Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria

In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract.


Introduction
The appearance of antibiotic-resistant bacteria has led to difficulties in treating infectious diseases. Therefore, new antibiotics have been developed, but bacteria rapidly evolve resistance to the new treatments, neutralizing their efficacy. Methicillin-resistant Staphylococcus aureus (MRSA) is one of such resistant strains. S. aureus is a bacterium commonly observed in nature as well as in normal skin, the nasal cavity, and the oral cavity. The bacterium causes diseases in many organs, including the skin when an individual is immunocompromised; this can result in serious diseases such as endocarditis and septicemia [1].
When antibiotics such as penicillin were developed, treatments for S. aureus infection were effective because of the high sensitivity of the bacterium to antibiotics; however, the frequency of resistant bacteria has increased with the utilization of penicillin [2,3]. The bacteria show resistance by producing -lactamase and inactivating antibiotics such as penicillin. Subsequently, methicillin, which is not inactivated by -lactamase, was developed and utilized for treating penicillin-resistant bacteria. However, since MRSA emerged in the early 1960s, its frequency has gradually increased [4]. MRSA is resistant to not only -lactamase antibiotics but also many other antibiotics, making effective treatment difficult [5,6]. Mechanisms of MRSA antibiotic resistance are not clearly understood. However, it is known that MRSA possesses mecA genes and produces PBP 2 , a derivative of penicillin-binding protein (PBP), which reduces the affinity of PBP to -lactamase antibiotics such as methicillin. In addition, resistance is reported to occur through the acquisition of resistant plasmid (R plasmid) with -lactamaseexpressing genes [2]. MRSA can form biofilms in implants that are injected into patients, in medical supplies or in medical devices [7,8]. Once biofilms are formed, they cannot be easily neutralized by antibiotics, paving the way to serious pathogenic infection [9]. MRSA can produce organic acids via carbohydrate metabolism pathways. It has been reported that organic acids produced by MRSA decrease pH and can facilitate biofilm formation [10,11]. MRSA possesses several virulence factors: sea encodes staphylococcal enterotoxin A (SEA). SEA, one of major virulence factors of S. aureus [12], is known to induce staphylococcal gastroenteritis, secrete Tcell-derived cytokines, and stimulate T-cell activation owing to the presence of immunomodulatory properties of superantigens. In S. aureus, production of virulence factors is regulated by global regulators such as agr and sarA [13].
MRSA has emerged to be one of the most important pathogenic bacteria because of its characteristics such as multidrug resistance and biofilm formation. Therefore, new drugs capable of treating an MRSA infection must be developed. Historically, many scientists have identified antibacterial substances in natural products, which is currently an active field of study [14,15].
Artemisia princeps (A. princeps), a perennial herb belonging to the Asteraceae family, is distributed throughout East Asia and is widely used to maintain hemostasis and in treating pain, hypermenorrhea, amenorrhea, uterine hemorrhage, hemorrhoids, inflammation, and menopausal diseases [16].
However, few studies have examined the effects of A. princeps on MRSA. In studies conducted to discover natural products with antibacterial effects against MRSA, it was found that ethanol extract of A. princeps showed antibacterial effects against MRSA. In this study, we investigated the inhibitory effect of A. princeps on proliferation, acid production, biofilm formation, and virulence gene expression of MRSA. Phytochemical analyses were also performed to investigate detailed chemical constituents of ethanol extract of A. princeps.

Materials and Methods
2.1. Material. Brain heart infusion (BHI) broth was purchased from Difco Laboratories (Detroit, MI, USA). Glucose and dimethyl sulfoxide (DMSO) were obtained from Sigma Co. (St. Louis, MO, USA). MRSA ATCC 33591 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Plant Material and Extraction. The leaves of A. princeps
were obtained from the oriental drug store Dae Hak Yak Kuk (Iksan, South Korea). The identity of the specimen was confirmed by Young-Hoi Kim at the College of Environmental and Bioresource sciences, Chonbuk National University (Jeonju, South Korea). A voucher specimen (number 8-10-13) has been deposited at the Herbarium of the Department of Oral Biochemistry in Wonkwang University. Dried leaves (250 g) of A. princeps were soaked in 3,500 mL of 70% ethanol for 72 h at room temperature. The extracted solution was filtered and evaporated under reduced pressure to yield an ethanol extract of 5.7 g (2.28%). After the extract was thoroughly dried to facilitate complete removal of the solvent, the dry extract was dissolved in DMSO to give the desired stock solution. The final concentration of DMSO applied to culture systems was adjusted to 0.1% (v/v), which did not interfere with the testing system. Control groups were treated with media containing 0.1% DMSO.

Bacterial Growth and Acid
Production. Bacterial growth was determined using a modification of a previously described method [3,8]. The growth of MRSA was examined at 37 ∘ C in 0.95 mL of BHI broth containing various concentrations of the ethanol extract of A. princeps. These tubes were inoculated with 0.05 mL of an overnight culture grown in BHI broth [final: 5 × 10 5 colony-forming units (CFU)/mL] and incubated at 37 ∘ C. After 24 h of incubation, the optical density (OD) of cells was measured spectrophotometrically at 550 nm, and the pH of the cultures was determined using a pH meter (Corning Inc., Corning, NY, USA). Three replicates were measured for each concentration of the test extract. NaF (0.1%) was used as a positive control.

Biofilm Assay.
The biofilm assay was based on a method described previously [6,11]. A. princeps extract was added to BHI broth containing 1% glucose in 35 mm polystyrene dishes, or 24-well plates (Nunc, Copenhagen, Denmark). The cultures were then inoculated with a seed culture of MRSA (final: 5 × 10 5 CFU/mL). After cultivating for 48 h at 37 ∘ C, the supernatant was removed completely, and the dishes, wells, or wells containing resin teeth were rinsed with distilled water. The amount of biofilm formed in the wells was measured by staining with 0.1% safranin. The bound safranin was released from the stained cells with 30% acetic acid, and the absorbance of the solution was measured at 530 nm. The biofilm formed on the surface of the resin teeth was also stained with 0.1% safranin and photographed.

Scanning Electron Microscopy (SEM).
The biofilm on 35 mm polystyrene dishes was also determined by SEM using a modification of a previously described method [4]. The biofilm formed on the dishes was rinsed with distilled water and fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) at 4 ∘ C for 24 h. After gradual dehydration with ethyl alcohol 60, 70, 80, 90, 95, and 100%, the sample was freeze-dried. The specimens were then sputter-coated with gold (108A sputter coater, Cressington Scientific Instruments Inc., Watford, UK). For observation, a JSM-6360 SEM (JEOL, Tokyo, Japan) was used.

Confocal Laser Scanning
Microscopy. Bactericidal effect of A. princeps extract was determined by confocal laser scanning microscopy. The cultured MRSA in BHI was diluted using BHI media to approximately 1 × 10 7 CFU/mL. The bacteria (1 × 10 7 CFU/mL) were treated with high concentrations (8-64 mg/mL) of A. princeps extract at 37 ∘ C under aerobic conditions. After 30 min of incubation, the bacteria were washed with PBS and stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA), prepared according to the manufacturer's instructions, for 15 min. Stained bacteria were observed confocal laser scanning microscopy (LSM 510, Zeiss, Germany). This method is based on two nucleic acid stains: green fluorescent SYTO . The primer pairs that were used in this study were described by previous report [17][18][19][20] and are listed in Table 1. 16S rRNA was used as an internal control.

Phytochemical
Screening. Phytochemical tests of the extract were performed as previously described [21,22]. Mayer's reagent was used for alkaloids, ferric chloride reagent for phenolics, Molish test for glycosides, Biuret reagent for peptides, Mg-HCl reagent for flavonoids, Liebermann-Burchard reagent for steroids, and silver nitrate reagent for organic acids.

Statistical Analysis.
All experiments were performed in triplicate. Data were analyzed using the Statistical Package for Social Sciences (SPSS, Chicago, IL, USA). The data are expressed as the mean ± standard deviation values. The differences between the means of the experimental and control groups were evaluated by Student's -test. Values of < 0.05 were considered statistically significant.

Results
In the present study, after performing ethanol extraction of A. princeps, the antibacterial effects against MRSA were tested. The results are shown in Figure 1. concentrations higher than 1 mg/mL. The positive control used in this study, 0.1% NaF, also showed antibacterial effects. The minimum inhibitory concentration (MIC) of ethanol extract of A. princeps against MRSA was confirmed to be 8 mg/mL. These results indicate that the ethanol extract of A. princeps has antibacterial effects against MRSA.
MRSA generates organic acids by metabolizing carbohydrates. To investigate whether the ethanol extract of A. princeps can suppress organic acid production in MRSA, the ethanol extract of A. princeps was added to MRSA culture medium and the change in pH was measured. The pH of the control culture medium was approximately 7.2 before the incubation, which decreased to approximately 5.87 after culturing (Table 2). However, this decrease in pH was suppressed in the group treated with the ethanol extract of A. princeps (1-8 mg/mL). The positive control, 0.1% NaF, also showed suppression in pH reduction. These results reveal that organic acid production in MRSA can be inhibited by the ethanol extract of A. princeps.
MRSA forms biofilms on implants, medical supplies, or on medical devices, which eventually increases the bacterium's antibiotic resistance. In this study, we examined whether an ethanol extract of A. princeps was able to inhibit the biofilm formation of MRSA. Using safranin staining, the ethanol extract of A. princeps at concentrations of 2-8 mg/mL was seen to inhibit MRSA biofilm formation (Figure 2). The positive control used in this study, 0.1% NaF, also inhibited  biofilm formation. These results were confirmed by SEM images (Figure 3), which showed similar outcomes to the results of safranin staining. In the control that was not treated with the ethanol extract of A. princeps, MRSA adhered densely to the surface of the polystyrene 35 mm dish and formed biofilms, whereas biofilm formation decreased in proportion to the ethanol extract concentration when the extract was introduced. Biofilm formation also decreased in the positive control. Using a confocal laser microscopy, the bactericidal effects of ethanol extract of A. princeps were examined. The ethanol extract of A. princeps was observed to be bactericidal in a dose-dependent manner (8-64 mg/mL) (Figure 4). Expression of mecA, an antibiotic-resistant gene of MRSA, as well that of sea, agrA, and sarA and virulence-factor genes was estimated using real-time PCR in sub-MIC. The ethanol extract of A. princeps inhibited mecA, sea, agrA, and sarA mRNA expression at concentrations greater than 1 mg/mL ( Figure 5).
Phytochemical analysis of princeps showed relatively high organic acid content, medium glycosides content, and weak phenolic content. Alkaloid, flavonoid, and peptides were detected only at very low levels ( Table 3).

Discussion
MRSA is a typical antibiotic-resistant strain. Development of new antibiotics is necessary for treating such strains. Natural products can be utilized as raw materials in the development of new antibacterial substances.
A. princeps is used as a digestive medicine, in treating fever, as an anthelmintic, and as an antihemorrhagic agent in oriental medicines and is known to be effective for treating gynecological and gastrointestinal diseases. In addition, its constituents are known to have various physiological effects such as insecticidal, antibacterial, and antitumor effects [23,24].
In this study, we investigated the antibacterial effects of an ethanol extract of A. princeps against MRSA. The ethanol extract of A. princeps at 1-8 mg/mL was found to inhibit MRSA growth. The evidence of antibacterial effects of the ethanol extract of A. princeps supports the use of A. princeps as a traditional medicine to treat patients with infectious diseases in Korea. According to previous studies on the aroma constituents of A. princeps, thujone, caryophyllene, and farnesol showed antibacterial effects against Escherichia coli, Enterobacter aerogenes, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus [23].
MRSA is known to produce organic acids through carbohydrate metabolism pathways [10,11]. Acetic acid is the main organic acid produced by MRSA, which lowers pH in the infected area; this lowered pH facilitates biofilm formation by microorganisms [10]. Our results showed that the ethanol extract of A. princeps inhibited the pH reduction induced by MRSA. This result indicates that the ethanol extract of A. princeps inhibits carbohydrate metabolism in MRSA.
MRSA adheres to and proliferates in damaged tissue, implanted medical and prosthetic devices, and is capable of forming biofilms [7,8]. Biofilms constitute bacterial communities that form on the surface of living and nonliving substances. Biofilms are surrounded by a self-produced extracellular matrix that consists of polysaccharides and proteins. These biofilms are very difficult to remove and are a cause of intractable infection. Biofilms formed on the surface of implanted medical devices cannot be removed by antibiotic injection and can only be removed by surgery in most cases. After excising the peripheral tissues of biofilms, antibiotics need to be utilized for a long period of time [7,8]. The most well-known method for analyzing biofilm formation is a tissue culture plate assay method [9]. In this study, biofilm formation was observed via safranin staining. The result showed that A. princeps inhibited biofilm formation of MRSA at concentrations of 1-8 mg/mL. Similar results were observed when MRSA biofilm formation was measured by SEM. A previous study reported that biofilm culture and planktonic culture showed different physiological characteristics, even when the same types of bacteria were cultured. Biofilm formation reportedly increases the immune response of bacteria and their resistance to antibacterial substances [17]. However, comparing biofilm culture data and planktonic culture data in the present study, there were no considerable differences in their resistance to ethanol extract of A. princeps between the two groups.
Bactericidal effects of ethanol extract of A. princeps were observed using confocal laser microscopy; the ethanol extract A. princeps showed bactericidal activity in a dose-dependent manner.
In MRSA, PBP 2 produced by the mecA gene has low affinity to -lactams antibiotics such as methicillin, and it is known to be antibiotic-resistant for cell-wall synthesis, even in the presence of -lactamase antibiotics [2]. In this study, mecA expression, an antibiotic-resistant gene of MRSA, was examined using real-time PCR. The ethanol extract of A. princeps inhibited mecA expression at concentrations higher than 1 mg/mL.
In MRSA, sea encodes SEA, which is a major virulence factor of S. aureus [12]. SEA induces staphylococcal gastroenteritis, secretion of T-cell-derived cytokines and Tcell activation because SEA possesses immunomodulatory properties of superantigens. The ethanol extract of A. princeps was observed to inhibit sea expression at concentrations higher than 1 mg/mL. NaF * * * * sea mRNA (relative quantity) (d) Figure 5: Real-time PCR analysis of expression of several virulence-factor genes. MRSA was cultured and treated with subminimal inhibitory concentration (1-4 mg/mL) of A. princeps extract, and real-time PCR analysis was then performed as described in Section 2. Expression of mecA, sea, and agrA was significantly inhibited at concentration higher than 1 mg/mL. Each value is expressed as a mean ± standard deviation. Significance was determined at * < 0.05 when compared with the control.
Production of virulence factors in S. aureus is controlled by global regulators such as agr and sarA [13]. agrA encodes accessory gene regulator A; when agrA expression is inhibited, production of virulence factors is also inhibited. In addition, sarA regulates the production of some matrix adhesion genes (e.g., fnbA) and exotoxin genes (e.g., hla), the virulence factors associated with adherence of S. aureus [25]. Our investigation of the expression of the agrA and sarA genes in this study showed that A. princeps inhibited sea expression at concentrations higher than 1 mg/mL.
According to a previous study, Artemisia spp. contain many components with strong antioxidant effects, such as caffeic acid, catechol, protocatechuic acid, vanillin, umbelliferone, and ferulic acid [26].
Based on our phytochemical analysis in the present study, the ethanol extract of A. princeps was confirmed to contain organic acids, glycosides, and phenolics. In particular, organic acids and glycosides showed an intense reaction and are thought to be the active components in the MRSA inhibition mechanisms. Therefore, further studies to examine the specific effects of the components present in A. princeps are necessary. Furthermore, additional studies should be conducted to identify antibacterial substances against MRSA.
In conclusion, we show that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract of A. princeps.