亚抑菌浓度抗生素对海水浴场来源肺炎克雷伯菌的接合及intI突变影响
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摘要
目的探讨不同水环境介质及亚抑菌浓度抗生素对肺炎克雷伯菌的生长、耐药表型及抗性基因转移的影响。方法对海水浴场来源的肺炎克雷伯菌进行不同亚抑菌浓度的氧氟沙星、头孢他啶抗生素传代培养、运用DNAstar分析I类整合子(IntI)突变类型及突变位点,以耐药菌为供体菌,EC600为受体菌进行耐青霉素、氧氟沙星的质粒接合实验。结果经亚抑菌浓度抗生素研究发现2种抗生素亚抑菌浓度海水与生理盐水传代培养下的细菌生物量均有明显差异。但头孢他啶亚抑菌浓度下,海水介质比生理盐水介质的影响大。传代培养240 h后,细菌耐药能力增强。亚抑菌浓度抗生素作用下耐药菌的IntI突变类型主要集中于氨基酸的缺失与插入,突变位点集中在262～273。此外,4株耐青霉素耐药基因肺炎克雷伯菌成功接合,接合效率分别是2.6×10~(-6)、4.2×10~(-6)、6.0×10~(-6)、3.6×10~(-6),1株氧氟沙星1/16mic亚抑菌浓度刺激下的肺炎克雷伯菌成功接合,接合效率是6.4×10~(-6)。结论亚抑菌浓度抗生素对肺炎克雷伯菌传代培养存在差异,有诱导自发性突变的可能;证明了耐药基因可伴随接合型质粒转移。
Objective To explore the effects of different water environment medium and sub-inhibitory concentrations of antibiotics on Klebsiella pneumoniae growth, drug resistance phenotype and resistance gene transfer. Methods Klebsiella pneumoniae from bathing beach was sub-cultured with ofloxacin and ceftazidime at different sub-inhibitory concentrations. The mutation type and mutation site of class I integron(IntI) were analyzed by DNAstar. The resistant strains were used as the donor strain, and EC600 was used as the recipient strain. The recipient bacteria performed a plasmid conjugation test for penicillin and ofloxacin. Results There were significant differences in bacterial biomass between the two sub-inhibitor antibiotic concentrations in seawater and physiological saline. However, under the concentration of ceftidine, the saline medium had greater influence than the seawater medium. After culture of 240 h, bacterial resistance increased. The IntI mutation type of antibiotic-resistant bacteria under the influence of sub-inhibitory concentrations of antibiotics was mainly concentrated on the deletion and insertion of amino acids, and the mutation sites were concentrated from 262 to 273. In addition, four Klebsiella pneumoniae-resistant penicillin-resistant pneumonia strains were successfully integrated at a binding efficiency of 2.6×10~(-6), 4.2×10~(-6), 6.0×10~(-6), and 3.6×10~(-6), respectively. The Klebsiella pneumoniae strain stimulated by a sub-anti-bacteria concentration of 1/16 mic was successfully integrated, and the conjugation efficiency was 6.4×10~(-6). Conclusion Sub-antibacterial concentrations of antibiotics differed in the subculture of Klebsiella pneumoniae, leading to the possibility of spontaneous mutations; it was demonstrated that drug-resistant genes could be transferred by the conjugative plasmid.
Objective To explore the effects of different water environment medium and sub-inhibitory concentrations of antibiotics on Klebsiella pneumoniae growth, drug resistance phenotype and resistance gene transfer. Methods Klebsiella pneumoniae from bathing beach was sub-cultured with ofloxacin and ceftazidime at different sub-inhibitory concentrations. The mutation type and mutation site of class I integron(IntI) were analyzed by DNAstar. The resistant strains were used as the donor strain, and EC600 was used as the recipient strain. The recipient bacteria performed a plasmid conjugation test for penicillin and ofloxacin. Results There were significant differences in bacterial biomass between the two sub-inhibitor antibiotic concentrations in seawater and physiological saline. However, under the concentration of ceftidine, the saline medium had greater influence than the seawater medium. After culture of 240 h, bacterial resistance increased. The IntI mutation type of antibiotic-resistant bacteria under the influence of sub-inhibitory concentrations of antibiotics was mainly concentrated on the deletion and insertion of amino acids, and the mutation sites were concentrated from 262 to 273. In addition, four Klebsiella pneumoniae-resistant penicillin-resistant pneumonia strains were successfully integrated at a binding efficiency of 2.6×10~(-6), 4.2×10~(-6), 6.0×10~(-6), and 3.6×10~(-6), respectively. The Klebsiella pneumoniae strain stimulated by a sub-anti-bacteria concentration of 1/16 mic was successfully integrated, and the conjugation efficiency was 6.4×10~(-6). Conclusion Sub-antibacterial concentrations of antibiotics differed in the subculture of Klebsiella pneumoniae, leading to the possibility of spontaneous mutations; it was demonstrated that drug-resistant genes could be transferred by the conjugative plasmid.
引文
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[19] 袁慧. 亚抑菌浓度抗生素对 E.coli-PA 接合反应的影响[D]. 广州: 广州中医药大学,2014.
[2] 袁经松, 方菁. 我国细菌对抗生素耐药性监测的研究进展[J]. 中国卫生检验杂志, 2015,25(4):605-608.
[3] 鲁曦, 金发光, 徐冬旸,等. 3种亚抑菌浓度抗生素对耐药基因水平传播的影响[J]. 中国抗生素杂志, 2014, 39(5):379-384.
[4] 蒋魁, 徐力文, 苏友禄,等. 2012年～2014年南海海水养殖鱼类病原菌哈维弧菌分离株的耐药性分析[J]. 南方水产科学, 2016, 12(6):99-107.
[5] 李情操, 魏取好, 卢雯君,等. 整合子与细菌耐药相关性研究进展[J]. 检验医学, 2016, 31(4):324-328.
[6] 张碧娇, 应建飞, 鲁勇. 临床分离阴沟肠杆菌耐药性与I类整合子关系研究[J]. 中国卫生检验杂志, 2017, 27(8):1197-1198.
[7] 国宪虎. 耐药整合子细菌在水环境中的散播调查及其多样性分析[D]. 济南,山东大学, 2011.
[8] Bulman ZP, Liang C, Walsh TJ, et al. Polymyxin Combinations Combat Escherichia coli Harboring mcr-1 and blaNDM-5: Preparation for a Postantibiotic Era[J]. Mbio, 2017, 8(4): e00540-17.
[9] 余中华, 刘家扬, 谌馥佳,等. 2株依赖海水细菌的分离培养和鉴定[J]. 热带农业科学, 2015, 35(6):50-55.
[10] 翁梦薇, 计徐, 郑卫江,等. 去甲肾上腺素对大肠杆菌耐药质粒接合转移的影响[J]. 南京农业大学学报, 2017, 40(5):901-908.
[11] 夏照帆, 吕开阳, 汤陈琪,等. 我国细菌耐药问题的现状和防控策略[J]. 中国工程科学, 2017, 19(2):106-111.
[12] 王冰, 何新宇, 周洋洋,等. 外源化学物质对副溶血性弧菌药物敏感性的影响[J]. 微生物学通报, 2017, 44(8):1785-1792.
[13] 熊家声, 陈亮, 余国荣,等. 上海市某医院污水与居民小区生活污水中耐热大肠菌群耐药性的对比研究[J]. 环境与职业医学, 2016, 33(2):167-170.
[14] 史惠卿. 亚-MIC头孢他啶、头孢哌酮对大肠埃希菌生物膜形成的影响及机制研究[D].重庆,第三军医大学,2013.
[15] 王方舟, 倪文涛, 崔俊昌. 抗菌药物亚抑菌浓度及其临床意义[J].中国感染与化疗杂志, 2017, 17(5):597-601.
[16] Ayrapetyan M, Williams TC, Oliver JD. Bridging the gap between viable but non-culturable and antibiotic persistent bacteria[J]. Trends Microbiol, 2015, 23(1): 7-13.
[17] 常爱英, 于艳妮, 宋文冲,等. 威海地区幽门螺杆菌耐药谱表型及遗传特征分析[J]. 中国微生态学杂志, 2015, 27(2) :182-183.
[18] Kubanov AA, Leinsoo AT, Chestkov AV, et al. Drug resistance mutations and susceptibility phenotypes of Neisseria gonorrhoeae isolates in Russia[J]. Mol Biol, 2017, 51(3): 431-441.
[19] 袁慧. 亚抑菌浓度抗生素对 E.coli-PA 接合反应的影响[D]. 广州: 广州中医药大学,2014.