Induction of Daptomycin Tolerance in Enterococcus faecalis by Fatty Acid Combinations

Abstract

Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid.IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

Keywords: Enterococcus faecalis; daptomycin; fatty acid; membrane fluidity.

FIG 1

Addition of a protective fatty acid rescues growth defects associated with palmitic acid or stearic acid. All fatty acids were added to a final concentration of 5 μg ml⁻¹, and ethanol (solvent control) was added to an equivalent final volume. (A) Addition of SLOP (stearic acid, linoleic acid, oleic acid, and palmitic acid), human serum (15%), or individual fatty acids as indicated. (B) Addition of stearic acid combinations. (C) Addition of palmitic acid combinations. (D) Palmitic acid was added at the time of dilution. Stasis is indicated by the black arrow. Oleic acid was added (indicated by arrows) at 30, 60, and 90 min poststasis (indicated according to the color legend). Note, data from the same biological replicates are replotted in panels B and C. For all experiments, n = 3.

FIG 2

Fatty acids induce altered morphology of OG1RF. Scanning electron micrographs of exponential-phase cells after long-term supplementation with fatty acids (5 μg ml⁻¹ of each) or solvent control (ethanol) are shown. Images were taken at a magnification of 45,000 and at 5.0 keV. Scale bar, 0.5 μm. Sample images of n = 2 biological replicates are shown, and a minimum of 10 fields per replicate were observed.

FIG 3

Membrane fluidity of OG1RF after short-term supplementation with fatty acids. Cells were grown to mid-log phase supplemented either with 5 μg ml⁻¹ of each fatty acid, 15% human serum, or an equivalent volume of ethanol (solvent control) for 30 min. Upon formation of protoplasts, anisotropy was determined using DPH at an excitation wavelength of 350 nm and emission wavelength of 428 nm. ****, P < 0.0001, as determined via Tukey’s range test (n = 3).

FIG 4

Oleic or linoleic acid can induce daptomycin tolerance in the presence of nonprotective fatty acids. (A) Supplementation with SLOP (stearic acid, linoleic acid, oleic acid, and palmitic acid), human serum (15%), oleic acid, or linoleic acid led to an increase in the survival rate for all time points (P < 0.0001). Cultures supplemented with oleic acid did not differ from cultures supplemented with oleic acid mixtures. (B) Supplementation with oleic acid or linoleic acid in combination with stearic acid led to an increase in the survival rate at all time points (P < 0.0001). (C) Supplementation with oleic acid or linoleic acid in combination with palmitic acid led to an increase in the survival rate at all time points (P < 0.0001). Note, data for the same biological replicates are replotted in the different panels (n = 3 biological replicates).