Department of Microbiology, Heersink School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
mBio. 2023 Jun 27;14(3):e0047223. doi: 10.1128/mbio.00472-23. Epub 2023 Apr 11.
Pf is a filamentous bacteriophage integrated in the chromosome of most clinical isolates of Pseudomonas aeruginosa. Under stress conditions, mutations occurring in the Pf genome result in the emergence of superinfective variants of Pf (SI-Pf) that are capable of circumventing phage immunity; therefore, SI-Pf can even infect Pf-lysogenized P. aeruginosa. Here, we identified specific mutations located between the repressor and the excisionase genes of Pf4 phage in the P. aeruginosa PAO1 strain that resulted in the emergence of SI-Pf. Based on these findings, we genetically engineered an SI-Pf (eSI-Pf) and tested it as a phage therapy tool for the treatment of life-threatening burn wound infections caused by PAO1. In validation experiments, eSI-Pf was able to infect PAO1 grown in a lawn as well as biofilms formed on polystyrene. eSI-Pf also infected PAO1 present in burned skin wounds on mice but was not capable of maintaining a sustained reduction in bacterial burden beyond 24 h. Despite not lowering bacterial burden in burned skin tissue, eSI-Pf treatment completely abolished the capability of P. aeruginosa to disseminate from the burn site to internal organs. Over the course of 10 days, this resulted in bacterial clearance and survival of all treated mice. We subsequently determined that eSI-Pf induced a small-colony variant of P. aeruginosa that was unable to disseminate systemically. This attenuated phenotype was due to profound changes in virulence determinant production and altered physiology. Our results suggest that eSI-Pf has potential as a phage therapy against highly recalcitrant antimicrobial-resistant P. aeruginosa infections of burn wounds. Pseudomonas aeruginosa is a major cause of burn-related infections. It is also the most likely bacterial infection to advance to sepsis and result in burn-linked death. Frequently, P. aeruginosa strains isolated from burn patients display a multidrug-resistant phenotype necessitating the development of new therapeutic strategies and prophylactic treatments. In this context, phage therapy using lytic phages has demonstrated exciting potential in the control P. aeruginosa infection. However, lytic phages can present a set of drawbacks during phage therapy, including the induction of bacterial resistance and limited bacteria-phage interactions . Here, we propose an alternative approach to interfere with P. aeruginosa pathogenesis in a burn infection model, i.e., by using an engineered superinfective filamentous phage. Our study demonstrates that treatment with the engineered Pf phage can prevent sepsis and death in a burn mouse model.
Pf 是一种整合在大多数铜绿假单胞菌临床分离株染色体中的丝状噬菌体。在应激条件下，Pf 基因组中发生的突变会导致 Pf 的超感染变体 (SI-Pf) 的出现，这些变体能够规避噬菌体免疫；因此，SI-Pf 甚至可以感染 Pf 溶原化的铜绿假单胞菌。在这里，我们鉴定了 Pf4 噬菌体在铜绿假单胞菌 PAO1 菌株中的阻遏蛋白和切除酶基因之间的特定突变，这些突变导致了 SI-Pf 的出现。基于这些发现，我们对一种 SI-Pf（eSI-Pf）进行了基因工程改造，并将其作为噬菌体治疗工具，用于治疗由 PAO1 引起的危及生命的烧伤感染。在验证实验中，eSI-Pf 能够感染在草坪上生长的 PAO1 以及在聚苯乙烯上形成的生物膜。eSI-Pf 还能感染感染小鼠烧伤皮肤伤口中的 PAO1，但不能持续降低细菌负荷超过 24 小时。尽管未能降低烧伤皮肤组织中的细菌负荷，但 eSI-Pf 治疗完全消除了铜绿假单胞菌从烧伤部位向内部器官传播的能力。在 10 天的时间里，这导致了所有治疗小鼠的细菌清除和存活。随后，我们确定 eSI-Pf 诱导了一种无法系统传播的铜绿假单胞菌小菌落变体。这种减毒表型是由于毒力决定因子产生的深刻变化和生理改变所致。我们的结果表明，eSI-Pf 具有作为治疗烧伤难治性耐多药铜绿假单胞菌感染的噬菌体治疗的潜力。铜绿假单胞菌是烧伤相关感染的主要原因。它也是最有可能发展为败血症并导致烧伤相关死亡的细菌感染。经常从烧伤患者中分离到的铜绿假单胞菌菌株表现出多药耐药表型，这需要开发新的治疗策略和预防性治疗。在这种情况下，溶菌噬菌体的噬菌体治疗在控制铜绿假单胞菌感染方面显示出了令人兴奋的潜力。然而，在噬菌体治疗过程中，溶菌噬菌体可能会带来一系列缺点，包括诱导细菌耐药性和有限的细菌噬菌体相互作用。在这里，我们提出了一种在烧伤感染模型中干扰铜绿假单胞菌发病机制的替代方法，即使用工程化的超感染丝状噬菌体。我们的研究表明，用工程化 Pf 噬菌体治疗可以预防烧伤小鼠模型中的败血症和死亡。
