Native Efflux Pumps Contribute Resistance to Antimicrobials of Skin and the Ability of Staphylococcus aureus to Colonize Skin

Lipids are important for energy storage, membrane structure, and cell signaling [1, 2]. Free fatty acids may be found in staphylococcal abscesses and are usually present on the skin surface where they may serve to limit skin colonization by bacteria such as Staphylococcus aureus and Staphylcococcus epidermidis [3–5]. Human sebum contains 16% free fatty acids, and sapienic acid is a sebum fatty acid that is unique to humans [6, 7]. The equivalent of sapienic acid in mouse sebum is palmitoleic acid, a monounsaturated fatty acid present in all tissues, including the skin surface. Lauric acid is a minor sebum component, occupying 1%–2% of total sebum free fatty acids. These fatty acids have antimicrobial properties and can cause a reduction in the growth of S. aureus and S. epidermidis [8, 9]. In human tissue, skin surface, and nasal fluid, the main bactericidal long chain unsaturated fatty acids are linoleic (C18:2), oleic (C18:1), palmitoleic (C16:1), and long chain saturated fatty acids are palmitic (C16:0), and stearic (C18:0) acids [10]. In addition, unsaturated fatty acids, such as undecanoic and undecenoic acids and their monacyl glycerols, are also active against S. aureus and Bacillus cereus [6, 11]. Antimicrobial polyamines are also present on skin [12, 13]. Polyamines are aliphatic compounds and exert pleiotropic effects on cellular physiology of all organisms. S. aureus lacks polyamine biosynthetic genes and cannot produce spermidine, spermine, or their precursors. Exogenous polyamines inhibit S. aureus growth and are bactericidal at concentrations that are nontoxic to host cells [14]. S. aureus has developed a number of strategies to survive on skin in the presence of a range of antimicrobial compounds [15]. Under iron-limited conditions, the surface protein IsdA contributes to resistance to fatty acids and antimicrobial peptides due to its ability to reduce binding or sequestration of these antimicrobial compounds [16]. Wall teichoic acids also appear to prevent penetration of hydrophobic antimicrobial fatty acids across the cell wall and reduce binding to the cytoplasmic membrane [17]. Most S. aureus strains are highly susceptible to polyamines, such as spermine and spermidine, by an uncertain mechanism, but some strains, such as the dominant community-associated strain USA300, are resistant to skin polyamines due to acquisition of the speG gene found on the mobile ACME element [18]. A number of efflux pumps of S. aureus have been identified to confer resistance to antimicrobial agents. The norB (resistance to quinolones), norD, and tet38 (resistance to tetracycline), are known to be selectively increased in subcutaneous abscesses, which contain antimicrobial fatty acids [4], and these pumps contribute to the ability of S. aureus to survive in the abscess environment [19, 20]. Linoleic acid has also been shown to induce expression of efflux transporter genes tet38, mdeA, and mmpL [21]. We thus evaluated the effects of five known efflux pumps, Tet38, NorA, NorB, NorC, and NorD on resistance to antimicrobial fatty acids and polyamines. We studied the effects of these compounds on pump gene expression, the ability of Tet38 to transport fatty acids, and their ability to promote survival on skin. NorB and NorD had little effect, but expression of tet38 was induced by fatty acids, and Tet38 conferred resistance to and transported select free fatty acids and contributed to S. aureus survival on mouse skin. Although NorC contributed to polyamine resistance, it did not affect skin colonization.