Abstract
Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.
Highlights
Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist
The rugose small-colony variants (RSCVs) phenotype is commonly caused by mutations in pathways that lead to elevated cyclic diguanylate monophosphate (c-di-GMP) [2]. c-di-GMP is a messenger molecule that signals the transition from planktonic to biofilm lifestyle in many bacteria [16]
Wounds were infected with two model strains (PA14-1 and PAO1-B11), three clinical isolates (B23-2, CF18-1, and S54485-1), and a water isolate (MSH10-2) (Table 1)
Summary
Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Several of the wsp mutant RSCVs acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was present in the inoculum These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent These recalcitrant variants are typically observed in chronic infections. When opportunistic pathogens leave an environmental reservoir to colonize a host, there are potentially many beneficial mutations available for selection in this environment Defining this new fitness landscape and the evolutionary dynamics of pathogen adaptation to the host environment is critical to understanding infection pathology, as the emergence and selection of adapted variants in an infection are often associated with worsening clinical outcomes [2]. Their importance in relation to other identified adaptive mutations and frequency in other chronic infections is unclear
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