Trans-translation is a unique bacterial ribosome rescue system that plays important roles in the tolerance to environmental stresses. It is composed of an ssrA-encoded tmRNA and a protein SmpB. In this study, we examined the role of trans-translation in antibiotic tolerance in Klebsiella pneumoniae and explored whether the inhibition of this mechanism could enhance the bactericidal activities of antibiotics. We found that deletion of the ssrA gene reduced the survival of K. pneumoniae after treatment with kanamycin, tobramycin, azithromycin, and ciprofloxacin, indicating an important role of the trans-translation in bacterial antibiotic tolerance. By using a modified ssrA gene with a 6×His tag we demonstrated that tobramycin suppressed the azithromycin and ciprofloxacin-elicited activation of trans-translation. The results were further confirmed with a trans-translation reporter system that is composed of a normal mCherry gene and a gfp gene without the stop codon. Compared to each individual antibiotic, combination of tobramycin with azithromycin or ciprofloxacin synergistically enhanced the killing activities against planktonic K. pneumoniae cells and improved bacterial clearance in a murine cutaneous abscess infection model. In addition, the combination of tobramycin and ciprofloxacin increased the bactericidal activities against biofilm-associated cells. Overall, our results suggest that the combination of tobramycin with azithromycin or ciprofloxacin is a promising strategy in combating K. pneumoniae infections.

反式翻译是一种独特的细菌核糖体救援系统，在耐受环境压力方面发挥着重要作用。它由ssrA编码的 tmRNA 和蛋白质 SmpB 组成。在本研究中，我们研究了反式翻译在肺炎克雷伯菌抗生素耐受中的作用，并探讨了抑制这一机制是否可以增强抗生素的杀菌活性。我们发现， ssrA基因的缺失降低了用卡那霉素、妥布霉素、阿奇霉素和环丙沙星治疗后肺炎克雷伯菌的存活率，表明反式翻译在细菌抗生素耐受性中的重要作用。通过使用带有 6×His 标签的修饰ssrA基因，我们证明妥布霉素抑制阿奇霉素和环丙沙星引起的反式翻译激活。该结果进一步通过由正常mCherry基因和不带终止密码子的gfp基因组成的反式翻译报告系统得到证实。与每种单独的抗生素相比，妥布霉素与阿奇霉素或环丙沙星的组合可协同增强对浮游肺炎克雷伯菌细胞的杀伤活性，并改善小鼠皮肤脓肿感染模型中的细菌清除率。此外，妥布霉素和环丙沙星的组合增加了对生物膜相关细胞的杀菌活性。总体而言，我们的结果表明，妥布霉素与阿奇霉素或环丙沙星的组合是对抗肺炎克雷伯菌感染的有前途的策略。