High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections

Abstract

Cystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistant Pseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity of P. aeruginosa populations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections.

Keywords: Pseudomonas aeruginosa; R-pyocin; bacteriocin; cystic fibrosis; lipopolysaccharide.

Fig 1

R2-pyocin inhibition zones for multiple cystic fibrosis (CF) variants from different patient sputum samples. The inhibition zones caused by R2-pyocin were applied to soft agar overlays containing clinical variants from the CF sputum samples listed on the left. PAO1∆rmlC serves as an R2-susceptible control and PAO1 wt serves as an R2-resistant control. Red boxes indicate patients with populations containing resistant variants. Up to 10 variants per sputum sample were tested. An inhibition zone indicates R2-pyocin susceptibility, while the absence of a visible zone indicates resistance.

Fig 2

R2-pyocin susceptibility varies among clinical P. aeruginosa populations and changes over time. Relative susceptibility to R2-pyocins differs among variants in each population and across populations. Susceptibility heterogeneity is observed across all five patients and within the same patient’s longitudinal samples. There is no trend of strains becoming more resistant to R2-pyocins in longitudinally sampled populations, up to 2 yr (consecutive numbers with collection number in parentheses). Two sputum samples contained at least one variant resistant to R2-pyocins (Relative Susceptibility = 0). Relative Susceptibility was determined by counting the number of serial dilutions (including neat) where inhibition zones appeared after R2-pyocins were applied to agar overlays. Means are shown with 95% confidence interval error bars.

Fig 3

Lipopolysaccharide (LPS) phenotype heterogeneity and lack of correlation with R2-susceptibility. A one-tailed Spearman correlation analysis for each phenotype revealed no correlations between R2-pyocin susceptibility and the absence of (A) common antigen (CPA), (C) O-specific antigen (OSA), or (E) an altered core (P > 0.05). CPA was absent in 42.73% of variants (B), OSA was absent in 76.36%, and (F) 27.27% of variants had altered LPS cores. Each data point represents the average relative susceptibility of a variant, and variants are colored based on their longitudinal collection.

Fig 4

No correlation between combined lipopolysaccharide (LPS) deficiency phenotypes and R2-pyocin susceptibility. An LPS Score of 0–3 was assigned to each variant from each sputum collection to assess the correlation between overall LPS phenotypes and R2-pyocin susceptibility. Variants from each population are shown as data points of the same color. No correlation was found between LPS score and R2-pyocin susceptibility (P > 0.05).