Rapid Bactericidal Activity of SC5005 Combined with Docosahexaenoic Acid against Multidrug-Resistant Staphylococcus aureus Persisters and Biofilms

ABSTRACT

Staphylococcus aureus can form persister cells and biofilms, making the treatment difficult and often leading to recurrent infections. In an effort to discover new anti-staphylococcal agents, we observed that oleic acid enhances the activity of a new antibacterial agent, SC5005, against S. aureus and MRSA strains. Subsequent studies showed that saturated or trans-form unsaturated fatty acids did not potentiate SC5005’s antibacterial activity. SC5005 only exhibits synergistic bactericidal activity with cis-form unsaturated fatty acids with 16 to 22 carbon atoms. In particular, docosahexaenoic acid (DHA) could reduce the MIC of SC5005 to the subng/mL range against different MRSA strains, including those resistant to second- and third-line antibiotics. However, we did not detect any significant shift in SC5005’s cytotoxicity toward four different mammalian cell lines, suggesting that the synergy of DHA and SC5005 is highly selective. Most importantly, this combination demonstrated fast-killing activity, completely eradicating MRSA USA300 planktonic and persister cells within 10 and 30 min, respectively, and removing nearly 98% of MRSA biofilms within 1 min. Together, our findings suggest that the combination of SC5005 and DHA has great potential as a new therapeutic for the treatment of infections caused by multidrug-resistant (MDR) S. aureus biofilms.

INTRODUCTION

Antibiotic resistance is a rising threat to public health worldwide. Infections caused by MDR bacteria are more difficult to treat and often lead to longer hospitalization times and higher mortality (1). It is estimated that by 2050, infections caused by antibiotic-resistant bacteria may result in 10 million deaths annually if no urgent action is taken (2). The World Health Organization (WHO) also highlighted that infections caused by several antibiotic-resistant pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), urgently need new antibiotics (3, 4).

S. aureus is a Gram-positive bacterium and the leading cause of hospital- and community-acquired infections in developed countries (5). It can produce various virulence factors, causing wound and skin structure infections and life-threatening diseases (5, 6). Additionally, S. aureus is capable of forming antibiotic-tolerant persister cells and biofilms, leading to chronic and recurrent infections (7). In the United States, the number of deaths due to S. aureus infection is greater than the sum of those due to HIV, viral hepatitis, tuberculosis, and influenza (8). In addition, S. aureus has acquired multiple resistance mechanisms to almost all antibiotics (9–12). Thus, the development of a new antibacterial therapy against MDR S. aureus has become an urgent need for public health.

Fatty acids (FAs) are amphiphilic molecules composed of hydrophobic carbon chains and hydrophilic carboxyl groups (13). FAs with no double bond in their carbon chains are known as saturated FAs, whereas those with double bond(s) are unsaturated FAs (13). Moreover, the double bond(s) may be either cis-form or trans-form, resulting in different physical properties. In nature, most unsaturated FAs are cis-form FAs, and trans-form FAs are more commonly manufactured via hydrogenation (14). Since 1911, FAs have been extensively demonstrated to exert antimicrobial activities against various pathogenic bacteria, including MRSA, by different modes of action (15). However, the lack of selective toxicity toward bacteria over host cells and the fact that they are effective only at a nonclinically relevant concentration limits the application of FAs in treating bacterial infections (13).

Previously, we discovered that SC5005, a derivative of the tyrosine kinase inhibitor sorafenib, possesses potent antibacterial activity against different staphylococcal species and MRSA strains by depolarizing and permeabilizing the bacterial membrane (16–18). Here, we found that the antibacterial activity of SC5005 can be significantly enhanced by oleic acid. Subsequent assessment of the synergism of SC5005 and various FAs indicated that docosahexaenoic acid (DHA) can dramatically potentiate SC5005’s activity against MRSA USA300 by more than 10,000-fold. Accordingly, the synergistic antibacterial activity of SC5005 and DHA against MDR S. aureus, persister cells, and biofilms is the focus of this investigation.

RESULTS

SC5005 exhibits synergistic antibacterial activity with cis-form monounsaturated FAs against MRSA USA300.

In developing the vehicle for in vivo efficacy evaluation, we unexpectedly discovered that SC5005 can act synergistically with oleic acid, a monosaturated FA (MUFA) (C18:1n9c) often used as an excipient in pharmaceuticals (19), to kill MRSA USA300 (Table 1 and Table S1). To gain insight into this synergy, we extended the test to combinations of SC5005 and various FAs having different numbers of carbon atoms, numbers of double bonds and geometrical properties (cis/trans). First, we found that despite having the same carbon length as oleic acid, saturated stearic acid (C18:0) did not show synergistic effects with SC5005 against MRSA USA300. A similar result was found for elaidic acid (C18:1n9t), which also has 18 carbon atoms but a trans-form double bond (Table 1). Our results showed that the cis-form double bond is important for the synergistic antibacterial activity of SC5005 and FAs.

TABLE 1.

Antibacterial activity of SC5005 in combination with fatty acids against MRSA USA300

Fatty acid (μg/mL)a	MRSA USA300
	MIC (μg/mL)
	FA	SC5005 (combination effect)b
		0	4	8	16
Myristoleic acid (C14:1n5c)	512	0.25	0.25 (NI)	0.25 (NI)	0.25 (NI)
Palmitoleic acid (C16:1n7c)	64	0.25	0.0625 (Syn)	0.03125 (Syn)	0.03125 (Syn)
Stearic acid (C18:0)	512	0.25	0.25 (NI)	0.25 (NI)	0.25 (NI)
Elaidic acid (C18:1n9t)	>1024	0.25	0.25 (NI)	0.25 (NI)	0.25 (NI)
Oleic acid (C18:1n9c)	512	0.25	0.03125 (Syn)	0.03125 (Syn)	0.03125 (Syn)
Linoleic acid (C18:2n6c)	1024	0.25	0.0156 (Syn)	0.0078 (Syn)	0.0078 (Syn)
α-Linolenic acid (C18:3n3c)	256	0.25	0.0625 (Syn)	0.0625 (Syn)	0.0156 (Syn)
Gondoic acid (C20:1n9c)	512	0.25	0.125 (NI)	0.25 (NI)	0.25 (NI)
Arachidonic acid (C20:4n6c)	128	0.25	0.0625 (Syn)	0.0625 (Syn)	0.03125 (Syn)
Eicosapentaenoic acid (C20:5n3c)	64	0.25	0.0625 (Syn)	0.0625 (Syn)	0.03125 (Syn)
Erucic acid (C22:1n9c)	128	0.25	0.125 (NI)	0.25 (NI)	0.25 (NI)
Docosahexaenoic acid (C22:6n3c)	1024	0.25	0.03125 (Syn)	0.000015 (Syn)	0.0000038 (Syn)
Nervonic acid (C24:1n9c)	128	0.25	0.25 (NI)	0.25 (NI)	0.25 (NI)

^aThe structure of individual fatty acids is represented by the form CA:DnEF, where A is the number of carbon atoms, D is the number of double bonds, E is the number of carbons from the methyl end to the first carbon in the double bond closest to the methyl end, and F denotes the geometric property of double bonds. c, cis or t, trans.

^bThe combination effect was measured by calculating the corresponding fractional inhibitory concentration index (FICI). Synergy (Syn) was defined as an FICI ≤0.5, no interaction (NI) was defined as an FICI > 0.5 and < 4.0, and antagonism (Ant) was defined as an FICI > 4.

Next, we investigated the effect of the carbon chain length of cis-form MUFAs on synergistic antibacterial activity with SC5005. The results of the MIC and MBC assays showed that the combined effect of SC5005 and the 16-carbon MUFA palmitoleic acid (C16:1n7c) was synergistic (Table 1 and Table S1). In contrast, the 20-carbon and 22-carbon MUFAs gondonic acid (C20:1n9c) and erucic acid (C22:1n9c), respectively, showed only a synergistic bactericidal effect with SC5005 but had no effect on the MIC of SC5005 against MRSA USA300 (Table 1 and Table S1). Moreover, 24-carbon nervonic acid (C24:1n9c) and 14-carbon myristoleic acid (C14:1n5c) did not show any synergistic activity with SC5005. Together, these findings indicate that SC5005 tends to have synergistic antibacterial activity with cis-form MUFAs having chain lengths in the range of 16 to 22 carbon atoms.

The number of double bonds is not correlated with the synergistic antibacterial activity between unsaturated FAs and SC5005.

The above evidence indicates that the cis-form double bond in the carbon chain plays an important role in the synergy of FAs and SC5005. Therefore, we further investigated whether the number of double bonds in the cis-form unsaturated FAs affects their synergistic antibacterial activity with SC5005. First, we evaluated the MIC and MBC of SC5005 against MRSA USA300 in the presence of cis-form polyunsaturated fatty acids (PUFAs) with 18 carbon atoms, including linoleic acid (C18:2n6c) and α-linolenic acid (C18:3n3c). The results showed that the antibacterial activity of SC5005 combined with linoleic acid was better than that with oleic acid or α-linoleic acid (Table 1 and Table S1). We also observed that although arachidonic acid (C20:4n6c) and eicosapentaenoic acid (C20:5n3c; EPA) have different numbers of double bonds, their effect on the antibacterial activity of SC5005 is the same (Table 1 and Table S1). The above findings indicate that the number of double bonds in the carbon chain of unsaturated FAs is not correlated with their synergistic antibacterial activity with SC5005.

Finally, we further assessed the antibacterial activity of SC5005 with docosahexaenoic acid (C22:6n3c; DHA), which has six double bonds and has been approved for treating cardiac diseases (20). In the presence of 8 μg/mL DHA, the MIC of SC5005 was dramatically reduced by more than 16,000-fold. Additionally, the killing activity of SC5005 was highly enhanced, as demonstrated by a 256-fold decrease in the SC5005 MBC against MRSA USA300 (Table 1 and Table S1). Together, our findings demonstrated that DHA has a superior potentiating effect on SC5005 among all FAs tested.

The combination of SC5005 and DHA is effective against S. aureus with resistance to second- and third-line antibiotics.

S. aureus is notorious for its ability to acquire resistance to different classes of antibiotics (9, 21). Therefore, we investigated whether the existing resistance mechanisms of S. aureus could confer cross-resistance to the combination of SC5005 and DHA. We assessed the activity of SC5005 combined with DHA against a collection of antibiotic-resistant S. aureus strains (Table S2). Despite possessing resistance to different classes of antibiotics, all bacterial strains tested exhibited the same susceptibility to DHA and SC5005, with MICs of 1024 μg/mL and 0.25 μg/mL, respectively (Table 2). Moreover, the combination of SC5005 and DHA also showed a synergistic antibacterial effect on all bacterial strains tested, but the susceptibility of individual strains to this combination was different. In the presence of 8 μg/mL DHA, we observed that MRSA ATCC49476 was most vulnerable to the antibacterial activity of SC5005 (MIC = 0.00000095 μg/mL), whereas SC5005’s MIC against S. aureus ATCC29213 was only 0.0039 μg/mL, implying that the number or the property of the target of the combination of SC5005 and DHA may vary among individual S. aureus strains. Nonetheless, our results still indicated that the synergistic antibacterial activity of SC5005 and DHA can overcome the mechanisms of S. aureus resistance to conventional antibiotics.

TABLE 2.

Antibacterial activity of SC5005 and PK150 in combination with DHA against a collection of S. aureus, MRSA, and VRSA strains

Bacterial strain	Resistance patterna	MIC (μg/mL)
		DHA	SC5005	PK150	+ 8 μg/mL DHA (combination effect)
					SC5005	PK150
S. aureus ATCC29213		1024	0.25	0.0625	0.0039 (Syn)	0.0039 (Syn)
S. aureus NCTC8325		1024	0.25	0.0625	0.0000019 (Syn)	0.000035 (Syn)
MRSA USA300	ERY, OXA, OFX, CIP	1024	0.25	0.0625	0.000015 (Syn)	0.00049 (Syn)
MRSA ATCC33591	ERY, OXA, TET	1024	0.25	0.0625	0.000061 (Syn)	0.000061 (Syn)
MRSA ATCC33592	ERY, OXA, TET, RIF	1024	0.25	0.0625	0.00049 (Syn)	0.00098 (Syn)
MRSA ATCC43300	ERY, OXA	1024	0.25	0.0625	0.00024 (Syn)	0.00098 (Syn)
MRSA ATCC49476	ERY, OXA, TET	1024	0.25	0.0625	0.00000095 (Syn)	0.000015 (Syn)
MRSA SCCmec VT	ERY, OXA, OFX	1024	0.25	0.125	0.0000019 (Syn)	0.00098 (Syn)
VRSA SJC1200	VAN	1024	0.25	0.125	0.0000019 (Syn)	0.00012 (Syn)
S. aureus DAP-NS	ERY, TET, OFX, CIP	1024	0.25	0.0625	0.0000019 (Syn)	0.0156 (Syn)

^aERY, erythromycin; OXA, oxacillin; TET, tetracycline; OFX, ofloxacin; CIP, ciprofloxacin; RIF, rifampicin; VAN, vancomycin.

PK150 also shows synergistic antibacterial activity with DHA.

Recently, Le et al. identified another sorafenib derivative, PK150, that possesses potent activity against S. aureus, Enterococcus faecalis, and Mycobacterium tuberculosis (22). By assaying the MIC of PK150 in CAMHB supplemented with 8 μg/mL DHA, we found that its antibacterial activities against S. aureus strains were highly enhanced (Table 2). Moreover, the combinatory effect of PK150 and DHA on individual bacterial strains was different, with MRSA ATCC49476 being the most susceptible to PK150, with an MIC of 0.000015 μg/mL, and the daptomycin-nonsusceptible isolate being the least susceptible (Table 2). In addition, the synergistic antibacterial activity of SC5005 along with DHA was higher than that of the combination of PK150 and DHA, as demonstrated by SC5005 having lower MIC values than PK150 against most S. aureus strains (Table 2). The above findings suggest that the synergistic antibacterial activity with DHA is conserved among SC5005 and PK150 and is independent of bacterial susceptibility to these compounds.

DHA does not increase the cytotoxicity of SC5005 toward mammalian cells.

The above results show that DHA dramatically increases SC5005’s antibacterial activity against MDR S. aureus. This finding raised the question of whether DHA also affects the cytotoxicity of SC5005 toward mammalian cells. To address this issue, we evaluated the antiproliferative activity of SC5005 toward four different mammalian cell lines, RAW264.7 murine macrophagic cells, HeLa human epithelial cells, HaCaT human keratinocytes, and HEK-293 human embryonic kidney cells, in the presence of escalating DHA concentrations. As shown, we did not observe a significant shift in the cytotoxicity of SC5005 toward the individual cell lines tested even in the presence of 16 μg/mL DHA (Table 3). In addition, we also did not detect significant hemolytic activity of SC5005 combined with 8 μg/mL DHA toward sheep red blood cells (RBCs), suggesting that this combination does not perturb the plasma membrane of eukaryotic cells (Fig. S1). Thus, this result suggests that the synergistic effect of SC5005 and DHA is specific to S. aureus without causing harm to mammalian cells.

TABLE 3.

Antiproliferative activity of SC5005 combined with DHA toward different mammalian cell lines

DHA (μg/mL)	SC5005 (μg/mL)
	RAW264.7		HeLa		HaCaT		HEK-293
	CC50	P valuea	CC50	P valuea	CC50	P valuea	CC50	P valuea
0	19.5 ± 0.6		22.5 ± 0.7		18.0 ± 1.7		14.1 ± 1.4
4	22.6 ± 0.8	0.09	24.2 ± 3.6	0.53	19.5 ± 1.7	0.63	15.6 ± 0.6	0.63
8	19.7 ± 2.5	1.00	21.9 ± 1.6	0.96	20.5 ± 0.3	0.21	14.9 ± 1.6	0.92
16	21.2 ± 2.3	0.53	21.4 ± 1.4	0.81	18.1 ± 1.6	1.00	14.0 ± 0.8	1.00

^aThe differences between the SC5005 + DHA treatment groups and the respective SC5005 alone treatment groups.

The combination of SC5005 and DHA rapidly eradicates S. aureus persister cells and biofilms.

To understand the mode of action of the synergistic antibacterial activity of SC5005 and DHA, we evaluated the killing kinetics of SC5005, DHA, and their combination against S. aureus NCTC8325 and MRSA USA300. Consistent with previous findings (16), we observed that SC5005 killed more than 99.9% of bacteria within 24 h (Fig. 1a and b). In contrast, DHA at 8 μg/mL exhibited only a moderate inhibitory effect on the growth of S. aureus NCTC8325 at the early stage of treatment, and then the growth of bacteria rebounded to the same level as that of the control group (Fig. 1a and b). It is worth noting that the combination of SC5005 and DHA completely eliminated all bacteria in 10 min, indicating that the synergistic antibacterial activity of SC5005 and DHA has a strong bactericidal effect (Fig. 1a and b).

FIG 1.

SC5005 combined with DHA effectively eradicates S. aureus persister cells. (a and b) The viability of S. aureus NCTC8325 (a) and MRSA USA300 (b) was assayed after exposure to SC5005 (4× MIC, 1 μg/mL; filled squares), DHA (8 μg/mL; filled inverted triangles), SC5005 combined with DHA (filled triangles), and vancomycin (VAN; 4× MIC, 4 μg/mL; filled circles). The number of viable bacteria in the broth after each exposure period was enumerated by a CFU assay and expressed as CFU/mL. The data shown are the mean ± SD of three independent experiments. (c and d) S. aureus NCTC8325 (c) and MRSA USA300 (d) were treated with vancomycin (VAN; 16× MIC, 16 μg/mL) in PBS for 12 h to trigger persistence, and then the bacterial cells were washed and exposed to SC5005 (16× MIC, 4 μg/mL; filled squares), DHA (8 μg/mL; filled inverted triangles), SC5005 combined with DHA (filled triangles), vancomycin (VAN; 16× MIC, 16 μg/mL; filled circle), and daptomycin (DAP; 16× MIC, 16 μg/mL; filled star). The number of viable bacteria at designated times was determined using a CFU assay. The data are representative of two independent experiments and are shown as the mean ± SD (n = 3 per group).

Recent evidence has revealed that S. aureus can enter a nongrowing state, the so-called persister form, and become tolerant to antibiotic treatment, leading to recurrent bacterial infections (23, 24). To isolate S. aureus persister cells, we treated bacteria with the bactericidal antibiotic vancomycin at a lethal concentration for 12 h in PBS buffer to eliminate nonpersister cells (25). As shown, the remaining bacterial cells were tolerant of the killing activity of vancomycin and another bactericidal antibiotic, daptomycin, indicating that this subpopulation of bacterial cells consisted of persister cells. In contrast, SC5005 and its combination with DHA were highly effective in killing persister cells, and the bacterial cells were eradicated within 24 h and 30 min, respectively (Fig. 1c and d). This finding suggests that the mechanism of action (MoA) of the combination of SC5005 and DHA is independent of bacterial metabolic activity.

When S. aureus attaches to medical implants or host tissue, it can establish biofilms that provide an environment that is conducive to the formation of persister cells and often associated with chronic infection and treatment failure (26, 27). To determine whether the combination of SC5005 and DHA can eradicate S. aureus persister cells in biofilms, we treated bacterial biofilms with escalating concentrations of SC5005 alone or along with 8 μg/mL DHA for 24 h. In line with our previous finding (18), SC5005 exhibited superior biofilm-eradicating activity compared with daptomycin and vancomycin (Fig. 2a and b). However, 8 μg/mL DHA did not enhance the biofilm-eradicating activity of SC5005 and even showed antagonism against MRSA USA300 biofilms, suggesting that the biofilm matrix, which is mainly composed of polysaccharides, proteins, and nucleic acids (28), might interfere with the synergy of SC5005 and DHA. Then, we elevated the concentration of DHA to 80 μg/mL and observed a 32-fold increase in the biofilm-eradicating activity of SC5005. Moreover, a subsequent time-line study showed that the combination of 1 μg/mL SC5005 and 80 μg/mL DHA removed nearly 98% of bacteria in biofilms in 1 min but had no detectable cytotoxicity toward RAW264.7 cells at up to 60 min (Fig. 2c and d). Collectively, our findings indicated that compared with second-line antibiotics, the combination of SC5005 and DHA exhibits superior and rapid bactericidal activity against both S. aureus persister cells and biofilms.

FIG 2.

SC5005 and DHA have synergistic and rapid eradication activity against S. aureus biofilms. Biofilms of S. aureus NCTC8325 (a) and MRSA USA300 (b) were treated with increasing concentrations of DHA, DAP, VAN, SC5005, and SC5005 combined with DHA for 24 h. The bacteria in the biofilms were harvested and then incubated in CAMHB for another 24 h. The number of viable bacteria was determined using a CFU assay. The data are representative of two independent experiments and are shown as the mean ± SD (n = 3 per group). ns, nonsignificant; P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (c) Biofilms of MRSA USA300 were treated with SC5005 along with 80 μg/mL DHA for 1, 3, or 10 min. The viable bacteria in the biofilms were harvested and enumerated using a CFU assay. The data are representative of two independent experiments and are shown as the mean ± SD (n = 3 per group). ns, nonsignificant; P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (d) RAW264.7 cells were exposed to increasing concentrations of SC5005 combined with 80 μg/mL DHA for 10, 30, or 60 min. The viability of cells was then determined using an MTT assay and expressed as a percentage relative to mock (DMSO and ethanol)-treated cells. Data shown are the mean ± SD (n = 3 per group).

DISCUSSION

The tolerance of S. aureus persister cells and biofilms to antibiotics underpins their role in chronic and recurrent infections. Patients with foreign implants and catheters often suffer from biofilm-associated diseases for which there is currently no effective antibacterial treatment (29). Here, we showed that, along with DHA, SC5005 can eradicate nearly 99% of S. aureus and MRSA biofilms in minutes at subμg/mL concentrations. This promising finding indicates that SC5005 and DHA can be developed as new therapeutics for diseases caused by S. aureus biofilms.

Although the combination of SC5005 and DHA exhibited strong bactericidal activity against MRSA in conventional growth media, their synergistic activity was highly attenuated in the presence of 2% FBS (data not shown), suggesting that the combination may not be suitable for the control of S. aureus systemic or skin infections. On the other hand, as previously found for their rapid eradication activity against S. aureus biofilms on microplates (Fig. 2c), we also observed that 1 μg/mL SC5005 combined with 40 μg/mL DHA can eliminate nearly 96% of S. aureus biofilms on silicone foley catheters (data not shown) but had no detectable cytotoxicity toward human RT4 bladder cells at up to 24 h (data not shown). Currently, we are working with clinicians to assess the activity of SC5005 and DHA against bacterial biofilms on foley catheters from patients with recurrent urinary tract infections, and to evaluate the potential of this combination to control catheter-associated urinary tract infections by infusing into patients’ bladders.

The antibacterial activity and mechanisms of different types of FAs vary (30–32). Our results showed that palmitoleic acid and EPA have the most potent activity against MRSA USA300, with MICs of 64 μg/mL (Table 1). However, their synergistic activity with SC5005 is much lower than that of DHA (Table 1), suggesting that the synergism of FAs and SC5005 is independent of the antibacterial activity of individual FAs. To elucidate the MoA of the combination of SC5005 and DHA, we sought to identify potential drug targets by analyzing mutations in the genomic DNA of drug-resistant isolates. However, even after exposure for 35 days, no significant decrease in the susceptibility of bacteria to this combination was observed (data not shown), suggesting that this combination has a low propensity to induce the development of resistance in S. aureus. In addition, we also observed no synergistic effect of SC5005 and DHA on bacterial membrane integrity and membrane potential (data not shown). The results suggested that the enhanced bactericidal activity of SC5005 by DHA is not mediated by increased perturbation of bacterial cytoplasmic membrane. Currently, new strategies to investigate the MoA of SC5005 combined with DHA are under way, including using RNA-seq techniques to assess the response of bacteria and the use of promoter-carrying mariner transposons to identify bacterial genes involved in their antibacterial activity. From a translational perspective, identifying the MoA of the combination of SC5005 and DHA will provide important information to guide the development of more potent derivatives of either agent for treating S. aureus infections.

MATERIALS AND METHODS

Bacterial strains.

The S. aureus strains ATCC29213 and BCRC12552 (also called NCTC8325), MRSA strain ATCC-BAA-1556 (also called USA300), ATCC33591, ATCC33592, ATCC43300, and ATCC49476, an SCCmec type V_T MRSA isolate (33), a vanA-mediated vancomycin-resistant S. aureus (VRSA) strain (34), and a daptomycin nonsusceptible (DAP-NS) S. aureus isolate were used in this study. Strains with the ATCC designation or BCRC designation were obtained from the American Type Culture Collection (ATCC) or the Bioresource Collection and Research Center (BCRC; Taiwan), respectively.

Cells.

The murine macrophage cell line RAW264.7 (BCRC) and human cervical epithelioid carcinoma cell line HeLa (BCRC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, New York, USA) supplemented with 10% heat-inactivated FBS (Gibco-BRL). The human embryonic kidney cell line HEK-293 (BCRC) and the immortalized nontumorigenic keratinocyte cell line HaCaT (obtained from Dr. Yung-Chiy Chang, National Taiwan University, Taiwan) were maintained in Roswell Park Memorial Institute (RPMI; Gibco-BRL) medium supplemented with 10% heat-inactivated FBS. Cells were cultured in T-75 flasks at 37°C in a 5% CO₂ atmosphere.

Reagents.

SC5005 and PK150 were synthesized as previously described (16, 22) and dissolved in DMSO to produce a stock solution (10 g/L). Oxacillin (10 g/L; Sigma–Aldrich, St. Louis, MO, USA), ofloxacin (1 g/L; Bio Basic, Markham ON, Canada), and vancomycin (10 g/L; Bio Basic) were dissolved in sterilized deionized water to produce stock solutions. Arachidonic acid, eicosapentaenoic acid, elaidic acid, erucic acid, gondoic acid, α-linolenic acid, linoleic acid, myristoleic acid, nervonic acid, oleic acid, palmitoleic acid, and stearic acid were purchased from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in 95% ethanol to produce stock solutions (10 g/L). Docosahexaenoic acid (40 g/L; Santa Cruz, TX, USA), erythromycin (10 g/L; Bio Basic), and tetracycline (10 g/L; Bio Basic) were dissolved in 95% ethanol to produce stock solutions. Ciprofloxacin (10 g/L; Sigma–Aldrich), daptomycin (10 g/L; Sigma–Aldrich), linezolid (10 g/L; Santa Cruz), rifampicin (10 g/L; Bio Basic), and sorafenib (10 g/L; kindly provided by Bayer Pharmaceuticals) were dissolved in DMSO to produce stock solutions.

MIC and MBC assays.

The MIC of individual test agents against each bacterial strain was determined using the broth microdilution method in accordance with CLSI guidelines (35). Briefly, overnight bacterial cultures in LB medium were diluted 1:50 in fresh LB medium and incubated at 37°C until reaching an OD₆₀₀ of 0.5 to 0.6. Bacterial suspensions were diluted to a final concentration of 5 × 10⁵ CFU/mL in fresh CAMHB and then exposed to each test agent at increasing doses in triplicate in 96-well plates for 24 h at 37°C. The MIC of each agent was defined as the lowest concentration without visible bacterial growth. The MBC was determined by subculturing 5 μL of broth dilutions at or above the MIC on LB agar plates for 24 h at 37°C. The lowest broth dilution that had no viable bacteria was considered the MBC.

To evaluate the effect of FAs on the antibacterial activities of SC5005 and PK150, bacterial suspensions were diluted to a final concentration of 5 × 10⁵ CFU/mL in fresh CAMHB supplemented with designated concentrations of FAs, followed by using the protocols described above to determine the MICs and MBCs of individual agents. Then, the synergy of each combination was measured by calculating their corresponding fractional inhibitory concentration index (FICI) with the equation ΣFIC (FICI) = FIC_A + FIC_B = (Comb_A/MIC_A) + (Comb_B/MIC_B), where MIC_A and MIC_B are the MICs of drugs A and B alone, respectively, and Comb_A and Comb_B are the concentrations of drugs A and B in combination, respectively. Synergy was defined as an FICI ≤ 0.5, no interaction was defined as an FICI > 0.5 to 4.0, and antagonism was defined as an FICI > 4 (36).

Antiproliferation assay.

The effects of SC5005 and DHA on the viability of mammalian cells were assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in six replicates per treatment group. Briefly, cells were seeded into 96-well, flat-bottomed plates at 10⁴ cells/well, cultured for 24 h, and then exposed to SC5005 at concentrations ranging from 5 to 30 μg/mL or the combination of SC5005 with designated concentrations of DHA for 24 h. Controls were treated with DMSO and ethanol vehicles at concentrations equal to those for drug-treated cells. At the end of treatments, the medium was replaced by 0.5 g/L MTT-containing culture medium, and the cells were incubated in a CO₂ incubator at 37°C for an additional 1.5 h. Supernatants were removed from the wells, and the reduced MTT dye was solubilized in DMSO followed by measuring the absorbance at 570 nm of each well using a VersaMax microplate reader (Molecular Devices, USA).

Hemolysis assay.

Sheep RBCs (TPM, Taiwan) were collected by centrifugation at 1,000 × g for 10 min and washed three times with normal saline (9 g/L NaCl). The cells were then suspended in normal saline to a final concentration of 4% (vol/vol) and exposed to DMSO (mock), 1% Triton X-100, or SC5005 at increasing doses (ranging from 0.25 to 64 μg/mL) with or without 8 μg/mL DHA in triplicate in 96-well plates for 1 h at 37°C. Then, the 96-well plates were centrifuged at 1,000 × g for 10 min to precipitate RBCs, and the absorbance of the supernatants at 540 nm was measured by a VersaMax microplate reader (Molecular Devices, USA). The results are expressed as percentages relative to those of 1% TX100-treated RBCs.

Persister killing assay.

Log-phase bacteria were adjusted to OD₆₀₀ = 0.2 and then treated with vancomycin at 16× MIC for 12 h at 37°C. Bacteria were collected by centrifugation at 4,000 × g for 10 min at 4°C, washed with ice-cold PBS, and suspended in PBS with test agents. Controls received DMSO and ethanol vehicles at concentrations equal to those for drug-treated bacteria. At the designated times, the number of viable bacteria was assessed by serial dilution of a 100-μL aliquot of bacterial suspension in PBS and then spread onto LB agar plates. After incubation at 37°C for 18 to 24 h, the number of colonies was counted and expressed as CFU/mL.

Biofilm eradication assay.

The eradication activities of daptomycin, vancomycin, SC5005, and the combination of SC5005 and DHA against bacterial biofilms were assessed as described previously (18). Briefly, bacteria were grown in tryptic soy broth (Becton, Dickinson, USA) supplemented with 1% glucose in a 96-well plate at 5 × 10⁵ CFU/mL to form biofilms on the pegs of the lid immersed in the bacterial culture. After 24 h, the pegs were washed twice with PBS and then transferred to a new 96-well plate containing test agents at concentrations ranging from 0.125 μg/mL to 1,024 μg/mL in each well. After incubating for 24 h at 37°C, the peg-lids were washed, placed onto a new 96-well plate with fresh CAMHB in each well and sonicated for 5 min to destroy the biofilms on the pegs, followed by assessment of the number of viable bacteria in each well by CFU assay.

Statistical analysis.

The data are expressed as the mean ± SD. Differences among group means were calculated using Prism software (v6.0, GraphPad, USA) using one-way ANOVA with Dunnett’s comparison and were considered significant at a P value <0.05.