主動(dòng)外排系統(tǒng)介導(dǎo)的鮑曼不動(dòng)桿菌耐藥機(jī)制研究
發(fā)布時(shí)間：2018-12-20 09:31
【摘要】： [目的]了解臨床分離54株鮑曼不動(dòng)桿菌的耐藥現(xiàn)狀及對(duì)外排泵底物的耐藥分析；探討主動(dòng)外排泵基因adeB、adeJ、abeM的在鮑曼不動(dòng)桿菌中的分布及其mRNA表達(dá)水平與該菌耐藥性的關(guān)系。 [方法]收集昆明醫(yī)學(xué)院第一附屬醫(yī)院臨床分離鮑曼不動(dòng)桿菌54株,采用K-B法檢測(cè)其對(duì)21種抗菌藥物的藥敏情況；用PCR方法擴(kuò)增鮑曼不動(dòng)桿菌外排泵編碼基因adeB、adeJ、abeM的片段；熒光RT-PCR法檢測(cè)外排系統(tǒng)基因mRNA的表達(dá)水平,采用分子克隆方法制作絕對(duì)定量標(biāo)準(zhǔn)品進(jìn)行熒光定量,并分析外排系統(tǒng)基因mRNA的表達(dá)水平與該菌耐藥性的關(guān)系。 [結(jié)果] 鮑曼不動(dòng)桿菌的耐藥檢測(cè)及外排泵底物的藥敏結(jié)果： 1、16種常用抗菌藥物藥敏結(jié)果顯示：慶大霉素(GEN)、甲氧芐啶／磺胺甲惡唑(SXT)、哌拉西林(PIP)、頭孢曲松(CRO)、四環(huán)素(TCY)、頭孢他啶(CAZ)、頭孢噻肟(CTX)、妥布霉素(TOB)、阿米卡星(AMK)的耐藥率高達(dá)70%左右,而對(duì)亞胺培南(IMP)、美洛培南(MEM)、頭孢哌酮／舒巴坦(CSL),仍保持較高的敏感性,其耐藥率均低于15%。54株鮑曼不動(dòng)桿菌耐5類抗菌藥物菌株15株(27.8%)、耐4類藥菌株15株(27.8%)、耐2類藥菌株9株(16.7%)、耐1類藥菌株9(16.7%)、敏感株6株(11.1%)。 2、54株鮑曼不動(dòng)桿菌對(duì)三種外排泵底物共10種抗菌藥物的耐藥率依次從高到低為林可霉素(LIN)100.0%、利福平(RIF)98.2%、氯霉素(CHL)96.4%、慶大霉素(GEN)75.0%、四環(huán)素(TET)71.4%、紅霉素(ERY)64.3%、阿米卡星(AMK)53.6%、妥布霉素(TOB)53.6%、環(huán)丙沙星(CIP)50.0%、左旋氧氟沙星(LVX)41.1%。 外排蛋白編碼基因adeB、adeJ、abeM的PCR擴(kuò)增及RT-PCR定量其mRNA的表達(dá)水平與該菌耐藥性分析結(jié)果： 1.adeB基因在54株鮑曼不動(dòng)桿菌中49株(90.7%)檢測(cè)到,未檢測(cè)到adeB基因的5株菌(9.3%)中,有4株是敏感株,1株為耐1類抗菌藥物株。多重耐藥株adeB基因mRNA的表達(dá)水平與敏感株、非多重耐藥株均有顯著性差異(P0.05)；非多重耐藥株與敏感株也有顯著性差異(P0.05)。外排泵AdeB的底物：慶大霉素、四環(huán)素、環(huán)丙沙星、左氧氟沙星,妥布霉素和阿米卡星在本次研究中,其mRNA表達(dá)水平耐藥株與敏感株之間有顯著性差異(P0.05)。 2、ade J基因本次研究顯示54株均檢測(cè)到。其mRNA表達(dá)水平耐藥株與敏感株之間無顯著性差異(P0.05)。外排泵底物藥物的耐藥株與敏感株之間也無顯著性差異(P0.05)。 3、abeM基因在54株中均檢測(cè)到。其mRNA表達(dá)水平耐藥菌株與敏感株有顯著性差異(P0.05)。外排泵AbeM的底物中環(huán)丙沙星、左氧氟沙星耐藥株與敏感株abeM基因mRNA表達(dá)水平有顯著性差異(P＜0.001)。[結(jié)論]鮑曼不動(dòng)桿菌耐藥形式嚴(yán)峻；其臨床分離株普遍存在外排泵編碼基因adeB、adeJ和abeM;主動(dòng)外排泵AdeABC和AbeM可能是鮑曼不動(dòng)桿菌發(fā)生耐藥的機(jī)制之一。
[Abstract]:[objective] to investigate the current situation of drug resistance of 54 strains of Acinetobacter baumannii and analysis of drug resistance of external pump substrates. To investigate the distribution of active efflux pump gene adeB,adeJ,abeM in Acinetobacter baumannii and the relationship between mRNA expression level and drug resistance of Acinetobacter baumannii. [methods] 54 strains of Acinetobacter baumannii isolated from the first affiliated Hospital of Kunming Medical College were collected and their susceptibility to 21 antimicrobial agents was detected by K-B method. The adeB,adeJ,abeM fragment of acinetobacter baumannii efflux pump coding gene was amplified by PCR. Fluorescence RT-PCR method was used to detect the expression level of efflux system gene mRNA, and molecular cloning method was used to make the absolute quantitative standard product for fluorescence quantification. The relationship between the expression level of efflux system gene mRNA and the drug resistance of the bacteria was analyzed. [results] the detection of drug resistance of Acinetobacter baumannii and the drug sensitivity of efflux pump substrates: 1. The results of 16 common antimicrobial agents showed that: gentamicin (GEN), trimethoprim / sulfamethoxazole (SXT), Piperacillin (PIP), ceftriaxone (CRO), tetracycline (TCY), ceftazidime (CAZ), cefotaxime (CTX), tobramycin (TOB), (AMK) resistance to imipenem (IMP), The sensitivity of (MEM), cefoperazone / sulbactam (CSL), was higher than 15.54 strains of Acinetobacter baumannii and 15 strains (27.8%) of Acinetobacter baumannii resistance. 15 strains (27.8%) were resistant to 4 kinds of drugs, 9 strains (16.7%) were resistant to 2 drugs, 9 (16.7%) were resistant to class 1, 6 strains (11.1%) were sensitive. (2) the drug resistance rates of Acinetobacter baumannii strains to three efflux pump substrates were lincomycin (LIN) 100.0g, rifampicin (RIF) 98.2i, chloramphenicol (CHL) 96.4B from high to low. Gentamycin (GEN) 75.0, tetracycline (TET) 71.4, erythromycin (ERY) 64.3, amicacin (AMK) 53.6, tobramycin (TOB) 53.6, ciprofloxacin (CIP) 50.0. Levofloxacin (LVX) 41.1. The results of PCR amplification and RT-PCR quantitative analysis of mRNA expression and drug resistance of efflux protein encoding gene adeB,adeJ,abeM showed that 1.adeB gene was detected in 49 strains (90.7%) of 54 Acinetobacter baumannii strains. Of the 5 strains (9.3%) with no adeB gene detected, 4 strains were sensitive and 1 strain was resistant to class 1 antimicrobial agents. The expression level of adeB gene mRNA in multidrug resistant strain was significantly higher than that in sensitive strain and non-multidrug resistant strain (P0.05), and there was also significant difference between non-multidrug resistant strain and sensitive strain (P0.05). In this study, the mRNA expression levels of gentamicin, tetracycline, ciprofloxacin, levofloxacin, tobramycin and amikacin were significantly different from those of sensitive strains (P0.05). 2Ade J gene was detected in 54 strains. There was no significant difference in mRNA expression between resistant and sensitive strains (P0.05). There was also no significant difference between the drug resistant strains and the sensitive strains of the efflux pump substrates (P0.05). The gene was detected in 54 strains. There was significant difference in mRNA expression between resistant strains and sensitive strains (P0.05). The mRNA expression of ciprofloxacin, levofloxacin resistant and sensitive AbeM substrates in efflux pump AbeM was significantly different (P < 0.001). [conclusion] the drug resistance of Acinetobacter baumannii is severe and the gene adeB,adeJ and abeM; active efflux pump AdeABC and AbeM may be one of the mechanisms of drug resistance of Acinetobacter baumannii.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378.99
本文編號(hào)：2387799
[Abstract]:[objective] to investigate the current situation of drug resistance of 54 strains of Acinetobacter baumannii and analysis of drug resistance of external pump substrates. To investigate the distribution of active efflux pump gene adeB,adeJ,abeM in Acinetobacter baumannii and the relationship between mRNA expression level and drug resistance of Acinetobacter baumannii. [methods] 54 strains of Acinetobacter baumannii isolated from the first affiliated Hospital of Kunming Medical College were collected and their susceptibility to 21 antimicrobial agents was detected by K-B method. The adeB,adeJ,abeM fragment of acinetobacter baumannii efflux pump coding gene was amplified by PCR. Fluorescence RT-PCR method was used to detect the expression level of efflux system gene mRNA, and molecular cloning method was used to make the absolute quantitative standard product for fluorescence quantification. The relationship between the expression level of efflux system gene mRNA and the drug resistance of the bacteria was analyzed. [results] the detection of drug resistance of Acinetobacter baumannii and the drug sensitivity of efflux pump substrates: 1. The results of 16 common antimicrobial agents showed that: gentamicin (GEN), trimethoprim / sulfamethoxazole (SXT), Piperacillin (PIP), ceftriaxone (CRO), tetracycline (TCY), ceftazidime (CAZ), cefotaxime (CTX), tobramycin (TOB), (AMK) resistance to imipenem (IMP), The sensitivity of (MEM), cefoperazone / sulbactam (CSL), was higher than 15.54 strains of Acinetobacter baumannii and 15 strains (27.8%) of Acinetobacter baumannii resistance. 15 strains (27.8%) were resistant to 4 kinds of drugs, 9 strains (16.7%) were resistant to 2 drugs, 9 (16.7%) were resistant to class 1, 6 strains (11.1%) were sensitive. (2) the drug resistance rates of Acinetobacter baumannii strains to three efflux pump substrates were lincomycin (LIN) 100.0g, rifampicin (RIF) 98.2i, chloramphenicol (CHL) 96.4B from high to low. Gentamycin (GEN) 75.0, tetracycline (TET) 71.4, erythromycin (ERY) 64.3, amicacin (AMK) 53.6, tobramycin (TOB) 53.6, ciprofloxacin (CIP) 50.0. Levofloxacin (LVX) 41.1. The results of PCR amplification and RT-PCR quantitative analysis of mRNA expression and drug resistance of efflux protein encoding gene adeB,adeJ,abeM showed that 1.adeB gene was detected in 49 strains (90.7%) of 54 Acinetobacter baumannii strains. Of the 5 strains (9.3%) with no adeB gene detected, 4 strains were sensitive and 1 strain was resistant to class 1 antimicrobial agents. The expression level of adeB gene mRNA in multidrug resistant strain was significantly higher than that in sensitive strain and non-multidrug resistant strain (P0.05), and there was also significant difference between non-multidrug resistant strain and sensitive strain (P0.05). In this study, the mRNA expression levels of gentamicin, tetracycline, ciprofloxacin, levofloxacin, tobramycin and amikacin were significantly different from those of sensitive strains (P0.05). 2Ade J gene was detected in 54 strains. There was no significant difference in mRNA expression between resistant and sensitive strains (P0.05). There was also no significant difference between the drug resistant strains and the sensitive strains of the efflux pump substrates (P0.05). The gene was detected in 54 strains. There was significant difference in mRNA expression between resistant strains and sensitive strains (P0.05). The mRNA expression of ciprofloxacin, levofloxacin resistant and sensitive AbeM substrates in efflux pump AbeM was significantly different (P < 0.001). [conclusion] the drug resistance of Acinetobacter baumannii is severe and the gene adeB,adeJ and abeM; active efflux pump AdeABC and AbeM may be one of the mechanisms of drug resistance of Acinetobacter baumannii.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378.99
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