In this study, we used methicillin-sensitive S. aureus (MSSA; ATCC 6538) and methicillin-resistant S. aureus (MRSA; MW2) strains. All experiments were performed at 37°C in Luria-Bertani medium (Silber et al., 2020). Thirty FAs, namely butanoic acid (C4:0), pentanoic acid (C5:0), hexanoic acid (C6:0), heptanoic acid (C7:0), octanoic acid (C8:0), nonanoic acid (C9:0), decanoic acid (C10:0), undecanoic acid (C11:0), lauric acid (C12:0), myristic acid (C14:0), myristoleic acid (C14:1), palmitic acid (C16:0), palmitoleic acid (C16:1 ω-5), heptadecanoic acid (C17:0), stearic acid (C18:0), vaccenic acid (C18:1 ω-7), oleic acid (C18:1 ω-9, cis), elaidic acid (C 18:1 ω-9, trans), petroselinic acid (C18:1 ω-12), linoleic acid (C18:2 ω-6), conjugated linoleic acid (C18:2 ω-6), α-linolenic acid (C18:3 ω-3), γ-linolenic acid (C18:3 ω-6), arachidonic acid (C20:4 ω-6), eicosapentaenoic acid (C20:5 ω-3), behenic acid (C22:0), erucic acid (C22:1 ω-9), docosahexaenoic acid (C22:6 ω-3), tricosanoic acid (C23:0), and nervonic acid (C24:1 ω-9) were purchased from Sigma-Aldrich (St. Louis, United States) or TCI Co. (Tokyo, Japan). All FAs except butanoic acid were dissolved in dimethyl sulfoxide (DMSO); butanoic acid was dissolved in water. DMSO at 0.1% v/v, which had no effect on bacterial growth or biofilm formation, was used as a control. Antibiotics viz. tobramycin, kanamycin, streptomycin, amoxicillin, vancomycin, tetracycline, erythromycin, rifampicin, gentamicin, ciprofloxacin, and methicillin were obtained from Sigma-Aldrich.

Planktonic cell growths and turbidities were measured at 600 nm using an Optizen 2120 UV spectrophotometer (Mecasys Co. Ltd., Daejeon, Korea). MIC was defined as the lowest concentration that visually inhibited the growth of planktonic cells, and MICs were also confirmed by colony counting and droplet assay modified from previous publication (Sieuwerts et al., 2008).

The combinatorial efficacies of FAs and antimicrobial agents were analyzed using the two S. aureus strains. Briefly, overnight cultures at an initial turbidity of OD 0.05 at 600 nm were inoculated into proper culture media (final volume 1,000 μl) with antibiotics and/or FAs. Cells were incubated with shaking for 1 h at 37°C, and cell mixtures were diluted serially in PBS (phosphate-buffered saline) and plated on LB agar plates. CFUs were determined by colony counting after incubation for 24 h at 37°C. Experiments were performed using two independent cultures in quadruplicate.

Biofilm formation by the two S. aureus strains was quantified in 96-well polystyrene plates (SPL Life Sciences, Korea), as previously described (Lee et al., 2019). Briefly, cells were inoculated into LB broth (300 μl) at an initial turbidity of 0.05 (3.5 × 10⁹ CFU/ml) at 600 nm and FAs were added at different concentrations and incubated for 24 h at 37°C in a static state. To quantify biofilm formation, cell cultures were rinsed three times with water (300 μl), and then biofilms were tinted with 0.1% crystal violet (300 μl) for 20 min, and dissolved in 300 μl of 95% ethanol. Absorbances were measured at 570 nm (OD₅₇₀) using a Multiskan EX microplate reader (Thermo Fisher Scientific, Waltham, USA). Static biofilm formation results are the averages of six replicate wells of two representative independent experiments.

To analyze biofilms, they were produced, as mentioned above, over 24 h at 37°C. After incubation, planktonic cells were discarded by gentle washing with distilled water three times and biofilms were analyzed by live imaging microscopy using the iRiS™ Digital Cell Imaging System (Logos BioSystems, Anyang, Korea) at different magnifications. Biofilm images were regenerated as color-coded 2D and 3D pictures using ImageJ.¹ Staphylococcus aureus biofilms on nylon filter membranes were also examined by SEM, as previously described (Kim et al., 2016). Briefly, a nylon filter membrane (Merck Millipore, Burlington, United States) was cut into 0.4 cm × 0.4 cm pieces, autoclaved, and placed in each well of 96-well plates containing cell culture and incubated with or without myristoleic acid and/or tobramycin for 24 h at 37°C. Biofilm cells on nylon membranes were washed with PBS, fixed with glutaraldehyde (2.5%) and formaldehyde (2%) for 24 h, post-fixed with OsO₄ (Osmium tetroxide), and dehydrated using an ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) and isoamyl acetate. After critical-point drying and sputter coating, cells on membranes were imaged using an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV.

Replication numbers for assays are provided above and results are expressed as means ± standard deviations. The statistical analysis was performed by one-way ANOVA followed by Dunnett’s test using SPSS Ver. 23 (SPSS Inc., Chicago, United States). The values of p < 0.05 were considered as significant, and asterisks indicate significant differences between treated and untreated samples.

The combinatorial antimicrobial activities of the 30 FAs (15 saturated and 15 unsaturated FAs) at 10 μg/ml were initially investigated against a methicillin-sensitive S. aureus strain (MSSA; ATCC 6538) in combination with the aminoglycoside tobramycin. Tobramycin treatment alone at a concentration of 10 μg/ml in 1 h decreased cell survival by 92%, whereas treatment with any of the FAs alone at the same concentration did not affect cell survival (Figure 1). Interestingly, co-treatment with tobramycin and FAs showed some synergistic antimicrobial effects with widely different efficiencies. Sixteen FAs, namely pentanoic (C5:0), heptanoic (C6:0), undecanoic (C11:0), lauric (C12:0), myristic (C14:0), myristoleic (C14:1), palmitoleic (C16:1 ω-5), oleic (C18:1 ω-9, cis), elaidic (C18:1 ω-9, trans), linoleic (C18:2 ω-6), conjugated linoleic (C18:2 ω-6), α-linolenic (C18:3 ω-3), γ-linolenic (C18:3 ω-6), arachidonic (C20:4 ω-6), erucic (C22:1 ω-9), and docosahexaenoic acid (C22:6 ω-3) significantly decreased cell survival as compared with tobramycin treatment alone (Figure 1A). Most notably, myristoleic acid plus tobramycin decreased cell survival by >5 log as compared with 1 log reduction by tobramycin alone. The synergistic antimicrobial efficacies of the six active FAs, namely, undecanoic, lauric, myristic, myristoleic, palmitoleic, and arachidonic acid, were also observed on a methicillin-resistant S. aureus strain (MRSA; MW2) and reduced cell survival by >1–3 log vs. tobramycin alone (Figure 1B). Hence, in subsequent studies, we focused on the effects of the most active FA, myristoleic acid, on both S. aureus strains.

The MICs of the 16 active FAs against MSSA ATCC 6538 are shown in Table 1. While the MICs of most FAs were > 400 μg/ml, the MICs of undecanoic, myristoleic, and linoleic acid were 200, 100, and 200 μg/ml, respectively. These results suggest that the antibacterial synergistic effect of FAs at 10 μg/ml was due to synergism between antibiotics and FAs but not to their antimicrobial activities.

To investigate which antibiotics, exhibit synergism with FAs, S. aureus was treated with 10 common antibiotics, namely kanamycin, streptomycin, amoxicillin, vancomycin, tetracycline, erythromycin, rifampicin, gentamicin, ciprofloxacin, and methicillin up to 200 μg/ml for 1 h. Three aminoglycosides, namely, kanamycin, gentamicin, and streptomycin significantly decreased cell survival, but the other seven antibiotics did not, indicating that these three had bactericidal effects (Supplementary Figure 1). Interestingly, unlike other antibiotics, tobramycin, kanamycin, gentamicin, and streptomycin are all aminoglycoside antibiotics.

As was expected, myristoleic acid in combination with kanamycin, gentamicin, or streptomycin dose-dependently decreased cell survival as compared with tobramycin treatment alone (Figure 2). For example, myristoleic acid (10 μg/ml) plus kanamycin (50 or 100 μg/ml) or gentamicin (5 or 10 μg/ml) decreased cell survival by >2 log while the synergistic effect of myristoleic acid was less when administered with streptomycin.

Staphylococcus aureus biofilm formation plays important roles in antimicrobial resistance and a variety of device-related infections (Stewart and Costerton, 2001), and sub-inhibitory concentrations of aminoglycosides often induce biofilm formation (Hoffman et al., 2005). Hence, we investigated the antibiofilm activities of tobramycin, kanamycin, gentamicin, and streptomycin alone and in combination with myristoleic acid. Treatment of MSSA ATCC 6538 with myristoleic acid alone dose-dependently reduced biofilm formation (Figure 3A), which is in agreement with a recent study which reported that myristoleic acid inhibited biofilm formation by Cutibacterium acnes alone or with S. aureus (Kim et al., 2021a). Low concentrations of myristoleic acid, tobramycin, and streptomycin slightly increased biofilm formation by S. aureus, whereas high doses decreased biofilm formation (Figure 3A). For example, streptomycin at 1 or 10 μg/ml increased S. aureus biofilm formation by 22% and 26%, respectively (Figure 3A).

To study the antibiofilm efficacies of myristoleic acid and antibiotic co-treatments, we administered antibiotics at concentrations that reduced biofilm formation by 16 to 75%. For example, tobramycin at 2 or 5 μg/ml, kanamycin at 50 μg/ml, gentamicin at 2 or 5 μg/ml, and streptomycin at 20 μg/ml were subjected to combinatorial assays.

Co-treatment with myristoleic acid and antibiotics enhanced antibiofilm activities. For example, tobramycin alone at 2 μg/ml reduced biofilm formation by 16%, whereas treatment with tobramycin (2 μg/ml) and myristoleic acid (5 μg/ml) reduced biofilm formation by 63% (Figure 3B). Similar results were obtained for tobramycin at 5 μg/ml (Figure 3C). Gentamicin alone at 2 μg/ml reduced biofilm formation by 28% and treatment with gentamicin (2 μg/ml) and myristoleic acid (5 μg/ml) reduced biofilm formation by 63% (Figure 3E). Similar results were also obtained for gentamicin at 5 ug/ml (Figure 3F). However, myristoleic acid had a smaller synergistic effect on the antibiofilm activities of kanamycin (Figure 3D) and streptomycin (Figure 3G). Fractional inhibitory concentration indices (FICI) for the antibiofilm activity were calculated to check the synergistic effects of myristoleic acid/antibiotic combinations. FICI values of tobramycin, gentamicin, kanamycin, and streptomycin were 0.2–0.3, 0.3–0.52, 1.5, and 0.6, respectively. Hence, combinatory effects were synergistic for tobramycin and gentamicin, additive for streptomycin, and indifferent for kanamycin.

In addition, combinatory antibiofilm efficacy trends of myristoleic acid and antibiotics against MRSA MW2 and MSSA ATCC 6538 were found to be similar, although MRSA MW2 produced less biofilm than MSSA ATCC 6538 (Figure 4).

Biofilm inhibition was analyzed using a live cell imaging system and by SEM. 3-D color mesh plots showed that tobramycin at 5 μg/ml or myristoleic acid at 20 μg/ml inhibited biofilm formation by S. aureus, but that in combination they had a markedly greater effect (Figure 5A). SEM analysis confirmed the inhibitory effect of myristoleic acid along with tobramycin and also showed the shape of S. aureus biofilm cells on the surface of nylon filter membrane. Treatment of myristoleic acid, tobramycin, or their combination markedly reduced the numbers of S. aureus cells. Tobramycin at 5 μg/ml or myristoleic acid at 20 μg/ml did not cause any surface defects or membrane damages. Interestingly, tobramycin or myristoleic acid produced few small cells, but in combination they produced many smaller cells (Figure 5B). Taken together, myristoleic acid co-treatment enhanced the antibiofilm effects of antibiotics on S. aureus.