Correspondence: Xuming Deng, Institute of Zoonoses, College of Animal Science and Veterinary Medicine, Jilin University, Xi'an Road 5333#, Changchun 130062, China. Tel.: +86 431 8783 6161; fax: +86 431 8783 6160; e-mail: email@example.com
In this study, the antibacterial activity of farrerol against Staphylococcus aureus was determined. The minimum inhibitory concentrations capable of inhibiting 35 S. aureus strains ranged from 4 to 16 μg mL−1. A haemolysis assay, Western blot and real-time reverse transcriptase-PCR assay were performed to identify the influence of subinhibitory concentrations of farrerol on the secretion of α-toxin by S. aureus. The results show that farrerol significantly decreased, in a dose-dependent manner, the production of α-toxin by both methicillin-sensitive S. aureus and methicillin-resistant S. aureus.
Staphylococcus aureus is a significant opportunistic pathogen that leads to a variety of infections. Treating such infections has been complicated by the widespread prevalence of methicillin-resistant S. aureus (MRSA) isolates. Therefore, there is an urgent need to develop novel and potent antimicrobial agents to treat life-threatening infections caused by MRSA strains.
Farrerol (Fig. 1) is a traditional Chinese medicine that has been commonly used as an antibechic. Additionally, farrerol exerts multiple biological activities, including anti-inflammatory, antibacterial and antioxidant activity for scavenging radicals and inhibiting a variety of enzymes (Zhu et al., 2007). However, to our knowledge, no studies have focused on its effects on S. aureus. In the present study, the anti-S. aureus activity of farrerol was evaluated, and the influence of subinhibitory concentrations of farrerol on α-toxin production by both methicillin-sensitive S. aureus (MSSA) and MRSA was determined.
Materials and methods
Bacterial strains and reagents
MSSA strain ATCC 29213 was obtained from the American Type Culture Collection (ATCC). Thirty-four S. aureus isolates, 14 MSSA and 20 MRSA (17 vancomycin-sensitive S. aureus and three vancomycin-intermediate S. aureus), were acquired from clinical samples at the First Hospital of Jilin University. These strains belong to four distinct pulsed field gel electrophoresis types. The clinical MRSA strains 2985 and 3701, which have the property to produce α-toxin, were subjected to further experimentation. Mueller–Hinton broth (MHB) was purchased from BD Biosciences Inc. (Sparks, MD). Farrerol (purity≥98%), oxacillin, vancomycin, gentamicin, erythromycin, clindamycin, tetracycline and ciprofloxacin were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and stock solutions of different concentrations were prepared in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, MO). Lipopolysaccharide (Escherichia coli 055:B5) and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY).
MTT assay for cell viability
The RAW264.7 mouse macrophage cell line was purchased from the China Cell Line Bank (Beijing, China). Cells were cultured in DMEM supplemented with 3 mM glutamine, antibiotics (100 U mL−1 penicillin and 100 U mL−1 streptomycin) and 10% heat-inactivated FBS. Cells were mechanically scraped, seeded in 96-well plates at 4 × 105 cells mL−1; following the addition of different concentrations of farrerol (4–32 μg mL−1), the macrophages were incubated in a 37 °C, 5% CO2 incubator for 48 h. Subsequently, 20 μL of 5 mg mL−1 MTT in FBS-free medium was added to each well and incubated for an additional 4 h. Cell-free supernatants were then removed and resolved with 150 μL DMSO. The OD570 nm was measured on a microplate reader.
Antimicrobial susceptibility testing
The minimal inhibitory concentration (MIC) of farrerol and other commonly used antibiotics for each isolate was determined using the broth microdilution method with an inoculum of 5 × 105 CFU mL−1 according to the Clinical and Laboratory Standards Institute guidelines, and incubated for 24 h at 37 °C (CLSI, 2005). All tests were performed in duplicate.
Bacteria were cultured in MHB at 37 °C, with graded subinhibitory concentrations of farrerol, until the postexponential growth phase (OD600 nm of 2.5) was reached. Culture supernatants were collected by centrifugation. Total haemolysis of culture supernatants were evaluated as described previously (Qiu et al., 2010b) using rabbit erythrocytes.
Staphylococcus aureus strains ATCC 29213, MRSA 2985 and MRSA 3701 were grown, and supernatants were prepared in the same manner as for the haemolysis assay. Samples (20 μL) of culture supernatants were boiled in Laemmli sample buffer and loaded on a 12% sodium dodecyl sulphate-polyacrylamide gel (Laemmli, 1970). Western blotting was performed as described by Xiang et al. (2010) and the product instructions for Amersham ECL Western blotting detection reagents (GE Healthcare, UK). Antibody to the α-toxin was obtained from Sigma-Aldrich.
Determinating proteolytic activity
A 100-μL volume of supernatant from the postexponential phase (OD600 nm of 2.5) cultures was added to 1 mL of azocasein (Sigma-Aldrich) and incubated at 37 °C for 1 h. After incubation, the reaction was stopped by adding 1 mL of 5% (w/v) trichloroacetic acid, and undigested azocasein was allowed to precipitate for 30 min. The mixture was then centrifuged at 10 000 g for 10 min, and A328 nm of the supernatant was read.
RNA isolation and real-time reverse transcriptase (RT-PCR)
Staphylococcus aureus strain ATCC 29213 was incubated with or without the addition of subinhibitory concentrations of farrerol in the same manner as for the haemolysis assay. Total RNA from the bacterial cultures was extracted as described previously (Qiu et al., 2010a). RNA was reverse transcribed into cDNA using the TaKaRa RNA PCR kit (AMV) Ver. 3.0 (Takara, Kyoto, Japan), according to the manufacturer's instructions. The primer pairs used in real-time RT-PCR are listed in Table 1. The PCR was performed using Sybr green. The reagents consisted of 12.5 μL 2 × SYBR Premix Ex Taq (Takara), 0.5 μL of each primer (10 μM) and 1 μL of sample cDNA in a final volume of 25 μL. The reactions were performed using the 7000 Sequence Detection System (Applied Biosystems, Courtaboeuf, France). Cycling conditions consisted of an initial denaturation step at 95 °C for 30 s, 35 cycles of 95 °C for 5 s, 55 °C for 30 s and 72 °C for 20 s. The melting curves for the PCR products were obtained by the stepwise increase of the temperature from 50 to 94 °C. All samples were analysed in triplicate, and the 16S rRNA housekeeping gene served as an internal control to normalize the expression levels between samples. The relative expression levels were analysed by the ΔΔCt method as described in the Applied Biosystems User Bulletin No. 2.
Table 1. Primers used in real-time RT-PCR
Location within gene
Stability studies of farrerol
The stability of farrerol in stock solution and culture medium was evaluated by HPLC analysis. The test was performed on an Agilent 1100 series (Agilent Technologies, Palo Alto, CA). Chromatography was performed through an ODS-3 analytical HPLC column (5 μm, 150 × 4.6 mm, Phenomenex, Torrance, CA). Elution was carried out with acetonitrile/ultrapure water (v/v, 70 : 30), operating at a flow rate of 1 mL min−1.
All statistical analyses were performed using spss 12.0 statistical software. Experimental data were expressed as the mean±SD. Statistical differences were examined using independent Student's t-test. A P-value of <0.05 indicated statistical significance.
The effect of farrerol on macrophage toxicity
Farrerol, at concentrations from 4 to 32 μg mL−1, did not display any cellular toxicity against RAW264.7 cells over 48 h, as determined by the MTT assay (data not shown).
Influence of farrerol on S. aureus growth
In this study, the antibacterial activity of farrerol against S. aureus was evaluated. The MICs of farrerol against 35 S. aureus strains ranged from 4 to 16 μg mL−1 (Table 2). The MIC value of strains ATCC 29213, MRSA 2985 and MRSA 3701 were 8 μg mL−1.
Table 2. MICs of farrerol, oxacillin, vancomycin, gentamicin, erythromycin, clindamycin, tetracycline and ciprofloxacin against 34 clinical isolates of Staphylococcus aureus
50% and 90%, MICs at which 50% and 90% of the isolates are inhibited, respectively.
Farrerol inhibits haemolysis of S. aureus by decreasing the level of α-toxin production
When cultured with 1/16 × MIC of farrerol, the haemolysis values of ATCC 29213, MRSA 2985 and MRSA 3701 culture supernatants were 52.7%, 90.5% and 86.9%, respectively, compared with a drug-free culture (Table 3). When at 1/2 × MIC, no haemolytic activity was observed. As expected, a dose-dependent (from 1/16 to 1/2 × MIC) attenuation of haemolysis was observed in all tested strains.
Table 3. Haemolytic activities in the supernatant of Staphylococcus aureus grown in increasing concentrations of farrerol
S. aureus strains
Haemolysis (%) of rabbit erythrocytes by culture supernatant†
None indicates no haemolytic activity was observed.
Values represent means±SD (n=3).
Haemolytic activity in untreated Staphylococcus aureus culture supernatants was set to 100%.
Farrerol decreased the production of α-toxin in a dose-dependent manner. Adding 1/16 × MIC of farrerol resulted in a recognizable reduction in α-toxin secretion; when at 1/4 × MIC or 1/2 × MIC, no immunoreactive protein was detected in supernatants from ATCC 29213, MRSA 2985 or MRSA 3701 cultures (Fig. 2).
The apparent reduction in secretion of α-toxin could result from an increase in protease secretion by S. aureus cultured in farrerol-containing medium. To address this possibility, extracellular proteases were quantified using azocasein. There was no significant effect on protease secretion by ATCC 29213, MRSA 2985 or MRSA 3701 cultured with 1/2 × MIC of farrerol.
Farrerol represses the transcription of hla and agrA by S. aureus
Real-time RT-PCR analysis was used to quantify mRNA levels of hla in S. aureus cultures after treatment with different concentrations of farrerol. As expression of hla is positively regulated by the agr locus (11), the transcription of agrA was also assessed. As expected, farrerol markedly decreased the transcription of hla and agrA in S. aureus strain ATCC 29213 in a dose-dependent manner (Fig. 3). When grown in the presence of 1/2 × MIC concentration of farrerol, the transcription levels of hla and agrA were decreased by 12.8-fold and 7.4-fold, respectively.
Stability of farrerol
Farrerol was stable in DMSO at 4 °C: after 10 days, the percentage of farrerol remaining was 98.8%. Furthermore, farrerol was stable in the culture medium (MHB) at 37 °C: after 48 h, the percentage of farrerol remaining was 97.9%.
In this study, we have demonstrated that farrerol is active against both MSSA and MRSA with MICs ranging from 4 to 16 μg mL−1. Consequently, farrerol may be used as a lead compound for the design of more potent antibacterial agents to be used in combating drug-resistant S. aureus strains.
Many toxin-encoding genes are coordinately regulated in response to a variety of global regulatory elements such as the accessory gene regulator (agr) and the staphylococcal accessory regulator (sar) (Novick, 2003). Previous studies have indicated that the inhibitory effects of antibiotics on S. aureus exotoxin production were secondary to the inhibition of translation of one or more global regulatory mRNAs (Herbert et al., 2001; Kuroda et al., 2007). Therefore, it is reasonable to speculate that the farrerol-induced inhibition of global regulators might lead to the decreased α-toxin production. Alpha-toxin is principally expressed during the postexponential growth phase, and is regulated by the agr locus (Recsei et al., 1986). Accordingly, we performed real-time RT-PCR to evaluate the influence of farrerol on the agr locus in S. aureus. Our data showed that farrerol significantly repressed the transcription of agrA in a dose-dependent fashion. However, the mechanism by which S. aureus controls virulence determinant gene expression is intricate and involves an interactive, hierarchical regulatory cascade involving the products of the agr and sar, as well as other components (Chan & Foster, 1998). Therefore, we presume that the reduction of α-toxin production observed in our study may be, in part, a consequence of farrerol-induced inhibition of the agr locus. The agr locus upregulates the expression of exotoxins genes while it downregulates the expression of surface-associated virulence factors. Therefore, in addition to α-toxin, the production of other exotoxins genes (e.g. enterotoxins and toxic shock syndrome toxin 1) may also be inhibited by farrerol. Meanwhile, farrerol might increase the expression of surface-related virulence factors (e.g. protein A).
This study was supported by National Key Project of Scientific and Technical Supporting Programs funded by Ministry of Science and Technology of China (No. 2006BAD31B05).