本文關(guān)鍵詞： 銅綠假單胞菌 瓊脂稀釋法 氨基糖苷類修飾酶基因 16SrRNA甲基化酶基因 聚類分析 整合子 多重耐藥 PCR RFLP 耐藥基因盒　出處：《廣州醫(yī)學(xué)院》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：銅綠假單胞菌( Pseudomonas aeruginosa, Pae)是院內(nèi)感染的主要病原菌之一。在中國重癥監(jiān)護病房分離的革蘭陰性菌中,銅綠假單胞菌位列第一。由于具有天然和獲得性耐藥,銅綠假單胞菌對很多抗菌藥物耐藥,給臨床治療帶來很大困難。β-內(nèi)酰胺類、氨基糖苷類、喹諾酮類抗菌藥物是臨床治療Pae感染的常用藥,近年來隨著新的耐藥基因的不斷出現(xiàn),Pae對這三類抗菌藥物的耐藥性普遍增加,甚至出現(xiàn)多藥耐藥(MDR)和泛耐藥(PDR)菌株。 因氨基糖苷類抗生素抗菌譜廣、療效卓越,在醫(yī)學(xué)臨床和畜牧獸醫(yī)業(yè)應(yīng)用廣泛,但由于濫用和過度使用氨基糖苷類藥物的耐藥問題隨之凸現(xiàn),細菌對該類藥物耐藥是因為細菌產(chǎn)生氨基糖苷類修飾酶(AMEs)、16SrRNA甲基化酶和氨基糖苷類抗生素作用靶位16S rRNA基因突變而致。氨基糖苷類修飾酶按功能可分成乙酰轉(zhuǎn)移酶(AAC)、磷酸轉(zhuǎn)移酶(APH)、核苷轉(zhuǎn)移酶(ANT)三類,分別修飾氨基糖苷藥物相應(yīng)位置上的- OH和- NH2 ,降低或喪失其對靶位核糖體的親和力。編碼AMEs的基因起源于抗生素產(chǎn)生菌,該類基因所表達的酶在抗生素產(chǎn)生菌中起到自我保護作用,許多研究表明抗藥性產(chǎn)生菌所存在的自我保護機制與臨床耐藥菌的耐藥機制相同。 整合子是一個能夠通過自身編碼的整合酶來獲取保外游離基因或基因片段并使之表達的遺傳元件系統(tǒng)。整合子既可位于細菌染色體上,又可位于質(zhì)粒上,還可位于轉(zhuǎn)座子上。對細菌抗生素耐藥機制的研究,導(dǎo)致了包括轉(zhuǎn)座子和共軛質(zhì)粒在內(nèi)的許多移動元件的發(fā)現(xiàn),而通過對這些元件序列進行比較分析后,最終發(fā)現(xiàn)了整合子的存在。 研究目的 銅綠假單胞菌是院內(nèi)感染的主要條件致病菌之一,可引起嚴(yán)重的院內(nèi)獲得性感染。本研究試圖了解臨床常用的七種抗生素對60株銅綠假單胞菌(30株分離自門診病人,30株分離自住院病人)的抗菌活性和多黏菌素B與其它六種抗菌藥物(慶大霉素、奈替米星、阿米卡星、妥布霉素、環(huán)丙沙星、亞胺培南)體外聯(lián)合抗菌效應(yīng),以及各種抗菌藥物對不同標(biāo)本來源菌株的抗菌效應(yīng)有無差別;了解氨基糖苷類耐藥相關(guān)基因和16SrRNA甲基化酶基因存在情況以及菌株之間的親緣性;另外通過對臨床分離的銅綠假單胞菌的耐藥特征進行分析、檢測相關(guān)耐藥基因和整合子攜帶情況等,初步探討銅綠假單胞菌對臨床常用抗菌藥物的分子耐藥機制,從而了解不同的耐藥機制間相互作用規(guī)律以及耐藥基因在菌株間水平傳播的特點,為臨床用藥提供實驗室依據(jù)和幫助。 研究方法 1、藥敏試驗和聯(lián)合藥敏試驗 60株銅綠假單胞菌均來自廣州醫(yī)學(xué)院第一附屬醫(yī)院的臨床標(biāo)本,對這些菌株采用瓊脂稀釋法測定7種臨床常用抗生素的最低抑菌濃度(Minimal inhibitory concentration,MIC)和藥敏情況,分析銅綠假單胞菌對這些抗生素的藥物敏感性;同時檢測多粘菌素B與其它6種藥物的聯(lián)合藥敏情況,分析它們之間的聯(lián)合藥敏情況,試圖開辟治療Pae感染新的途徑,在用藥選擇、降低用藥量和毒副作用方面起到一定作用。 2、多藥耐藥銅綠假單胞菌氨基糖苷類修飾酶和16SrRNA甲基化酶基因分析 針對臨床分離的Pae菌株,采用聚合酶鏈反應(yīng)分析氨基糖苷類修飾酶、16SrRNA甲基化酶基因型及其它基因型,運用SPSS統(tǒng)計分析軟件對菌株樣本親緣性做聚類分析。 3、整合子的鑒定與分析 利用PCR方法,通過擴增整合子恒定區(qū)的方法,篩選攜帶整合子的菌株,并利用限制性片段多態(tài)性性(RFLP)酶切分析的方法對這些整合子進行分類;對整合子恒定區(qū)擴增陽性的菌株進行測序和序列分析。探討整合子攜帶的耐藥基因盒是否介導(dǎo)銅綠假單胞菌的多重耐藥。 研究結(jié)果 1、銅綠假單胞菌對三類六種抗生素耐藥率普遍較高,分別為奈替米星68.3%、妥布霉素65.0%、慶大霉素60.0%、環(huán)丙沙星46.7%、亞氨培南40.0%、阿米卡星13.3%,而多黏菌素B的耐藥率為0;這些菌株中多重耐藥和泛耐藥菌的檢出率分別為55.0%(33/60)和31.7%(19/60)。多黏菌素B與環(huán)丙沙星、亞氨培南有明顯聯(lián)合抗菌效應(yīng),多黏菌素B與奈替米星聯(lián)合,能明顯降低彼此的MICG。各種抗生素對住院病例分離菌株和門診病例分離菌株,在MICG和差異顯著性方面表現(xiàn)各不相同。 2、21株氨基糖苷類耐藥菌株中(其中20株為多藥耐藥菌株),氨基糖苷類耐藥基因型aac(6,)-Ⅰ陽性13株(61.9%)、aac(6,)-Ⅱ陽性13株(61.9%)、ant(2,,)-Ⅰ陽性10株(47.6%)、ant(3,,)-Ⅰ陽性9株(42.9%)、aac(3)-Ⅱ陽性的有1株(4.8%),另有1株菌oprD2基因缺失,未檢出基因型aac(6,)-Ⅰae、aph(3,)-Ⅲ、aac(6,)-aph(2,,)和ant(4,)-Ⅰ;16SrRNA甲基化酶基因rmtA基因型陽性19株(90.4%)、armA基因型陽性有8株(38.1%),未檢出基因型rmtC、rmtD。聚類分析結(jié)果顯示分離的菌株中存在克隆傳播。 3、30株臨床分離銅綠假單胞菌中16株(53.3%)整合子恒定區(qū)和可變區(qū)擴增陽性,片段長度在0.7-4kb之間。共檢出5種不同的整合子組合形式,含有編碼對氨基糖苷類、β-內(nèi)酰胺類和喹喏酮類抗菌藥耐藥的基因,其中有2例為新型基因盒組合形式,包括aacA4-VIM2和aadA2-OXA10-aacA4-blaIMP9-aatI1,Genbank基因庫登陸號分別為GQ890658和GU122165,另外3例分別與Genbank基因庫登錄號為FJ917747、FJ817423、GU367339的序列吻合。 4、I類整合子3′-末端qacEΔ1-sul1和intI1類整合酶基因的陽性率和陽性菌株相同。 結(jié)論 1、臨床檢出的銅綠假單胞菌對常用抗生素多表現(xiàn)為多重耐藥。多黏菌素B單用有很好的抗銅綠假單胞菌效果,與環(huán)丙沙星(喹諾酮類)、亞氨培南(β-內(nèi)酰胺類)聯(lián)合應(yīng)用也有明顯的聯(lián)合抗菌效應(yīng)。對多藥耐藥和泛耐藥菌株,為減少用藥量和毒副作用,提高抗菌效果,可考慮多黏菌素B與這些抗生素聯(lián)合用藥。 2、大部分測試的銅綠假單胞菌對臨床常用的銅綠假單胞菌抗感染藥物已產(chǎn)生廣泛耐藥,尤其對氨基糖苷類抗生素。這些菌株的氨基糖苷類修飾酶常見耐藥基因型檢出率高, 16SrRNA甲基化酶基因型rmtA和armA的檢出率亦較高。30株測試菌株中存在克隆傳播。 3、本研究中攜帶intI1類整合子的16株P(guān)ae對氨基糖苷類全部耐藥,對喹喏酮類耐藥的有13株,對亞氨培南耐藥的有10株,對所有三類抗生素全部耐藥的有8株占50%,說明目前引起Pae耐藥的機制中,整合子耐藥機制越來越起著重要的作用。銅綠假單胞菌的耐藥和多重耐藥與整合子密切相關(guān),主要攜帶氨基糖苷類和β內(nèi)酰胺類抗生素耐藥基因,首次發(fā)現(xiàn)2種攜帶新型基因盒組合形式的整合子。 4、I類整合子3′-末端qacEΔ1-sul1和intI1類整合酶基因的陽性率和陽性菌株相同,表明這2個基因是緊密伴隨intI1而存在的,可作為I類整合子的標(biāo)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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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