Planktonic aggregates of Staphylococcus aureus protect against common antibiotics

Abstract

Bacterial cells are mostly studied during planktonic growth although in their natural habitats they are often found in communities such as biofilms with dramatically different physiological properties. We have examined another type of community namely cellular aggregates observed in strains of the human pathogen Staphylococcus aureus. By laser-diffraction particle-size analysis (LDA) we show, for strains forming visible aggregates, that the aggregation starts already in the early exponential growth phase and proceeds until post-exponential phase where more than 90% of the population is part of the aggregate community. Similar to some types of biofilm, the structural component of S. aureus aggregates is the polysaccharide intercellular adhesin (PIA). Importantly, PIA production correlates with the level of aggregation whether altered through mutations or exposure to sub-inhibitory concentrations of selected antibiotics. While some properties of aggregates resemble those of biofilms including increased mutation frequency and survival during antibiotic treatment, aggregated cells displayed higher metabolic activity than planktonic cells or cells in biofilm. Thus, our data indicate that the properties of cells in aggregates differ in some aspects from those in biofilms. It is generally accepted that the biofilm life style protects pathogens against antibiotics and the hostile environment of the host. We speculate that in aggregate communities S. aureus increases its tolerance to hazardous environments and that the combination of a biofilm-like environment with mobility has substantial practical and clinical importance.

Figure 1. Aggregate visualized by SEM.

Aggregates from a post-exponential 8325-4 culture were fixed and visualized using Scanning Electron Microscopy (SEM). Panels A-D represent increasing magnification and red squares indicate the area magnified in the following panel. Overview of an aggregate, which is visible to the naked eye (A). Zooming in reveals pores and crevices in a topographical landscape of aggregated clusters (B). At higher magnifications it is revealed that some clusters are embedded in a web-like matrix (black arrows) while some are not (white arrows) (C, D).

Figure 2. Aggregate size distribution measured by LDA.

Four different S. aureus strains were investigated for their level of aggregation in post-exponential growth phase (OD₆₀₀ = 2) (A). The two aggregating strains were followed through exponential growth and the percentage of cells in aggregates (>6 µm, solid bars) relative to the non-aggregating fraction (open bars) is shown (B). % total biovolume is calculated as the percentage of cells of a given size relative to the total suspended cell mass. Error bars indicate standard deviation (n = 5).

Figure 3. Kinetic of aggregate formation.

8325-4 cells were mixed 1∶1 with 8325-4 cells expressing YFP to OD₆₀₀ of 0.01 and examined using CLSM every 30 min through a growth cycle. Small clusters of cells dominate until OD₆₀₀ = 0.1 (A) at which time they start to fuse (B) and form large aggregates around OD₆₀₀ = 0.5 (C). Note different sizes of scale bars.

Figure 4. Aggregation is influenced by the presence of sub-inhibitory concentrations of antibiotics.

8325-4 (panel A) and 15981 (panel B) were cultivated to OD₆₀₀ of 2 in TSB (solid circle) or in TSB added 1/25×MIC of erythromycin (open triangles), cefuroxime (open circles) or rifampicin (open squares). The size distribution of planktonic aggregates was examined using LDA. Error bars indicate standard deviation (n = 5).

Figure 5. Polysaccharide constitutes the extracellular matrix of 8325-4 aggregates.

Aggregates of 8325-4 cells were treated with DNase (tube 2), proteinase K (tube 3) or sodium metaperiodate (tube 5) at 37°C for 18 hours. Tubes 1 and 4 are untreated controls.

Figure 6. Production of the Polysaccharide Intercellular Adhesin (PIA).

Dot blot and immune-detection was used to determine the amount of PIA produced by different strains in post-exponential growth phase (A) or by strain 8325-4 after exposure to 1/25 MIC of different antibiotics (B).

Figure 7. Metabolic activity is significantly higher in aggregated compared to dispersed cells.

Metabolic activity was determined in a post-exponential (OD₆₀₀ = 2) culture of 8325-4 by measuring reduced XTT (arbitrary units) normalized to mg dry weight (A). Error bars indicate standard deviation (n = 3). The distribution of active cells (green) and cells with low membrane potential (red) in aggregates was determined using LIVE/DEAD staining and CLSM (B).

Figure 8. Aggregated cells have increased survival after antibiotic treatment.

Cells in aggregates (solid bars) survive better than dispersed cells (open bars) following treatment with 25×MIC of kanamycin (KAN), ciprofloxacin (CIP), erythromycin (ERM) or vancomycin (VAN). Survival was calculated as CFU present in the aggregate and dispersed fractions after treatment with antibiotics relative to CFU measured before antibiotic exposure. Error bars indicate standard deviation (n = 3).