Synergistic Effect between Cryptotanshinone and Antibiotics against Clinic Methicillin and Vancomycin-Resistant Staphylococcus aureus

Cryptotanshinone (CT), a major tanshinone of medicinal plant Salvia miltiorrhiza Bunge, demonstrated strong antibacterial activity against clinic isolated methicillin and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) in this experiment. The CT was determined against clinic isolated MRSA 1–16 with MIC and MBC values ranging from 4 to 32 and 8 to 128 μg/mL; for MSSA 1-2 from 16 to 32 μg/mL and 64 to 128 μg/mL; for VRSA 1-2 from 2 to 4 μg/mL and 4 to 16 μg/mL, respectively. The range of MIC50 and MIC90 of CT was 0.5–8 μg/mL and 4–64 μg/mL, respectively. The combination effects of CT with antibiotics were synergistic (FIC index <0.5) against most of tested clinic isolated MRSA, MSSA, and VRSA except additive, MRSA 4 and 16 in oxacillin, MRSA 6, 12, and 15 in ampicillin, and MRSA 6, 11, and 15 in vancomycin (FIC index < 0.75–1.0). Furthermore, a time-kill study showed that the growth of the tested bacteria was completely attenuated after 2–6 h of treatment with the 1/2 MIC of CT, regardless of whether it was administered alone or with ampicillin, oxacillin, or vancomycin. The results suggest that CT could be employed as a natural antibacterial agent against multidrug-resistant pathogens infection.


Introduction
Staphylococcus aureus (S. aureus) is one of the most important pathogens in both hospitals and the community and can cause numerous syndromes in humans, such as furuncle, carbuncle, abscess, pneumonia, meningitis, bacterial arthritis, myocarditis, endocarditis, osteomyelitis, and septicemia [1][2][3]. Methicillin-resistant Staphylococcus aureus (MRSA) is a significant problem in hospitals and communities worldwide, and awareness of MRSA in animals and reports of its zoonotic spread have increased in recent years [3,4]. The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has led to widespread, increased use of vancomycin [5,6]. Subsequently, numerous reports of elevated minimum inhibitory concentrations (MIC) to vancomycin among MRSA isolates have surfaced, concomitant with increased global vancomycin exposure to MRSA [7,8]. Greater than 60% of S. aureus isolates are now resistant to methicillin (oxacillin), and some strains have developed resistance to more than 20 different antimicrobial agents; new agents are therefore needed for the treatment of S. aureus [9,10]. Plant medicines are used on a worldwide scale to prevent and treat infectious diseases. They are of great demand both in the developed and developing countries for the primary health care needs due to their wide biological and medicinal activities, higher safety margin and lesser costs [11,12]. Plants are rich in a wide variety of secondary metabolites such as tannins, alkaloids, terpenoids, and flavonoids having been found in vitro since they have antimicrobial properties and may serve as an alternative, effective, cheap, and safe antimicrobial for the treatment of microbial infections [12][13][14][15]. At the same time,  because of the difficulty in developing chemical synthetic drugs and because of their side effects, scientists are making more efforts to search for new drugs from plant resources to combat clinical multidrug-resistant microbial infections [11,12,16]. The main components of S. miltiorrhiza can be divided into two groups: hydrophilic compounds such as salvianolic acids and lipophilic chemicals, including diterpenoid and tanshinones [17]. The second group of components, labeled as tanshinone I, tanshinone II, and cryptotanshinone, are the major bioactive constituents and have various kinds of pharmacological effects including antibacterial, antioxidant, and antitumor activities and prevention of angina pectoris and myocardial infarction [18][19][20]. The cryptotanshinone (CT) exhibits antimicrobial activity against a broad range of Gram-positive bacteria, including S. aureus, and Gramnegative bacteria as well as other microorganisms [18,21]. The hexane and chloroform fractions of S. miltiorrhiza evidenced profound antimicrobial activity and inhibited resistant gene expression against Staphylococcus aureus and MRSA (methicillin-resistant Staphylococcus aureus) [21,22].
In this study, the antimicrobial activities of cryptotanshinone (CT) against methicillin and vancomycin-resistant Staphylococcus aureus isolated in a clinic were assessed using broth microdilution method and the checkerboard and timekill methods for synergistic effect of the combination with antibiotics.

Plant Extraction and Isolation.
The air-dried roots (2.0 kg) of S. miltiorrhiza were crushed and extracted with MeOH (8 L × 3) at room temperature. The MeOH extracts (210 g) were evaporated and suspended in distilled water and partitioned sequentially with CH 2 Cl 2 , EtOAc, and n-BuOH. The CH 2 Cl 2 soluble fraction was concentrated in vacuo, and its crude extract (28 g) was subjected to silica gel column chromatography using a CHCl 3 -MeOH gradient solvent system to provide ten fractions (A1-A10). Fraction A4 was further subjected to silica gel column chromatography with a CHCl 3 -MeOH (10 : 1) to yield five fractions (B1-B5). Fraction B3 was purified on recycling prep-HPLC (JAIGEL GS column and 220 nm) eluted with MeOH (4.0 mL/min) to yield cryptotanshinone (98 mg). The structure of cryptotanshinone was identified by comparing its spectral data with those published [23,24].        (Table 1). Antibiotic susceptibility was determined in testing the inhibition zones (inoculums 0.5 McFarland suspension, 1.5 × 10 8 CFU/mL) and MIC/MBC (inoculums 5 × 10 5 CFU/mL) for strains, measured as described in the National Committee for Clinical Laboratory Standards (NCCLS, 1999). Briefly, the growth of bacteria was examined at 37 ∘ C in 0.95 mL of BHI broth containing various concentrations of CT. These tubes were inoculated with 5 × 10 5 colony-forming units (CFU)/mL of an overnight culture grown in BHI broth and incubated at 37 ∘ C. After 24 h of incubation, the optical density (OD) was measured spectrophotometrically at 550 nm. Three replicates were measured for each concentration of tested drugs.

Time-Kill Curves.
The bactericidal activities of the drugs evaluated in this study were also evaluated using time-kill curves constructed using the isolated and reference strains.
Cultures with an initial cell density of 1-5 × 10 6 CFU/mL were exposed to the MIC of CT alone or CT (1/2 MIC) plus oxacillin or ampicillin or vancomycin (1/2 MIC). Viable counts were conducted at 0, 0.5, 1, 2, 3, 4, 5, 6, 12, and 24 h by plating aliquots of the samples on agar and subsequent incubation for 24 hours at 37 ∘ C. All experiments were repeated several times and colony counts were conducted in duplicate, after which the means were determined.

Results and Discussion
Many researchers are studying natural products that could be used as antibiotics against MRSA and are employing novel dosing regimens and antimicrobials that would be advantageous for combating the therapeutic problems associated with S. aureus [10,13,14,16,27]. The main bioactive constituents of S. miltiorrhiza include water-soluble phenolic acids and lipophilic diterpenoid tanshinones [28][29][30]. Cryptotanshinone was isolated from dried S. miltiorrhiza roots and identified via comparison of their spectral data with the data reported in the literature [23,24].   Evaluation of in vivo effectiveness of the antimicrobial combinations is necessary to generate data that can be extrapolated to the clinical situation as well as predicting relevant concentration of optimal dosing regimens for both agents of the combinations [31,32]. Combination antibiotic therapy has been studied to promote the effective use of antibiotics in increasing in vivo activity of antibiotics, in preventing the spread of drug-resistant strains, and in minimizing toxicity [32,33]. The combination of oxacillin and CT resulted in a reduction in the MICs/MBCs for isolates VRSA 1-2 and MSSA 1-2, with the MICs/MBCs of 2/16 or 8/128 g/mL, for oxacillin becoming 0.125/0.125-0.25 g/mL and reduced by ≥4-fold, evidencing a synergistic effect as defined by a FICI and FBCI of ≤0.5 ( Table 2). The combination of oxacillin and CT resulted in a reduction against isolates MRSA 1-16, with the MICs/MBCs values of 0.5-16/2-32 g/mL, for oxacillin becoming 1-128/4-256 g/mL and reduced by ≥4fold, evidencing a synergistic effect as defined by a FICI and FBCI of ≤0.5 except MRSA 4 and 16 of additive (FICI ≥ 0.5) and MRSA 1, 7, and 15 of additive (FBCI ≥ 0.5). The combination of ampicillin and CT resulted in a reduction in the MICs/MBCs for isolates VRSA 1-2 and MSSA 1-2, with the MICs/MBCs of 0.5/1 or 2/4 g/mL and 4/8 or 8/16, for ampicillin becoming 32/128 or 64/256 g/mL and 16/64 or 32/128 g/mL and reduced by ≥4-fold, evidencing a synergistic effect as defined by a FICI and FBCI of ≤0.5 and in most of MRSA tested were reduced by ≥4-fold evidencing a synergistic effect as defined by a FICI and FBCI of ≤0.5 except MRSA 6, 12, and 15 (FICI ≥ 0.5) and MRSA 6, 11, and 13 (FBCI ≥ 0.5), respectively ( Table 3). The combination of vancomycin and CT resulted in a reduction against isolates MRSA 1-16, with the MICs/MBCs values of 1-16/2-32 g/mL, for vancomycin becoming 0.125-0.5/0.5-2 g/mL and reduced by ≥4-fold, evidencing a synergistic effect as defined by a FICI and FBCI of ≤ 0.5 except MRSA 6, 11, and 15 of additive (FICI ≥ 0.5) and MRSA 1 and 9 of additive (FBCI ≥ 0.5) and for isolates VRSA 1-2 and MSSA 1-2, with the MICs/MBCs of 0.5/1 or 1/4 g/mL and 4/8 or 16/16, for vancomycin becoming 4/8 or 8/16 g/mL and 0.25/2 or 0.25/1 g/mL and reduced by ≥4fold, evidencing a synergistic effect as defined by a FICI and FBCI of ≤0.5 except MSSA 1 and 2 of additive (FBCI ≥ 0.5) ( Table 4).
The effects of CT administered in combination with oxacillin and/or ampicillin and/or vancomycin against standard (MSSA and MRSA) and clinical isolates of MSSA (1, 2), VRSA (1,2), and MRSA (MRSA 1-16) were confirmed by time-kill curve experiments (Figures 1, 2, 3, 4, 5, 6, 7, and 8). Cultures of each strain of bacteria with a cell density of 10 6 CFU/mL were exposed to the MIC of CT and antibiotics alone or CT (1/2 MIC) with oxacillin (1/2 MIC), ampicillin (1/2 MIC), or vancomycin (1/2 MIC). We observed that 30 minutes of CT treatment with ampicillin, oxacillin, or vancomycin resulted in an increased rate of killing as compared to that observed with CT (MIC) alone. A profound bactericidal effect was exerted when a combination of drugs was utilized. The growth of the tested bacteria was completely attenuated after 2-5 h of treatment with the 1/2 MIC of CT, regardless of whether it was administered alone or with oxacillin (1/2 MIC), ampicillin (1/2 MIC), or vancomycin (1/2 MIC) (Figures 1-8).
It has been reported that some plant-derived compounds can improve the in vitro activity of some cell-wall inhibiting antibiotics by directly attacking the same target site, that is, peptidoglycan [7,9,34]. CT exhibits antimicrobial activity against a broad range of Gram-positive bacteria, including S. aureus, and Gram-negative bacteria as well as other microorganisms [21,22]. Despite the pharmacological activities, potential risks regarding combination use of S. miltiorrhiza and drugs have been observed [28,35,36]. Scientific findings concluded S. miltiorrhiza fraction, and its components (cryptotanshinone and dihydrotanshinone I) showed antibacterial activity against a broad range of bacteria, including the broad range of Gram-positive bacteria and Gram-negative bacteria, and superoxide radicals are considered important in the antibacterial actions of the agents [18]. The Gram-positive bacteria-specific properties of CT are caused by the inhibition of RNA and protein synthesis, rather than by attacking the bacterial membrane [21,22].
In conclusion, CT of S. miltiorrhiza is expected to be recognized as natural sources for the development of new functional drugs against multiresistant S. aureus, MRSA and VRSA.