Host Polyunsaturated Fatty Acids Potentiate Aminoglycoside Killing of Staphylococcus aureus

Abstract

Aminoglycoside antibiotics rely on the proton motive force to enter the bacterial cell, and facultative anaerobes like Staphylococcus aureus can shift energy generation from respiration to fermentation, becoming tolerant of aminoglycosides. Following this metabolic shift, high concentrations of aminoglycosides are required to eradicate S. aureus infections, which endangers the host due to the toxicity of aminoglycosides. Membrane-disrupting molecules prevent aminoglycoside tolerance in S. aureus by facilitating passive entry of the drug through the membrane. Polyunsaturated fatty acids (PUFAs) increase membrane permeability when incorporated into S. aureus. Here, we report that the abundant host-derived PUFA arachidonic acid increases the susceptibility of S. aureus to aminoglycosides, decreasing the aminoglycoside concentration needed to kill S. aureus. We demonstrate that PUFAs and aminoglycosides synergize to kill multiple strains of S. aureus, including both methicillin-resistant and -susceptible S. aureus. We also present data showing that PUFAs and aminoglycosides effectively kill S. aureus small colony variants, strains that are particularly recalcitrant to killing by many antibiotics. We conclude that cotreatment with PUFAs, which are molecules with low host toxicity, and aminoglycosides decreases the aminoglycoside concentration necessary to kill S. aureus, lowering the toxic side effects to the host associated with prolonged aminoglycoside exposure. IMPORTANCE Staphylococcus aureus infects every niche of the human host, and these infections are the leading cause of Gram-positive sepsis. Aminoglycoside antibiotics are inexpensive, stable, and effective against many bacterial infections. However, S. aureus can shift its metabolism to become tolerant of aminoglycosides, requiring increased concentrations and/or longer courses of treatment, which can cause severe host toxicity. Here, we report that polyunsaturated fatty acids (PUFAs), which have low host toxicity, disrupt the S. aureus membrane, making the pathogen susceptible to aminoglycosides. Additionally, cotreatment with aminoglycosides is effective at killing S. aureus small colony variants, strains that are difficult to treat with antibiotics. Taken together, the data presented herein show the promise of PUFA cotreatment to increase the efficacy of aminoglycosides against S. aureus infections and decrease the risk to the human host of antibiotic-induced toxicity.

Keywords: MRSA; PUFA; Staphylococcus aureus; aminoglycoside; antibiotic tolerance; arachidonic acid; gentamicin; linoleic acid; persister; polyunsaturated fatty acid; small colony variant.

FIG 1

Polyunsaturated fatty acids potentiate gentamicin activity against S. aureus. (A) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM AA, or gentamicin and AA. Bacterial growth was monitored by optical density at 600 nm (OD₆₀₀) every 30 min for 24 h. Data are the mean values ± standard errors of the means for measurements acquired in biological triplicate. (B) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM AA, or gentamicin and AA for 3 h at 37°C. After incubation, viable bacteria were quantified by dilution plating on solid medium. Data are presented as mean values, and each point represents a single biological replicate. Gray symbols represent replicates with the number of viable bacterial colonies below the limit of detection. (C) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM PA, or gentamicin and PA. Bacterial growth was monitored by OD₆₀₀ every 30 min for 24 h. Data are mean values ± standard errors of the means for measurements acquired in biological triplicate. (D) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM PA, or gentamicin and PA for 3 h at 37°C. After incubation, viable bacteria were quantified by dilution plating on solid medium. Data are presented as mean values, and each point represents a single biological replicate. (E) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM LA, or gentamicin and LA. Bacterial growth was monitored by OD₆₀₀ every 30 min for 24 h. Data are mean values ± standard errors of the means for measurements acquired in biological triplicate. (F) JE2 was treated with vehicle, 1 μg/mL gentamicin, 50 μM LA, or gentamicin and LA for 3 h at 37°C. After incubation, viable bacteria were quantified by dilution plating on solid medium. Data are presented as mean values, and each point represents the value for a single biological replicate. P values were calculated by one-way analysis of variance (ANOVA). Nonsignificant (ns), P > 0.05; ****, P < 0.0001.

FIG 2

Cotreatment with AA and gentamicin kills S. aureus small colony variants. (A) JE2 and NE1345 (menD) were treated with vehicle or 5 μM menadione. Bacterial growth was monitored by OD₆₀₀ every 30 min for 24 h. Data are mean values ± standard errors of the means for measurements acquired in biological triplicate. (B) JE2 was treated with vehicle, 15 μM AA, 16 μg/mL gentamicin, or gentamicin and AA. Bacterial growth was monitored by OD₆₀₀ every 30 min for 24 h. Data are mean values ± standard errors of the means for measurements acquired in biological triplicate. (C) NE1345 (menD) was treated with vehicle, 15 μM AA, 16 μg/mL gentamicin, or gentamicin and AA. Bacterial growth was monitored by OD₆₀₀ every 30 min for 24 h. Data are mean values ± standard errors of the means for measurements acquired in biological triplicate. (D) NE1345 (menD) was treated with vehicle, 15 μM AA, 16 μg/mL gentamicin, or gentamicin and AA for 18 h at 37°C. After incubation, viable bacteria were quantified by dilution plating on solid medium. Data are presented as mean values, and each point represents the value for a single biological replicate. P values were calculated by one-way ANOVA. ****, P < 0.0001.