Menashe Ofir, Kaganskaya Elena, Baasov Timor, Yaron Sima
Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Antimicrob Agents Chemother. 2008 Mar;52(3):920-6. doi: 10.1128/AAC.00382-07. Epub 2008 Jan 2.
The high antibacterial activity and selectivity of aminoglycosides and their low activity against intracellular bacteria associated with eukaryotic cells make them the antibiotics of choice for the elimination of extracellular bacteria during intracellular studies. Given the evidence that aminoglycosides can penetrate the eukaryotic cell membrane, the goal of this study was to examine the influence of aminoglycosides on macrophage-associated Salmonella. Herein, we show that gentamicin, kanamycin, and tobramycin at concentrations between 15 to 150 microg ml(-1) do not kill intracellular Salmonella but have other effects on the bacterial physiology. By using Salmonella enterica serovars Typhimurium and Virchow harboring luciferase reporter plasmid, we observed that the light produced by intracellular Salmonella declined immediately upon exposure to aminoglycosides, indicating that the bacteria were under stress. The extent of this effect was dependent on the macrophage host, on the identity of the aminoglycoside and its concentration, on the exposure time, and on the Salmonella serovar. Salmonella associated with Nramp1-negative macrophages, in which the phagosomal pH is higher, were more susceptible to aminoglycosides than Salmonella associated with Nramp1-expressing macrophages. These results verify that aminoglycosides affect intracellular bacteria and that the extent of this effect is dependent on the acidity level within the phagosome, suggesting that for the study of intracellular bacteria, the aminoglycoside concentration should be limited to two to five times the MIC for the bacterial strain studied. This precaution should guarantee the complete execution of extracellular bacteria with minimal effects on the intracellular bacteria and the host cells.
氨基糖苷类抗生素具有高抗菌活性和选择性，且对与真核细胞相关的细胞内细菌活性较低，这使得它们成为细胞内研究中消除细胞外细菌的首选抗生素。鉴于有证据表明氨基糖苷类抗生素可穿透真核细胞膜，本研究的目的是考察氨基糖苷类抗生素对巨噬细胞相关沙门氏菌的影响。在此，我们表明庆大霉素、卡那霉素和妥布霉素在浓度为15至150微克/毫升时不会杀死细胞内的沙门氏菌，但会对细菌生理学产生其他影响。通过使用携带荧光素酶报告质粒的鼠伤寒沙门氏菌和维尔肖沙门氏菌血清型，我们观察到细胞内沙门氏菌在接触氨基糖苷类抗生素后立即发出的光减弱，这表明细菌处于应激状态。这种影响的程度取决于巨噬细胞宿主、氨基糖苷类抗生素的种类及其浓度、暴露时间以及沙门氏菌血清型。与Nramp1阴性巨噬细胞相关的沙门氏菌（其吞噬体pH值较高）比与表达Nramp1的巨噬细胞相关的沙门氏菌对氨基糖苷类抗生素更敏感。这些结果证实氨基糖苷类抗生素会影响细胞内细菌，且这种影响的程度取决于吞噬体内的酸度水平，这表明对于细胞内细菌的研究，氨基糖苷类抗生素的浓度应限制在所研究细菌菌株最低抑菌浓度的两到五倍。这种预防措施应能确保在对细胞内细菌和宿主细胞影响最小的情况下完全清除细胞外细菌。
