Inhibition of Staphylococcus aureus Biofilm Formation and Virulence Factor Production by Petroselinic Acid and Other Unsaturated C18 Fatty Acids

Abstract

Staphylococcus aureus is a major human pathogen that secretes several toxins associated with the pathogenesis of sepsis and pneumonia. Its antibiotic resistance is notorious, and its biofilms play a critical role in antibiotic tolerance. We hypothesized fatty acids might inhibit S. aureus biofilm formation and the expressions of its virulence factors. Initially, the antibiofilm activities of 27 fatty acids against a methicillin-sensitive S. aureus strain were investigated. Of the fatty acids tested, three C18 unsaturated fatty acids, that is, petroselinic, vaccenic, and oleic acids at 100 μg/mL, inhibited S. aureus biofilm formation by more than 65% without affecting its planktonic cell growth (MICs were all > 400 μg/mL). Notably, petroselinic acid significantly inhibited biofilm formation of two methicillin-resistant S. aureus strains and two methicillin-sensitive S. aureus strains. In addition, petroselinic acid significantly suppressed the production of three virulence factors, namely, staphyloxanthin, lipase, and α-hemolysin. Transcriptional analysis showed that petroselinic acid repressed the gene expressions of quorum sensing regulator agrA, effector of quorum sensing RNAIII, α-hemolysin hla, nucleases nuc1 and nuc2, and the virulence regulator saeR. Furthermore, petroselinic acid dose-dependently inhibited S. aureus biofilm formation on abiotic surfaces and porcine skin. These findings suggest that fatty acids, particularly petroselinic acid, are potentially useful for controlling biofilm formation by S. aureus. IMPORTANCE Fatty acids with a long carbon chain have recently attracted attention because of their antibiofilm activities against microbes. Here, we report the antibiofilm activities of 27 fatty acids against S. aureus. Of the fatty acids tested, three C18 unsaturated fatty acids (petroselinic, vaccenic, and oleic acids) significantly inhibited biofilm formation by S. aureus. Furthermore, petroselinic acid inhibited the production of several virulence factors in S. aureus. The study also reveals that the action mechanism of petroselinic acid involves repression of quorum-sensing-related and virulence regulator genes. These findings show that natural and nontoxic petroselinic acid has potential use as a treatment for S. aureus infections, including infections by methicillin-resistant S. aureus strains, and in food processing facilities.

Keywords: Staphylococcus aureus; biofilm; fatty acids; hemolysis; petroselinic acid.

FIG 1

Antibiofilm activity of petroselinic acid against S. aureus. Biofilm formation by S. aureus MSSA 6538 in the presence of petroselinic acid (A, CLSM; B, Crystal violet assay). Planktonic cell growth in the presence of petroselinic acid (C) was assessed for 24 h and COMSTAT analysis was performed based on CLSM results (D–F). Scale bars represent 100 μm.

FIG 2

Antibiofilm activity of petroselinic acid against three other S. aureus strains, MSSA 25923 (A), MRSA MW2 (B), and MRSA 33591 (C). Color-coded 3-D images of S. aureus biofilms were generated in the presence of petroselinic acid (D–F).

FIG 3

Inhibition of S. aureus biofilm formation by petroselinic acid on an abiotic surface. SEM images of S. aureus MSSA 6538 biofilms formed in the presence or absence of petroselinic acid on nylon membranes. Yellow and green scale bars represent 10 and 1.5 μm, respectively.

FIG 4

Effect of petroselinic acid on the production of staphyloxanthin (A), lipase (B), and hemolysin (C) in S. aureus MSSA 6538.

FIG 5

Relative transcriptional profiles of S. aureus cells treated with petroselinic acid at 100 μg/mL for 6 h. Transcriptional profiles were acquired by qRT-PCR. Fold changes delineate changes in the gene transcriptions of treated versus non-treated S. aureus MSSA 6538 as determined by qRT-PCR. P < 0.05 versus nontreated cells (None).

FIG 6

Antibiofilm effect of petroselinic acid on porcine skin. SEM images of S. aureus MSSA 6538 biofilms formed on porcine skin over 24 h. The blank shows the result obtained without bacterial treatment, and None indicates no treatment with fatty acid. Scale bars represent 10 μm.

FIG 7

Ball and stick models of fatty acids and a schematic of the suggested mode of action of petroselinic acid in S. aureus. Chemical structures of six fatty acids (A). Putative mechanism for the effects of petroselinic acid in S. aureus (B).