Increased resistance of a methicillin-resistant Staphylococcus aureus Δagr mutant with modified control in fatty acid metabolism

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to β-lactam antibiotics remains unclear. In the present study, we found the Δagr mutant strain having higher resistance to high concentrations of b-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The Δagr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.

Combination Therapy Using Low-Concentration Oxacillin with Palmitic Acid and Span85 to Control Clinical Methicillin-Resistant Staphylococcus aureus

“…In a previous study, we found that palmitic acid, which is the most common saturated fatty acid found in animals, plants, and microorganisms, as well as a component in soap, was capable of inhibiting biofilm formation of community-associated MRSA (CA-MRSA) LAC and its Δ agr mutant, which has a higher oxacillin minimum inhibitory concentration (MIC) (>200 μg/mL) [ 9 ]. Concomitant treatment with palmitic acid and oxacillin led to a dramatic increase in the efficacy of oxacillin.…”

“…As a possible solution, drug combination is considered a simple method to avoid the complex resistance mechanism of S. aureus [ 8 ]. This is because multiple antibacterial agents, such as antibiotics, free fatty acids, flavonoids, and surfactants, can exert synergistic effects arising from the complex activities of their combinations [ 9 , 10 ].…”

“…Agents other than antibiotics, such as free fatty acids, polyunsaturated fatty acids (PUFAs), flavonoids, triclosan, and surfactants, have been found to evade the bacterial resistance mechanism [ 9 , 11 , 12 , 13 ]. Among these, free fatty acids are natural antibacterial compounds found in human tissues and fluids [ 14 ].…”

“…Specifically, palmitic acid at a concentration of over 1.6 mg/mL inhibited MRSA in vitro, and it inhibited the growth of MRSA in infected cells in vivo [ 15 ]. Moreover, in a previous study, palmitic acid was shown to eliminate the biofilm of MRSA and increase the effectiveness of oxacillin [ 9 ].…”

“…Sorbitan trioleate (Span85) is a non-ionic detergent used as an emulsifier in cosmetics, medicines, and nanoparticles and contains three oleic acid chains per molecule compared to palmitic acid [ 19 ]. Span85 has also shown effectiveness against MRSA by inhibiting biofilm formation [ 9 ].…”