Quantitative NMR metabolite profiling of methicillin-resistant and methicillin-susceptible Staphylococcus aureus discriminates between biofilm and planktonic phenotypes

Abstract

Wound bioburden in the form of colonizing biofilms is a major contributor to nonhealing wounds. Staphylococcus aureus is a Gram-positive, facultative anaerobe commonly found in chronic wounds; however, much remains unknown about the basic physiology of this opportunistic pathogen, especially with regard to the biofilm phenotype. Transcriptomic and proteomic analysis of S. aureus biofilms have suggested that S. aureus biofilms exhibit an altered metabolic state relative to the planktonic phenotype. Herein, comparisons of extracellular and intracellular metabolite profiles detected by (1)H NMR were conducted for methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains grown as biofilm and planktonic cultures. Principal component analysis distinguished the biofilm phenotype from the planktonic phenotype, and factor loadings analysis identified metabolites that contributed to the statistical separation of the biofilm from the planktonic phenotype, suggesting that key features distinguishing biofilm from planktonic growth include selective amino acid uptake, lipid catabolism, butanediol fermentation, and a shift in metabolism from energy production to assembly of cell-wall components and matrix deposition. These metabolite profiles provide a basis for the development of metabolite biomarkers that distinguish between biofilm and planktonic phenotypes in S. aureus and have the potential for improved diagnostic and therapeutic use in chronic wounds.

Figure 1

Two phylogenetically distinct strains of S. aureus have unique pigmentation. (A) Phylogenetic separation of S. aureus 6538 and S. aureus 10943 indicates that S. aureus 10943 is most closely related to common CA-MRSA strains, while S. aureus 6538 is most closely related to non-MRSA strains. Blue lines indicate phylogenetic branches associated with methicillin resistance and red lines indicate phylogenetic branches associated with S. aureus species with no known antibiotic resistance. Black lines indicate parent phylogenetic branches. (B) Two phylogenetically dissimilar strains of S. aureus were inoculated onto tissue culture inserts and grown as biofilms in six-well tissue culture plates at 37 °C. Every 24 hours, feeder medium was refreshed within the plate well. Images represent mature biofilms that have been cultured a total of 72 h to reach linear growth phase (referred to as the T0 biofilm growth time point). Left panel depicts the chronic wound isolate, MRSA strain S. aureus 10943 and right panel depicts the lab-adapted, MSSA strain S. aureus “Rosenbach” ATCC 6538.

Figure 2

Changes in glucose concentration and pH in the bulk media correspond to transitions between growth phases in S. aureus cultures. (A) Growth curves for S. aureus 6538 and S. aureus 10943 planktonic and biofilm cultures were plotted for all growth time periods. Cultures were inoculated with an equal amount of cell mass, as determined by comparable absorbance readings of seed flasks (i.e., OD_600 nm). Time zero refers to mature biofilms grown up to 72 h (T0) and inoculum for planktonic cultures, respectively. Error bars have been calculated from growth curve measurements of duplicate biological replicates for each growth condition and growth time point. (B) Decreasing glucose concentration in the media for planktonic cultures corresponds in time to transition from exponential to stationary phase of growth. For biofilm cultures, consumption of glucose is below the detection limit of ¹H 1D NMR for all time points of growth. (C) Changes in pH profile for planktonic cultures correspond in time to changes in growth rate, with significant pH differences between strains observed for biofilm cultures. Slight differences in bulk pH were detected between the two strains based on growth phase and growth phenotype.

Figure 3

Principal component analysis (PCA) comparison of two S. aureus strains. 2D PCA scores plots indicate statistically significant sample separations along the first dimension (PC1) between the metabolite profiles of S. aureus 10943 (blue lines) and S. aureus 6538 (red lines) for both intracellular and extracellular metabolites of planktonic cultures (panels A and B, respectively) and biofilm extracellular metabolites (panel C, as detected by 1D ¹H NMR). In contrast, metabolite profiles for biofilm intracellular metabolites cluster nearly identically (panel D). Dark colors (red and blue) indicate planktonic cultures, and light colors (red and blue) indicate biofilm cultures. Numbers correspond to hours postinoculation for planktonic cultures (e.g., T2, T4, T6, etc.) and hours past reaching linear growth in biofilms (e.g., T24, T48, T72).

Figure 4

PCA analysis of intracellular metabolites for S. aureus 10943 and 6538 biofilm and planktonic cultures results in statistical clustering of biofilm phenotype. PCA analysis of intracellular metabolites in S. aureus 10943 and 6538 biofilm and planktonic cultures detected by 1D ¹H NMR separates the biofilm phenotype into a single quadrant of the 2D PCA scores plot as a result of significant statistical separations from S. aureus 6538 planktonic cultures along PC1 and from S. aureus 10943 planktonic cultures along PC2.

Figure 5

PCA analysis of extracellular metabolites for S. aureus 10943 and 6538 biofilm and planktonic cultures results in statistical separation of the biofilm phenotype from its planktonic counterpart. PCA analysis of extracellular metabolite profiles in S. aureus 10943 and 6538 biofilm and planktonic cultures detected by 1D ¹H NMR separates the biofilm phenotype into a single quadrant of the 2D PCA score plots as a result of statistically significant separations from S. aureus 10943 planktonic cultures along PC1 and from S. aureus 6538 planktonic cultures along PC2.

Figure 6

Schematic representation of central metabolism and secondary metabolic activity in S. aureus characteristic of the biofilm phenotype. Bar charts represent fold changes in metabolite concentrations for biofilm and stationary planktonic samples, respectively, normalized to the metabolite concentrations in each respective exponential planktonic culture for select intracellular and extracellular biofilm metabolites that contribute to statistical separation of the biofilm phenotype from the planktonic phenotype irrespective of strain. S. aureus 10943 is indicated by blue bars and S. aureus 6538 is indicated by red bars. Dark bars indicate planktonic samples and light bars indicate biofilm samples. Data shown are representative samples from duplicate experiments.