Untargeted Metabolomics Reveals Species-Specific Metabolite Production and Shared Nutrient Consumption by Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

While bacterial metabolism is known to impact antibiotic efficacy and virulence, the metabolic capacities of individual microbes in cystic fibrosis lung infections are difficult to disentangle from sputum samples. Here, we show that untargeted metabolomic profiling of supernatants of multiple strains of Pseudomonas aeruginosa and Staphylococcus aureus grown in monoculture in synthetic cystic fibrosis media (SCFM) reveals distinct species-specific metabolic signatures despite intraspecies metabolic variability. We identify a set of 15 metabolites that were significantly consumed by both P. aeruginosa and S. aureus, suggesting that nutrient competition has the potential to impact community dynamics even in the absence of other pathogen-pathogen interactions. Finally, metabolites that were uniquely produced by one species or the other were identified. Specifically, the virulence factor precursor anthranilic acid, as well as the quinoline 2,4-quinolinediol (DHQ), were robustly produced across all tested strains of P. aeruginosa. Through the direct comparison of the extracellular metabolism of P. aeruginosa and S. aureus in a physiologically relevant environment, this work provides insight toward the potential for metabolic interactions in vivo and supports the development of species-specific diagnostic markers of infection. IMPORTANCE Interactions between P. aeruginosa and S. aureus can impact pathogenicity and antimicrobial efficacy. In this study, we aim to better understand the potential for metabolic interactions between P. aeruginosa and S. aureus in an environment resembling the cystic fibrosis lung. We find that S. aureus and P. aeruginosa consume many of the same nutrients, suggesting that metabolic competition may play an important role in community dynamics during coinfection. We further identify metabolites uniquely produced by either organism with the potential to be developed into species-specific biomarkers of infection in the cystic fibrosis lung.

Keywords: LC-MS; Pseudomonas aeruginosa; Staphylococcus aureus; metabolism.

FIG 1

Summary of experimental design and bacterial strains used in this study. (A) Five strains of P. aeruginosa (green) and four strains of S. aureus (yellow) were each grown individually in SCFM for 24 h. Cultures were filter sterilized, and LC-MS was performed on supernatants. SCFM medium was also profiled as a control. Strain-specific metabolic profiles, cross-species metabolite consumption, and species-specific metabolite production were identified. (B and D) Phylogenetic trees of P. aeruginosa (B) and S. aureus (D) strains. Reference genomes were used for laboratory strains UCBPP-PA14, ATCC 29213, and USA300. A single reference genome was used for isogenic and SCFM-evolved UCBPP-PA14 given their shared history and highly similar metabolic functionality. (C and E) Antimicrobial susceptibility profiles of P. aeruginosa (C) and S. aureus (E) strains.

FIG 2

Metabolic profiles of P. aeruginosa and S. aureus grown in monoculture in SCFM. (A) Metabolomics profiles of conditioned SCFM. Metabolites significantly altered relative to SCFM in at least one condition are shown (P < 0.05). Metabolites clustered by Euclidean distance with complete linkage. Significance determined by Wilcoxon rank sum test with Benjamini-Hochberg correction. (B to D) PCoA of Bray-Curtis distances calculated using all raw peak areas for all replicates of all strains (B), raw peak areas for P. aeruginosa strains only (C), or S. aureus strains only (D). Percent variance is shown for each principal coordinate. n = 7 replicates for all conditions.

FIG 3

Intraspecies strain-specific metabolite production and consumption. Number of significant metabolites produced (P < 0.05, log₂FC > 0) or consumed (P < 0.05, log₂FC < 0) by subsets of P. aeruginosa strains (A and B) or S. aureus strains (C and D). Metabolites produced by all strains of a species are shown in red. Metabolites consumed by all strains of a species are shown in blue.

FIG 4

Shared metabolite consumption between P. aeruginosa and S. aureus in a lung-like medium. (A) Number of significant metabolites robustly consumed across all tested monocultures of P. aeruginosa and S. aureus (P < 0.05, log₂FC < 0). Log₂FCs in metabolites consumed by both P. aeruginosa (B) and S. aureus (C) relative to SCFM. Crossbar denotes median change across strains.

FIG 5

P. aeruginosa and S. aureus specific metabolite production in a lung-like medium. (A) Number of metabolites that were always or never significantly produced across strains of each species (P < 0.05, log₂FC > 0). Metabolites uniquely produced by P. aeruginosa strains are shown in green. Metabolites uniquely produced by S. aureus strains are shown in yellow. Log₂FCs in metabolites produced by only P. aeruginosa (B) or S. aureus (C) strains. Only metabolites with a minimum log₂FC of ≥1 across all strains within a species are shown. Metabolites produced by P. aeruginosa without an associated KEGG ID were excluded. Crossbar denotes median change of each metabolite across strains.