Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.
Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
Clin Microbiol Infect. 2024 Jun;30(6):787-794. doi: 10.1016/j.cmi.2024.03.015. Epub 2024 Mar 22.
Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics.
Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles.
We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin.
Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.
噬菌体（噬菌体）治疗是一种有前途的抗感染选择，可对抗抗菌药物耐药性。然而，噬菌体治疗的临床应用受到噬菌体耐药性出现的严重影响。尽管某些噬菌体耐药机制可以恢复细菌对某些抗生素的敏感性，但缺乏对噬菌体耐药机制的了解会阻碍噬菌体的最佳利用及其与抗生素的联合应用。
用肺炎克雷伯氏菌 MKP103 的突变文库进行全基因组转座子筛选，以鉴定噬菌体 pKMKP103_1 抗性突变体。通过时间杀伤动力学和效率平板测定评估噬菌体抗性表型。通过吸附、一步生长和突变频率测定研究噬菌体耐药机制。用肉汤微量稀释法和种群分析谱测定抗生素敏感性。
我们观察到肺炎克雷伯氏菌中有一系列噬菌体耐药机制，如噬菌体结合中断（fhuA::Tn 和 tonB::Tn）、噬菌体潜伏期延长（mnmE::Tn 和 rpoN::Tn）和突变频率增加（mutS::Tn 和 mutL::Tn）。值得注意的是，与噬菌体耐药重新使抗生素耐药细菌敏感的普遍观点相反，我们观察到对细菌抗生素敏感性的双向引导效应。具体而言，rpoN::Tn 增加了对粘菌素的敏感性，而 mutS::Tn 和 mutL::Tn 增加了对利福平霉素和粘菌素的耐药性。
我们的发现表明，肺炎克雷伯氏菌采用多种策略来克服噬菌体感染，这可能导致抗生素敏感性增强或降低。应将机制导向的噬菌体引导纳入噬菌体治疗中，以便更好地为噬菌体-抗生素联合应用的临床决策提供信息。
