Institute of Medical Microbiology, RWTH Aachen University Hospital, 52074 Aachen, Germany.
Electron Microscopy Facility, RWTH Aachen University Hospital, 52074 Aachen, Germany.
Virus Res. 2024 Sep;347:199417. doi: 10.1016/j.virusres.2024.199417. Epub 2024 Jun 20.
Due to the spread of multidrug resistance there is a renewed interest in using bacteriophages (briefly: phages) for controlling bacterial pathogens. The objective of this study was the characterization of a newly isolated phage (i.e. phage LAPAZ, vB_KpnD-LAPAZ), its antimicrobial activity against multidrug resistant Klebsiella pneumoniae and potential synergistic interactions with antibiotics. LAPAZ belongs to the family Drexlerviridae (genus: Webervirus) and lysed 30 % of tested strains, whereby four distinct capsular types can be infected. The genome consists of 51,689 bp and encodes 84 ORFs. The latent period is 30 min with an average burst size of 27 PFU/cell. Long-term storage experiments show that LAPAZ is significantly more stable in wastewater compared to laboratory media. A phage titre of 90 % persists up to 30 min at 50 ˚C and entire phage loss was seen only at temperatures > 66 ˚C. Besides stability against UV-C, antibacterial activity in liquid culture medium was consistent at pH values ranging from 4 to 10. Unlike exposure to phage or antibiotic alone, synergistic interactions and a complete bacterial eradication was achieved when combining LAPAZ with meropenem. In addition, synergism with the co-presence of ciprofloxacin was observed and phage resistance emergence could be delayed. Without co-addition of the antibiotic, phage resistant mutants readily emerged and showed a mixed pattern of drug sensitivity alterations. Around 88 % became less sensitive towards ceftazidime, meropenem and gentamicin. Conversely, around 44 % showed decreased resistance levels against ciprofloxacin. Whole genome analysis of a phage-resistant mutant with a 16-fold increased sensitivity towards ciprofloxacin revealed one de novo frameshift mutation leading to a gene fusion affecting two transport proteins belonging to the major facilitator-superfamily (MFS). Apparently, this mutation compromises ciprofloxacin efflux efficiency and further studies are warranted to understand how the non-mutated protein might be involved in phage-host adsorption.
由于多药耐药性的传播，人们重新对利用噬菌体（简称噬菌体）来控制细菌病原体产生了兴趣。本研究的目的是对一种新分离的噬菌体（即噬菌体 LAPAZ，vB_KpnD-LAPAZ）进行表征，研究其对多药耐药性肺炎克雷伯菌的抗菌活性以及与抗生素的潜在协同作用。LAPAZ 属于 Drexlerviridae 科（属：Webervirus），可裂解 30%的测试菌株，其中可感染 4 种不同的荚膜型。基因组由 51689bp 组成，编码 84 个 ORF。潜伏期为 30 分钟，平均爆发量为 27PFU/细胞。长期储存实验表明，与实验室培养基相比，LAPAZ 在废水中的稳定性显著提高。噬菌体效价为 90%时，在 50°C 下可保持 30 分钟，只有在温度高于 66°C 时才会完全丧失噬菌体。除了对 UV-C 的稳定性外，在 pH 值为 4 到 10 的液体培养基中，抗菌活性也保持一致。与单独暴露于噬菌体或抗生素不同，当将 LAPAZ 与美罗培南联合使用时，可实现协同作用并完全清除细菌。此外，还观察到与环丙沙星共存的协同作用，并可延迟噬菌体耐药性的出现。没有抗生素的共同添加，噬菌体耐药突变体很容易出现，并显示出药物敏感性改变的混合模式。约 88%的突变体对头孢他啶、美罗培南和庆大霉素的敏感性降低。相反，约 44%的突变体对环丙沙星的耐药水平降低。对一个对环丙沙星的敏感性增加 16 倍的噬菌体耐药突变体进行全基因组分析，发现一个新的移码突变导致基因融合，影响属于主要易化子超家族（MFS）的两个转运蛋白。显然，这种突变会降低环丙沙星的外排效率，需要进一步的研究来了解未突变的蛋白如何参与噬菌体-宿主吸附。
