Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions

Abstract

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O₂]). As [O₂] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O₂] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O₂] S. epidermidis limits its growth and develops the ability to form biofilms.

Keywords: Biofilms; Fermentation; Metabolism; Oxygen concentration; Rate of oxygen consumption; Staphylococcus epidermidis.

Fig. 1

Rate of oxygen consumption by S. epidermidis in the presence of different respiratory substrates. Experimental conditions: 10 mM HEPES (pH 7.4). As indicated, substrates were: L: 10 mM lactate; G: 40 mM glucose or E: 33 mM ethanol. Cells were grown at different [O₂] as follows: aerobic (empty bars), microaerobic (gray bars) and anaerobic (black bars)

Fig. 2

Fermentation by S. epidermidis grown at different [O₂]. Cytoplasmic extracts were obtained from S. epidermidis grown under aerobic, microaerobic or anaerobic conditions. Fermentation by cell cytoplasmic extracts (0.5 mg prot. mL⁻¹) was measured using a 20 mM glucose or b 20 mM glycerol. Samples were incubated at 30 °C for: 2.5 min (black columns), 5 min (gray columns) or 10 min (white columns). Results are reported as μmol ethanol per mg protein. Tukey’s comparison test was used to determine significant differences (*P < 0.05)

Fig. 3

Intracellular ATP concentrations in S. epidermidis grown at different [O₂]. Cells were grown at different [O₂] in LB plus glucose. Cytoplasmic extracts were obtained from each of these cultures and used to measure intracellular ATP. ATP concentration was estimated using luciferase and interpolating into a standard curve (see “Materials and methods”). The average of three experiments is shown with SD. * indicates significant difference P < 0.05

Fig. 4

H₂O₂ effect on cellular viability. S. epidermidis susceptibility to hydrogen peroxide was determined using 0, 0.5, 1, 5, 10 or 25 mM H₂O₂ in each group: aerobiosis (black bar), microaerobiosis (gray bar) or anaerobiosis (white bar). After 30 min of incubation with H₂O₂, the samples were diluted 1:1000, 10 µL were taken and plated in LB plus 2% glucose-agar. CFU/mL were counted. Samples without treatment were assigned as 100% viable cells. The average of three experiments is shown with SD. Significance *P < 0.0001

Fig. 5

In vitro biofilm inhibition assay. S. epidermidis was grown under microaerobic conditions. a Different metabolic inhibitors were added as indicated: 100 µM NaCN, 1 mM B1,4BP or both inhibitors. b Different concentrations of the uncoupler CCCP (0.5, 1.0 and 1.5 µM) were added to deplete ATP. After 24 h of incubation biofilm generation was evaluated by measuring the absorbance at 492 nm with a microplate reader. Each sample was compared with the control (without additions). Statistics were applied using ANOVA and Dunnett’s post hoc test. Significance *P < 0.0001

Fig. 6

Cartoon depicting the shift that Staphylococcus epidermidis makes when [O₂] decreases in the growth medium. When high oxygen concentrations are found in the medium, S. epidermidis are planktonic cells and flow with the blood (top). In contrast, under micro- or anaerobic conditions cells shift to a fermentative metabolism and accumulate ATP adhering to a suitable surface (e.g. epithelia, catheters, artificial valves) and eventually forming a biofilm. In this state the cells exhibit more resistance to H₂O₂ mediated damage. Excess ATP is probably used to produce adhesion proteins and poly-N-acetylglucosamine (gray fibers in the illustration) (bottom)