3、整合子的鉴定与分析利用PCR方法,通过扩增整合子恒定区的方法,筛选携带整合子的菌株,并利用限制性片段多态性性(RFLP)酶切分析的方法对这些整合子进行分类;对整合子恒定区扩增阳性的菌株进行测序和序列分析。探讨整合子携带的耐药基因盒是否介导铜绿假单胞菌的多重耐药。研究结果 1、铜绿假单胞菌对三类六种抗生素耐药率普遍较高,分别为奈替米星68.3%、妥布霉素65.0%、庆大霉素60.0%、环丙沙星46.7%、亚氨培南40.0%、阿米卡星13.3%,而多黏菌素B的耐药率为0;这些菌株中多重耐药和泛耐药菌的检出率分别为55.0%(33/60)和31.7%(19/60)。多黏菌素B与环丙沙星、亚氨培南有明显联合抗菌效应,多黏菌素B与奈替米星联合,能明显降低彼此的MICG。各种抗生素对住院病例分离菌株和门诊病例分离菌株,在MICG和差异显著性方面表现各不相同。 2、21株氨基糖苷类耐药菌株中(其中20株为多药耐药菌株),氨基糖苷类耐药基因型aac(6,)-Ⅰ阳性13株(61.9%)、aac(6,)-Ⅱ阳性13株(61.9%)、ant(2,,)-Ⅰ阳性10株(47.6%)、ant(3,,)-Ⅰ阳性9株(42.9%)、aac(3)-Ⅱ阳性的有1株(4.8%),另有1株菌oprD2基因缺失,未检出基因型aac(6,)-Ⅰae、aph(3,)-Ⅲ、aac(6,)-aph(2,,)和ant(4,)-Ⅰ;16SrRNA甲基化酶基因rmtA基因型阳性19株(90.4%)、armA基因型阳性有8株(38.1%),未检出基因型rmtC、rmtD。聚类分析结果显示分离的菌株中存在克隆传播。 3、30株临床分离铜绿假单胞菌中16株(53.3%)整合子恒定区和可变区扩增阳性,片段长度在0.7-4kb之间。共检出5种不同的整合子组合形式,含有编码对氨基糖苷类、β-内酰胺类和喹喏酮类抗菌药耐药的基因,其中有2例为新型基因盒组合形式,包括aacA4-VIM2和aadA2-OXA10-aacA4-blaIMP9-aatI1,Genbank基因库登陆号分别为GQ890658和GU122165,另外3例分别与Genbank基因库登录号为FJ917747、FJ817423、GU367339的序列吻合。 4、I类整合子3′-末端qacEΔ1-sul1和intI1类整合酶基因的阳性率和阳性菌株相同。结论 1、临床检出的铜绿假单胞菌对常用抗生素多表现为多重耐药。多黏菌素B单用有很好的抗铜绿假单胞菌效果,与环丙沙星(喹诺酮类)、亚氨培南(β-内酰胺类)联合应用也有明显的联合抗菌效应。对多药耐药和泛耐药菌株,为减少用药量和毒副作用,提高抗菌效果,可考虑多黏菌素B与这些抗生素联合用药。 2、大部分测试的铜绿假单胞菌对临床常用的铜绿假单胞菌抗感染药物已产生广泛耐药,尤其对氨基糖苷类抗生素。这些菌株的氨基糖苷类修饰酶常见耐药基因型检出率高, 16SrRNA甲基化酶基因型rmtA和armA的检出率亦较高。30株测试菌株中存在克隆传播。 3、本研究中携带intI1类整合子的16株Pae对氨基糖苷类全部耐药,对喹喏酮类耐药的有13株,对亚氨培南耐药的有10株,对所有三类抗生素全部耐药的有8株占50%,说明目前引起Pae耐药的机制中,整合子耐药机制越来越起着重要的作用。铜绿假单胞菌的耐药和多重耐药与整合子密切相关,主要携带氨基糖苷类和β内酰胺类抗生素耐药基因,首次发现2种携带新型基因盒组合形式的整合子。 4、I类整合子3′-末端qacEΔ1-sul1和intI1类整合酶基因的阳性率和阳性菌株相同,表明这2个基因是紧密伴随intI1而存在的,可作为I类整合子的标志基因。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa Pae) is one of the primary pathogens of nosocomial infections in ICU. The separation of Chinese gram negative bacteria, Pseudomonas aeruginosa ranked first. Because of the natural and acquired drug resistance of Pseudomonas aeruginosa to many antibiotics, which brings great difficulties to the clinical treatment. Beta lactams, aminoglycosides, quinolones are commonly used drugs for clinical treatment of Pae infection, in recent years, with the emergence of new resistance genes, Pae of the three kinds of antimicrobial drug resistance is increasing, and even the emergence of multidrug resistance (MDR) and pan drug resistant (PDR) strains.
Due to a broad spectrum antibacterial aminoglycoside antibiotics in clinical medicine, the curative effect is remarkable, and the animal husbandry and veterinary medicine widely used in industry, but because of the abuse and overuse of aminoglycoside resistance increasing, bacterial resistance to the drugs is because of the bacteria producing aminoglycoside modifying enzymes (AMEs), 16SrRNA methylase and aminoglycoside antibiotics target 16S rRNA gene mutation. Aminoglycoside modifying enzymes according to the functions can be divided into acetyltransferase (AAC), phosphate transferase (APH), nucleoside transferase (ANT) three, respectively modified aminoglycoside drugs corresponding to the position of the - OH and - NH2, reduce or lose the target a ribosomal affinity. The genes encoding AMEs originated in the antibiotic producing strains, the expression of the gene of the enzyme producing strains play the role of self-protection in antibiotics, many studies have shown that resistant bacteria generated by the existence of self protection The mechanism of drug resistance is the same as that of clinical drug resistant bacteria.
Integration through a combination of enzyme and its encoding to obtain for free genes or gene fragments and the expression of the genetic component system. The integration can be located in the bacterial chromosome, and was located on but also on the transposon. On the mechanisms of bacterial antibiotic resistance, resulting in a number of mobile devices including transposons and plasmid conjugation, discovery, and through a comparative analysis of sequences of these components, finally found the presence of integrons.
research objective
Pseudomonas aeruginosa is main pathogen of nosocomial infection, can cause serious nosocomial infections. This study attempts to understand the clinical commonly used seven antibiotics against 60 strains of Pseudomonas aeruginosa (30 strains isolated from outpatients, 30 strains isolated from inpatients) and the antibacterial activity of polymyxin B and other six kinds of antibiotics (gentamicin, netilmicin, Amikacin, tobramycin, ciprofloxacin, imipenem) combined antibacterial effect in vitro, and the antibacterial effect of antibiotics to these isolates from different sources. There is no difference; the solution of aminoglycoside resistance related genes and 16SrRNA methylase genes between strains and the affinity; the characteristic of drug resistance in Pseudomonas aeruginosa clinical isolates were analyzed to detect drug resistance related genes and integrons carrying case, preliminary study of Pseudomonas aeruginosa The mechanism of molecular resistance of commonly used antibiotics is understood, so as to understand the interaction rule of different drug resistance mechanisms and the characteristics of drug resistance genes spreading at the level of strains, so as to provide laboratory evidence and help for clinical medication.
research method
1, drug sensitivity test and combined drug sensitivity test
The clinical specimens of 60 strains of Pseudomonas aeruginosa were from the First Affiliated Hospital of Guangzhou Medical College, the minimum inhibitory concentration of 7 kinds of commonly used antibiotics in clinic of these strains by agar dilution method (Minimal inhibitory concentration, MIC) and drug sensitivity analysis of these drugs, antibiotic susceptibility of Pseudomonas aeruginosa; simultaneous detection of combined drug sensitivity situation polymyxin B and other 6 kinds of drugs, analysis of drug sensitivity between them, trying to open up a new way in the treatment of Pae infection, drug selection, reduce the dosage and side effect play a role.
2, analysis of aminoglycoside modifier and 16SrRNA methylase gene of Pseudomonas aeruginosa in multidrug resistance
For clinical isolates of Pae, polymerase chain reaction was used to analyze aminoglycoside modifying enzymes, 16SrRNA methylase genotypes and other genotypes. Cluster analysis was carried out on the affinity of strain samples by SPSS statistical analysis software.
3, identification and analysis of integrons
By using the PCR method, amplified integrons constant region, selection of strains carrying integrons and use of restriction fragment length polymorphism (RFLP) enzyme analysis method to classify these integrons; of integron positive strains amplified constant region sequencing and sequence analysis of resistance gene cassette integration carry zygotic multi - drug resistance mediated by Pseudomonas aeruginosa.
Research results
1, Pseudomonas aeruginosa three kinds of six kinds of antibiotics were generally higher, netilmicin 68.3%, 65% tobramycin, gentamicin 60%, ciprofloxacin 46.7%, sub imipenem 40%, Amikacin 13.3%, and resistance to polymyxin B was 0; the detection of these strains of multi drug resistant and pan resistant bacteria rates were 55% (33/60) and 31.7% (19/60). Polymyxin B and imipenem ciprofloxacin, Asia has obvious combined antibacterial effect of polymyxin B with netilmicin combined with each other, can significantly reduce the MICG. of various antibiotics of hospitalized patients and outpatients in isolated strains isolated strains in MICG and significant differences in performance vary.
2,21 strains of aminoglycoside resistant strains (including 20 strains of multi drug resistant strains), aminoglycoside resistance gene AAC (6) of 13 strains were positive (61.9%), AAC (6) of 13 strains were positive (61.9%), ant (2, 1) - positive 10 strains (47.6%), ant (3, 1) - positive 9 strains (42.9%), AAC (3) - II positive 1 strains (4.8%), and 1 strains of oprD2 gene deletion were not detected in genotype AAC (6, AE) - 1, APH (3) - III, AAC (6) -aph (2,) and ant (4) - 1; 16SrRNA methyltransferase gene rmtA was positive in 19 strains (90.4%), armA genotypes were 8 strains (38.1%), were not detected in genotype rmtC, rmtD. cluster analysis showed that clonal spread of isolates present.
3,30 clinical isolates of 16 strains of Pseudomonas aeruginosa (53.3%) integron constant and variable regions of amplified fragment length, between 0.7-4kb. There were 5 kinds of different forms of integron containing combination encoding of aminoglycosides, beta lactam and quinolone antibacterial drug resistance genes, which there were 2 cases of new gene cassette combinations, including aacA4-VIM2 and aadA2-OXA10-aacA4-blaIMP9-aatI1, Genbank gene bank accession number were GQ890658 and GU122165, the other 3 cases of Genbank respectively with the GenBank accession number is FJ917747, FJ817423, GU367339 series of anastomosis.
4, the positive rate of I integron 3 '- terminal qacE Delta 1-sul1 and intI1 integrase gene was the same as that of the positive strain.
conclusion
1, the detection of Pseudomonas aeruginosa to commonly used antibiotics showed multidrug resistance more. Polymyxin B with anti Pseudomonas aeruginosa to good effect, and ciprofloxacin (quinolones), and imipenem (beta lactam) combined have combined antibacterial effect on multi drug obviously. Resistance and pan resistant strains, in order to reduce the dosage and side effects, improve the antibacterial effect of polymyxin B can be considered with the combination of these antibiotics.
2, most of the test of Pseudomonas aeruginosa to commonly used Pseudomonas aeruginosa has generated widespread anti infective drug, especially for aminoglycoside antibiotics. These strains of aminoglycoside modifying enzyme common resistance genes in high detection rate, 16SrRNA methyltransferase genotype rmtA and armA detection rate of clonal spread there the higher the.30 test strains.
3, in the study of class intI1 integrons in 16 strains of Pae are resistant to aminoglycosides, quinolones resistant to 13 strains of imipenem resistant to 10 strains of all three types of all antibiotics resistant 8 strains accounted for 50%, indicating that the current caused by the mechanisms of resistance to Pae. The resistance mechanism of zygote increasingly plays an important role. The resistance of Pseudomonas aeruginosa and multi drug resistance and integrons are closely related, mainly carrying aminoglycosides and beta lactam antibiotic resistance gene, first found 2 carrying new array of gene cassettes in the form of integration.
4, the positive rate of I class integron 3 '- terminal qacE Delta 1-sul1 and intI1 class integrase gene is the same as that of the positive strain, indicating that these 2 genes are closely related to intI1, and can be used as the marker genes of I class integrons.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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