Evolution of Pseudomonas aeruginosa PAO1 in a chronic wound model

Six strains of Pseudomonas aeruginosa, ranging from laboratory to environmental and clinical isolates and containing a diverse prophage assortment, were co-inoculated into a porcine full-thickness burn wound model. Hyperbiofilm producing stable variants were isolated 3, 14, and 28 days post infection. Of the six strains inoculated into the wound, only the laboratory strains, PA14 and PAO1, evolved phenotypic variants over the course of the experiment. Short read genome sequencing revealed a remarkable level of mutational parallelism in the frequently observed wsp pathway in PA14 (PRJNA491911). Mutations in this operon increased the intracellular levels of cyclic-di-GMP that in turn generated a hyperbiofilm forming phenotype. However, no driver mutations were detected by short read genome sequencing in many of the evolved PAO1 variants. Instead, long read sequencing and de novo assemblies revealed that interstrain movement of prophage elements from the clinical and environmental strains occurred, with PAO1 as the primary recipient strain. Parallelism was observed in these recombination events, where the sites of phage insertion disrupted genes encoding phosphodiesterases and global regulators, again leading to increased cyclic-di-GMP levels and subsequently a hyperbiofilm forming phenotype. The implications of prophage-mediated adaptation are broad, as even transient members of microbial communities can cause the evolution of persistent phenotypes associated with poor clinical outcomes.

选取6株铜绿假单胞菌（Pseudomonas aeruginosa），涵盖实验室菌株、环境分离株与临床分离株，且携带有多样化的前噬菌体组合，将其共同接种至猪全层烧伤创面模型中。于感染后第3、14、28天，分离得到高产生物膜的稳定变异体。在接种至创面的6株菌株中，仅实验室菌株PA14与PAO1在实验全过程中演化出了表型变异体。短读长基因组测序（short read genome sequencing）结果显示，在PA14的wsp通路（wsp pathway，测序项目编号PRJNA491911）中存在显著的突变平行性。该操纵子上的突变可提升细胞内环二鸟苷酸（cyclic-di-GMP）的水平，进而诱导产生高产生物膜的表型。然而，针对多数演化后的PAO1变异体，短读长基因组测序并未检测到驱动突变。取而代之的是，长读长测序与从头组装（de novo assemblies）结果显示，前噬菌体元件从临床与环境菌株发生了跨菌株转移，PAO1为主要的受体菌株。在这类重组事件中同样观测到了平行性：噬菌体的插入位点破坏了编码磷酸二酯酶与全局调控因子的基因，同样导致环二鸟苷酸水平升高，最终诱导产生高产生物膜的表型。前噬菌体介导的适应性演化具有广泛的研究价值：微生物群落中即使是短暂存在的成员，也可引发与不良临床结局相关的持久性表型演化。