孔雀石绿对整合子捕获耐药性基因盒频率的影响
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摘要
目的通过测定添加与不添加孔雀石绿影响的大肠杆菌BL21(DE3)中整合子的整合频率,研究消毒剂孔雀石绿对整合子捕获耐药性基因盒频率的影响。方法将含有整合子和氨基糖苷腺苷转移酶(aad A2)基因盒的质粒和高表达整合酶的质粒同时导入大肠杆菌工程菌株BL21(DE3)中,命名为HS2。试验组将20株HS2菌株接种于含有20μg/ml孔雀石绿的LB液体培养基中37℃培养16 h,对照组将20株HS2菌株接种于普通LB液体培养基中在37℃下培养16 h。用荧光定量PCR法分别测定两组发生整合的整合子拷贝数和总整合子拷贝数,两数之比即为整合频率。比较两组大肠杆菌的整合频率。结果荧光定量PCR测定对照组的HS2菌株整合频率为(8. 80±2. 61)×10-5,试验组整合频率为(3. 97±3. 79)×10-5。两组差异具有统计学意义(P <0. 05)。结论孔雀石绿可使整合子捕获耐药基因盒频率下降。
Objective To study the effect of disinfectant malachite green on the frequency of integron-capturing antibiotic resistance gene cassette by measuring the integration frequency of integron in E. coli BL21( DE3) with or without malachite green. Methods A plasmid containing the integron and aminoglycoside adenosyltransferase( aad A2) gene cassette and plasmids overexpressing the integrase were transformed into E. coli BL21( DE3) strains,which was named as HS2. The 20 HS2 strains were added to LB liquid medium containing 20 μg/ml malachite green for 16 h at 37 ℃ in test group. The 20 HS2 strains were added to ordinary LB liquid medium in37 ℃ for 16 h in control group. The copy numbers of total integrons and the copy number of integrated aad A2 of integrons were determined using real-time PCR,and the integration frequency was the result of the former divided by the latter. The integration frequency of E. coli between two groups. Results The integration frequency of HS2 strains was( 8. 80 ± 2. 61) × 10-5 in control group and( 3. 97 ±3. 79) × 10-5 in test group,and the difference was statistically significant( P < 0. 05). Conclusion Malachite green can reduce the frequency of integron-capturing antibiotic resistance gene cassette.
Objective To study the effect of disinfectant malachite green on the frequency of integron-capturing antibiotic resistance gene cassette by measuring the integration frequency of integron in E. coli BL21( DE3) with or without malachite green. Methods A plasmid containing the integron and aminoglycoside adenosyltransferase( aad A2) gene cassette and plasmids overexpressing the integrase were transformed into E. coli BL21( DE3) strains,which was named as HS2. The 20 HS2 strains were added to LB liquid medium containing 20 μg/ml malachite green for 16 h at 37 ℃ in test group. The 20 HS2 strains were added to ordinary LB liquid medium in37 ℃ for 16 h in control group. The copy numbers of total integrons and the copy number of integrated aad A2 of integrons were determined using real-time PCR,and the integration frequency was the result of the former divided by the latter. The integration frequency of E. coli between two groups. Results The integration frequency of HS2 strains was( 8. 80 ± 2. 61) × 10-5 in control group and( 3. 97 ±3. 79) × 10-5 in test group,and the difference was statistically significant( P < 0. 05). Conclusion Malachite green can reduce the frequency of integron-capturing antibiotic resistance gene cassette.
引文
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[8]黄永茂,吴志鹃,陈枫,等.大肠埃希菌Ⅰ类整合子与多药耐药的相关性探讨[J].中华医院感染学杂志,2011,21(14):2882-2885.
[9] Zhang Y,Gu AZ,He M,et al. Subinhibitory concentrations of disinfectants promote the horizotal transfer of multidrug resistance genes within and across genera[J]. Environ Sci Technol,2017,51:570-580.
[10]季宇彬,孙莹,张青扬,等.新型孔雀石绿衍生物的合成及其生物活性研究[J].中国医药生物技术,2018,13(1):24-30.
[11] Alderman DJ. Malachite green:a review[J]. J Fish Dis,1985,8(3):289-298.
[12] Boulos RA,Man NY,Lengkeek NA. Inspiration from old dyes:tris(stilbene)compounds as potent gram-positive antibacterial agents[J]. Chemistry,2013,19(52):17980-17988.
[2] Escudero JA,Loot C,Nivina A,et al. The integron:adaptation on demand[J]. Microbiol Spectr,2015,3(2):19-41.
[3]杨泽华,胡敏,周双艳,等.抗菌药物浓度对整合子内耐药基因盒重组效率影响的研究[J].中华检验医学杂志,2018,41(7):531-535.
[4]胡敏,杨泽华,赵克斌.饥饿应激对整合子捕获耐药性基因盒频率影响的研究[J].中国药物与临床,2013,13(7):837-840.
[5]王卓,王修银,赖沛炼,等.多重耐药铜绿假单胞菌整合子和ISCR1结构及基因型分型研究[J].医学检验与临床,2017,28(2):20-22.
[6] Hall R M. Integrons and gene cassettes:hotspots of diversity in bacterial genomes[J]. Ann NY Acad Sci,2012,1267(1):71-78.
[7]罗永鸿,黄永茂,林雁,等.耐多药大肠埃希菌整合子分布与细菌耐药关系分析[J].西南军医,2018,20(3):319-322.
[8]黄永茂,吴志鹃,陈枫,等.大肠埃希菌Ⅰ类整合子与多药耐药的相关性探讨[J].中华医院感染学杂志,2011,21(14):2882-2885.
[9] Zhang Y,Gu AZ,He M,et al. Subinhibitory concentrations of disinfectants promote the horizotal transfer of multidrug resistance genes within and across genera[J]. Environ Sci Technol,2017,51:570-580.
[10]季宇彬,孙莹,张青扬,等.新型孔雀石绿衍生物的合成及其生物活性研究[J].中国医药生物技术,2018,13(1):24-30.
[11] Alderman DJ. Malachite green:a review[J]. J Fish Dis,1985,8(3):289-298.
[12] Boulos RA,Man NY,Lengkeek NA. Inspiration from old dyes:tris(stilbene)compounds as potent gram-positive antibacterial agents[J]. Chemistry,2013,19(52):17980-17988.