1. Introduction
Presently, the global spread of methicillin-resistant Staphylococcus aureus (MRSA) is of great concern in the treatment of Staphylococcal infections, since it has quickly acquired resistance to all clinical antibacterial agents. Even a glycopeptides resistant strain, i.e., Vancomycin resistant S. aureus (VRSA) has also been reported [1]. MRSA has become the most common cause of infections among many pathogenic bacteria, leading to many life-threatening diseases such as endocarditis, pneumonia and toxin shock syndrome. In our hospital, MRSA could be found in over 80 percent of sputum samples of pneumonia elderly patients in the intensive care unit (ICU). Therefore, the developing of agents with novel modes of action is vital for overcoming this troublesome pathogen. Synergy of photochemicals with antibiotics has been viewed as approaching a new generation of phytopharmaceuticals [2,3]. We are devoting effort to search for novel anti-MRSA agents from plant sources with use in traditional Chinese medicine (TCM) in recent years [4–8].
Hypericum japonicum Thunb. ex Murray (Di-er-cao or Tian-ji-huang in Chinese; Guttiferae) was first recorded in Zhiwu Mingshi Tukao (or Illustrated Catalogue of Plants) which dates back to 1848 [9,10]. This medicinal plant has been used to treat viral hepatitis and other infectious diseases for over one hundred years. Bioassay-guided fractionation of the alcoholic extract of the aerial parts of Hypericum japonicum led us to identify an anti-MRSA xanthone Isojacareubin (ISJ). The present report investigates the anti-MRSA activity of this compound alone and its synergistic effects in combination with conventional antibacterial agents.
2. Results and Discussion
ISJ was isolated through bioassay-guided fractionation of extracts from H. japonicum (Table 1) [4]. Its structure was elucidated and identified mainly by spectral analysis and compared with a previous report (Scheme 1) [11]. Ten MRSA isolates, which were used for the evaluation of antibacterial activities, were characterized [4–8], and the presence of the mecA gene and SCCmec genotypes (Figure 1) was demonstrated through multiplex PCR experiments [12].
The in vitro anti-MRSA activities of ISJ and four conventional antibacterial agents, i.e., Ampicillin (AMP), Ceftazidime (CAZ), Levofloxacin (LEV) and Azithromycin (AZM) both alone and combined, against the 10 clinical MRSA strains of SCCmec III type [12] are shown in Table 2. The ranges of minimal inhibitory concentrations/minimal bactericidal concentrations (MICs/MBCs, μg/mL) alone were 4~16/16~64 for ISJ, within the order of potency as ISJ (4~16/16~64) = LEV (4~16/8~64) > AMP (16~128/64~512) > CAZ (128~512/128~ >1024) >> AZM (>1024), so the potency of ISJ and LEV were nearly equivalent. In the control experiments, MICs and MBCs of Vancomycin and ISJ against the methicillin-susceptible S. aureus (MSSA, ATCC 25923) strain were 1/2 and 8/16 μg/mL, respectively.
Tested by the chequerboard method [13], ISJ showed synergy when it was combined with various antibacterials, with the values of 50% of the fractional inhibitory concentration indices (FICI50) at 0.37 (AMP), 0.25 (CAZ) and 0.37 (LEV), respectively. However, the ISJ and AZM combination showed indifference (FICI50 = 1.50). The combination of ISJ with CAZ was observed as the most effective, with their values of MIC50 (μg/mL) reduced from 8 (ISJ alone) and 512 (CAZ alone) to 1 + 32 (ISJ + CAZ). Combination of ISJ and LEV was observed as less effective, with their values of MIC50 (μg/mL) reduced from 8 (ISJ alone) and 16 (LEV alone) to both 2 (ISJ + LEV). According to the CLSI interpretive standard for Staphylococcus spp., these values were close to the MICs (μg/mL) of breakpoints of LEV (≤1 for susceptible and ≥4 for resistant) and CAZ (≤8 for susceptible and ≥32 for resistant), respectively, which demonstrated significant potentiation of anti-MRSA effects (or even reversion of the resistance of the corresponding antibacterial agents against MRSA) [14].
Time-kill curves (Figure 2) showed ISJ alone was the most effective at reducing the viable counts by 4.66 log10CFU/mL (−4.66, synergy) of MRSA at 24 h of incubation at the concentration of 1 × MIC, while the counts (log10CFU/mL) of combinations of ISJ with AMP (−0.82, indifference), CAZ (−0.92, indifference), LEV (−1.5, additivity) and AZM (+0.53, indifference) were also observed against MR004 (one of the ten isolates) (data not shown). Therefore, the combined bactericidal activities were not as potent as those of bacteriostatic at the concentration of 1 × MIC [15]. Concentrations of higher MIC times of ISJ might produce better combinatory bactericidal effects.
H. japonicum is a Chinese herb used in TCM. Its 85% ethanol treated water extract was documented in Chinese Pharmacopoeia as an injection for the treatment of viral hepatitis [16]. In Europe, a plant of the same genus, i.e., H. perforatum is known by the name St John’s wort, which is a first-line herbal medicine used for mild to moderate depression [17]. These records demonstrated their safety of therapeutic properties. ISJ is a characteristic constituent in H. japonicum and was first found in the plant by Ishiguro et al. [11]. Although many xanthone derivatives have been studied for their antimicrobial (including anti-MRSA) activities, the limited distribution in plant resources has meant that ISJ remained as yet to be investigated to the best of our knowledge [18–20]. For the first time we studied its anti-MRSA activities in this report.
As the clinical MRSA which was originally called the “superbug” has been haunting us in recent years and has become an increasingly pressing clinical problem worldwide, anti-MRSA synergistic effects between plant natural compounds and conventional antibacterial agents has further been demonstrated here as a promising way of overcoming current antibiotic resistances [3].
3. Experimental Section
3.1. Plant Materials
The aerial parts of H. japonicum were purchased from the crude drug mart in Kunming, China. They were identified at the Botany Department, Kunming Institute of Botany (KIB), the Chinese Academy of Sciences. The voucher specimen is deposited at the herbarium of KIB.
3.2. Bacterial Strains and Media
MRSA strains (ten isolates with SCCmec III genotype) were obtained and characterized from the infectious sputum samples of critically ill patients in Kunming General Hospital [14,21,22]. The presence of mecA gene and SCCmec genotypes were determined by multiplex PCR method (Figure 1) [12]. ATCC 25923 was used as the control strain. Standard Mueller-Hinton agar and broth (MHA and MHB, Tianhe Microbial Agents Co., Hangzhou, China) were used as bacterial culture media. MHB was used for all susceptibility testing and time–kill experiments. Colony counts were determined using MHA plates.
3.3. Antibacterial Agents
Four antibacterial agents representing four conventional agents used in clinic were purchased from the manufacturers, i.e., Ampicillin (AMP) (North China pharmaceutical Co., Ltd., Shijiazhuang, China), Ceftazidime (CAZ) (Jida pharmaceutical Co., Ltd., Kunming, China), Azithromycin (AZM) and Levofloxacin (LEV) (Yangzhijiang pharmaceutical Co., Ltd., Taizhou, China). Vancomycin (Eli Lilly Japan K. K., Seishin Laboratories) was used as the positive control agent. Standard Cefoxitin disks were purchased from Tiantan biological products Co., Ltd. (Beijing, China).
3.4. Bioassay-Guided Isolation and Identification of ISJ
Powdered aerial parts of H. japonicum (4.7 kg) were extracted with 80% ethanol and the extract (500 g) was successively fractionated between water and petroleum ether (P), ethyl acetate (E) and n-butanol to afford four fractions. The E fraction (90 g), which showed the most activity by the agar diffusion test described in Section 3.5 below (Table 1) [4], was subjected to column chromatography over silica gel (300–400 mesh; Qingdao, China) and eluted (200 mL/fraction) with ethyl acetate. TLC monitored the eluates and combined to afford three sub-fractions (Frs. A–C1-6), gradient elution of Fr. C2 with P-E-M (methanol) (4:1:0.2–1:1:0.5) to furnish 20 mg of pure ISJ: light-yellow powder; C18H14O6, ESI-MS: m/z 327 ([M + 1]+); 1H NMR (400 MHz) δ: 7.58 (1H, d, J = 8.8 Hz, H-8), 7.05 (1H, d, J = 10.0 Hz, H-4′), 6.92 (1H, d, J = 8.8 Hz, H-7), 6.15 (1H, s, H-2), 5.72 (1H, d, J = 10.0 Hz, H-3′), 1.44 (6H, s, 2′, 2Me); 13C NMR (100 MHz) δ: 162.8 (C-1), 98.8 (C-2), 160.2 (C-3), 102.7 (C-4), 51.7 (C-4a), 146.5 (C-4b), 132.9 (C-5), 152.9 (C-6), 115.4 (C-7), 116.6 (C-8), 113.3 (C-8a), 180.4 (C-9), 101.4 (C-9a), 78.6 (C-2′), 127.8 (C-3′), 113.8 (C-4′), 28.3 (C-2′-Me). All the spectral data were identical with the literature [11].
3.5. Susceptibility Testing
The inhibition zone diameters (IZDs, mm) of the extracts (30 mg/mL in dimethyl sulfoxide) were determined by the agar diffusion method on MHA plates with inoculums of 1.5 × 108 CFU/mL of the microorganisms following the previous report [5]. MICs/MBCs were determined by standardized broth microdilution techniques with the final inoculums of 5 × 105 CFU/mL in each well of the 96-well plate according to the CLSI guidelines and incubated at 35 °C for 24 h [23,24]. All the experiments were performed in duplicate, with concentrations ranging up to 1024 mg/L for AZM.
3.6. Synergy Testing
Potential anti-MRSA synergy was measured by FICIs from the chequerboard method and by time-kill curves as previous report [13]. The FIC of the combination was calculated by dividing the MIC of the ISJ-antibacterial agent combination by the MIC of ISJ or of the antibacterial agent used alone. The FICI was obtained by adding the FIC of the ISJ and that of the antibacterial agent. The FICI results were interpreted as follows: FICI ≤ 0.5, synergy; 0.5 < FICI ≤ 1, additivity; and 1 < FICI < 2, indifference (or no effect) and FICI ≥ 2, antagonism [13,25]. In the killing curves, synergy was defined as ≥2 log10 CFU/mL increase in killing at 24 h with the combination, in comparison with the killing by the most active single drug. Additivity was defined as a 1–2 log10 CFU/mL increase in kill with the combination in comparison with the most active single agent. Indifference was defined as ±1 log10 CFU/mL killing or growth. Combinations that resulted in >1 log10 CFU/mL bacterial growth in comparison with the least active single agent were considered to represent antagonism [15,26]. All experiments were performed in triplicate.