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Introduction

Staphylococcus aureus (S. aureus), a bacterium that grows in the human nose and skin and a major pathogen for skin and soft-tissue infections, has previously been treated with methicillin. S. aureus is a gram-positive bacterium and a major nosocomial agent, responsible for several hospital-acquired infections, including bacteremia, skin infections and septic shock (1–3). Hospital-acquired infections comprise a serious problem, particularly due to the increased prevalence of methicillin-resistant S. aureus (MRSA) (4,5). This pathogen is associated with various infectious diseases and has a mortality rate of 36–50% (6,7). With increasing resistance to various antibiotics, combination therapy appears to be a useful option, particularly in developing countries where antibiotic availability is limited (8–10). Furthermore, MRSA strains have been found to be resistant not only to β-lactam antibiotics, but also to fluoroquinolones and other types of antibiotics (8).

Luteolin (LUT), a polyphenolic flavonoid compound, is present in numerous plant groups, including Magnoliophyta, Pinophyta, Pteridophyta and Bryophyta species. Dietary sources of LUT include carrots, peppers, celery, olive oil, peppermint, thyme, rosemary and oregano. LUT is considered to have diverse biological benefits, including cardioprotective, antioxidant, anti-inflammatory and anticancer activities (11–14). In addition, LUT has exhibited a strong antiproliferative activity against various human cancer cell lines, such as lung cancer cell lines, and is a widely used ingredient in nutritional supplements (13,14). However, the antimicrobial activity of LUT against S. aureus has not yet been fully elucidated, but merits investigation.

The aim of the present study was to investigate the potential of using LUT in combination with antibiotics as an alternative therapeutic regime to overcome the drug-resistance of MRSA strains, including reference strains and clinical isolates.

Materials and methods

Reagents

Ampicillin (AM), oxacillin (OX), gentamicin (GT) and luteolin (≥98.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The structure of luteolin is shown in Fig. 1.

Figure 1. Structure of luteolin.

Bacterial strains and growth conditions

Five clinical MRSA isolates (DPS-1 to −5) were obtained from 5 different patients admitted to the Wonkwang University Hospital (Iksan, South Korea), and two reference strains were acquired from American Type Culture Collection (ATCC; Manassas, VA, USA): S. aureus ATCC 33591 (MRSA) and S. aureus ATCC 25923 (methicillin-sensitive S. aureus; MSSA). Prior to each experiment, bacteria were stored in 30% glycerol and frozen at −70°C. They were then cultured in Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA; Difco; BD Biosciences, Sparks, MD, USA) by incubating at 37°C for 24 h.

Minimum inhibitory concentration (MIC)

MICs were determined using the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (15). Briefly, a microorganism suspension was prepared by growing bacteria in broth for 24 h, and adjusting to a 0.5 McFarland standard turbidity (~1.5×108 CFU/ml). Final inoculums were adjusted to 1.5×106 CFU/ml. Serially diluted antibiotics and/or LUT were then incubated with the inoculum at 37°C for 18 h. The MIC was the lowest effective concentration of antimicrobial agent. At the end of the incubation period, the wells were visually examined for turbidity. Cloudiness indicated that bacterial growth had not been inhibited at the concentration of antimicrobial agent in the medium.

Checkerboard dilution test

Synergistic combinations were investigated using the preliminary checkerboard method (16). The MIC was defined as the lowest drug concentration, either alone or in combination, that inhibited visible bacterial growth. In vitro interactions were quantified by the fractional inhibitory concentration (FIC) index, which was calculated as FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone). FIC indices (FICI) were interpreted as follows: ≤0.5, synergy; <0.5-≤0.75, partial synergy; >0.75-≤1.0, additive effect; >1.0-≤4.0, indifference; and >4.0, antagonism. Finally, the varying rates of synergy between two given agents were determined (17). All experiments were conducted in triplicate.

Methyl thiazolyl tetrazolium assay (MTT) colorimetric assay

An MTT colorimetric assay for rapid detection of the presence of bacteria was performed as previously described (18–20). Briefly, a stock solution of 5 mg/ml MTT (Sigma-Aldrich) was prepared in phosphate-buffered saline and kept at −70°C. A final concentration of 1 mg/ml MTT was used in the assay. Following incubation of the bacteria for 24 h at 37°C, 20 µl yellow MTT was added to a 96-well microtiter plate and incubation was continued for an additional 20 min. Blue color indicated the presence of bacteria.

Time-kill curve assay

A time-kill curve assay was performed as previously described (21) to study the effects of antimicrobial agent concentration on bacterial growth over time. A standard inoculum of ~106 CFU/ml was used. LUT (0.5 MIC) was used with antibiotics (0.5 MIC) in various combinations. A test plate containing only MHB plus inoculum served as the control. Viable strains were counted at different intervals up to 24 h at 37°C. The rate and extent of bacterial death were determined by plotting viable colony counts (CFU/ml) against time in MHA. All experiments were conducted in triplicate.

Statistical analysis

All experiments were performed at least in triplicate. The data obtained from the experiments are presented as the mean ± standard error. The statistical analysis was performed using one-way analysis of variance followed by Dunnett's t-test (SPSS software 19.0; IBM Corp., Armonk, NY, USA). P<0.01 was considered to indicate a statistically significant difference.

Results

MICs and synergistic effect

Against all strains, the MIC of LUT was 62.5 µg/ml. The synergistic effects of LUT with various antibiotics were evaluated in MRSA strains using a checkerboard dilution assay. The antibacterial effects of LUT alone, each antibiotic alone, and LUT combined with one of the three antibiotics (AM, OX and GT) are shown in Tables I–III. The presence of LUT reduced the MIC of these antibiotics markedly against S. aureus strains. All strains were found to be resistant to AM, OX and GT, with MIC values ranging from 15.63 to 1,000 µg/ml. LUT + antibiotic combinations exhibited markedly decreased MICs. In combination with LUT, the MICs of AM, OX and GT underwent 2–16-, 0–16- and 4–16-fold reductions, respectively. None of the aforementioned combinations exhibited an antagonistic effect. These findings demonstrate the promising potential of LUT plus antibiotic combination therapy in the suppression of MRSA growth.

Table I. Interpreted FICI response for LUT + AM combinations against MRSA and MSSA strains.

Table I.

Interpreted FICI response for LUT + AM combinations against MRSA and MSSA strains.

	MIC (µg/ml)
	LUT		AM
Strain	Alone	+AM	Alone	+LUT	FICI
ATCC 33591	62.5	3.9	500	250	0.562
ATCC 25923	62.5	3.9	15.63	0.975	0.125
DPS-1	62.5	3.9	1,000	62.5	0.125
DPS-2	62.5	3.9	1,000	62.5	0.125
DPS-3	62.5	3.9	250	15.63	0.125
DPS-4	62.5	3.9	250	15.63	0.125
DPS-5	62.5	3.9	125	7.81	0.125

[i] FICI, fractional inhibitory concentration index; LUT, luteolin; AM, ampicillin; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive S. aureus; MIC, minimum inhibitory concentration.

Table III. Interpreted FICI response for LUT + GT combinations against MRSA and MSSA strains.

Table III.

Interpreted FICI response for LUT + GT combinations against MRSA and MSSA strains.

	MIC (µg/ml)
	LUT		GT
Strain	Alone	+GT	Alone	+LUT	FICI
ATCC33591	62.5	3.9	62.5	3.9	0.125
ATCC25923	62.5	3.9	62.5	3.9	0.125
DPS-1	62.5	3.9	250	62.5	0.312
DPS-2	62.5	31.25	500	31.25	0.562
DPS-3	62.5	3.9	500	31.25	0.125
DPS-4	62.5	3.9	500	31.25	0.125
DPS-5	62.5	3.9	500	62.5	0.187

[i] FICI, fractional inhibitory concentration index; LUT, luteolin; GT, gentamicin; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, meticillin-sensitive S. aureus; MIC, minimum inhibitory concentration.

The combined use of LUT and AM, OX or GT antibiotics against the ATCC 33591 MRSA strain resulted in a FICI of 0.125 or 0.562 (Tables I–III). None of the combinations exhibited an antagonistic effect (FICI, >4.0). These results demonstrate that combinations of LUT with antibiotics could be used for the suppression of MRSA growth.

Time-kill curve assay

Time-kill tests were performed to examine the synergistic effects of LUT and antibiotics over time. The control did not exhibit a reduction in CFU counts, and LUT or antibiotic alone did not induce cell death after 24 h. When used together, LUT and antibiotics markedly reduced bacterial counts. As shown in Figs. 2 and 3, the combination of ½MIC LUT + ½MIC antibiotic completely Notably, the combination of ½MIC LUT + ½MIC GT, completely inhibited the growth of S. aureus after 16 h.

Figure 2. Time-kill curves of LUT and OX against a clinical MRSA strain (DPS-2). Data represent the mean of three independent experiments. *P<0.001 vs. OX alone. LUT, luteolin; OX, oxacillin; MRSA, methicillin-resistant Staphylococcus aureus; MIC, minimum inhibitory concentration.

Figure 3. Time-kill curves of LUT and GT against clinical MRSA strains (DPS-3 and DPS-4). Data represent the mean of three independent experiments. *P<0.001 vs. GT alone. LUT, luteolin; GT, gentamicin; MRSA, methicillin-resistant Staphylococcus aureus. MIC, minimum inhibitory concentration.

Discussion

The resistance of S. aureus to drugs, including synthetic penicillin and other conventional antibiotics, is a major obstacle in the treatment MRSA infection, which is an increasingly common type of infection, particularly among hospitalized patients (22). While vancomycin is often used as the only remaining effective antibiotic against MRSA, even vancomycin-resistant species of S. aureus have now been reported (22,23). In the absence of any effective antibiotic treatment for multi-resistant infections, the development of alternative methods for the prevention and treatment of these diseases is required (22–24).

A consequence of the increase in the prevalence of resistant bacteria is that novel antimicrobial drugs active against infectious diseases are urgently required. Novel antimicrobial agents with no associated toxic or side effects may be developed from natural products. Combination therapy is the most commonly recommended empirical treatment for bacterial infections in intensive care units, since not all potential pathogens are susceptible to monotherapy; in addition, it may aid the prevention of antibacterial resistance (25). The aim of the present study was to investigate combination therapy with LUT and antibiotics against S. aureus, in order to find a solution to the problem of multi-drug resistance.

To the best of our knowledge, this is the first study investigating the potential of using antibiotics in combination with LUT in the treatment of MRSA and MSSA. As an indicator of the potency of LUT and antibiotic combinations against resistant strains of S. aureus, MIC values were determined. The antimicrobial activity of LUT was found to be moderate (MIC, 62.5 µg/ml). Checkerboard dilution tests were performed to determine the action of LUT alone, as well as its synergistic action with antibiotics against the 7 strains. Although LUT alone had only a moderate inhibitory effect on MRSA growth, when a non-growth inhibitory dose of LUT (62.5 µg/ml) or antibiotic was used, the combination was shown to be highly effective, with a FICI of 0.125–0.562. Similar effects were observed in the MSSA strain. This experimental method clearly showed that LUT was a potent MRSA growth inhibitor. The use of three different antibiotics, namely AM, OX and GT, in combination with LUT clearly suppressed MRSA growth, enabling the dose of the antibiotics to be reduced. The time-kill curves confirmed the ability of LUT to increase the antibacterial effects of the antibiotics, synergistically reducing the bacterial counts below the lowest detectable limit after 8–24 h. The present study demonstrated the potential of LUT as an effective therapeutic agent against MRSA, reinforcing the possibility of substantially reducing the use of existing antibiotics. The results obtained for the combinations tested in the current study suggest that LUT can increase susceptibility to antibacterial action, as well as reduce the inducible antibiotic resistance of bacteria.

In conclusion, LUT may have potential as an antibacterial drug candidate for clinical use against MRSA and MSSA. The results of the present study are promising and may support the use of drugs derived from natural products.

Acknowledgements

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea, the Ministry of Education, Science and Technology (grant nos. 2012-0004337 and 2008-0062484), and the Cooperative Research Program for Agriculture Science & Technology Development (project no. PJ00962202), Rural Development Administration, Republic of Korea.

References