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

Conjugation and mutations affection of intI of subinhibitory concentration antibiotics on the source of Klebsiella pneumonia

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.