In vitro synergism of magnolol and honokiol in combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a problematic pathogen posing a serious therapeutic challenge in the clinic. It is often multidrug-resistant (MDR) to conventional classes of antibacterial agents and there is an urgent need to develop new agents or strategies for treatment. Magnolol (ML) and honokiol (HL) are two naturally occurring diallylbiphenols which have been reported to show inhibition of MRSA. In this study their synergistic effects with antibacterial agents were further evaluated via checkerboard and time-kill assays. Methods The susceptibility spectrum of clinical MRSA strains was tested by the disk diffusion method. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of ML and HL were assayed by broth microdilution. The synergy was evaluated through checkerboard microdilution and time-killing experiments. Results ML and HL showed similar activity against both MSSA and MRSA with MIC/MBC at 16 ~ 64 mg/L, with potency similar to amikacin (AMK) and gentamicin (GEN). When they were used in combination with conventional antibacterial agents, they showed bacteriostatic synergy with FICIs between 0.25 ~ 0.5, leading to the combined MICs decreasing to as low as 1 ~ 2 and 1 ~ 16 mg/L for ML (HL) and the agents, respectively. MIC50 of the combinations decreased from 16 mg/L to 1 ~ 4 mg/L for ML (HL) and 8 ~ 128 mg/L to 2 ~ 64 mg/L for the antibacterial agents, which exhibited a broad spectrum of synergistic action with aminoglycosides (AMK, etilmicin (ETM) and GEN), floroquinolones (levofloxacin (LEV), ciprofloxacin and norfloxacin), fosfomycin (FOS) and piperacillin. The times of dilution (TOD, the extent of decreasing in MIC value) were determined up to 16 for the combined MIC. A more significant synergy after combining was determined as ML (HL) with AMK, ETM, GEN and FOS. ML (HL) combined with antibacterial agents did not show antagonistic effects on any of the ten MRSA strains. Reversal effects of MRSA resistance to AMK and GEN by ML and HL were also observed, respectively. All the combinations also showed better dynamic bactericidal activity against MRSA than any of single ML (HL) or the agents at 24 h incubation. The more significant synergy of combinations were determined as HL (ML) + ETM, HL + LEV and HL + AMK (GEN or FOS), with △LC24 of 2.02 ~ 2.25. Conclusion ML and HL showed synergistic potentiation of antibacterial agents against clinical isolates of MRSA and warrant further pharmacological investigation.


Background
The opportunistic pathogen Staphylococcus aureus (SA) is a leading cause of bacterial infections of people, causing a broad spectrum of pathology ranging from common skin infections to deep-seated fatal disease [1]. Antibiotic treatment of SA has once contributed greatly to human health for decades. However, due to long, wide and irrational applications of antibacterial agents in treatments in various fields other than in the clinic, methicillin-resistant Staphylococcus aureus (MRSA) has evolved as a problematic pathogen and has posed a serious therapeutic challenge in clinic [1,2]. Nowadays, MRSA infections can be monitored both in hospitals [healthcare-acquired /associated (HA) MRSA] and community [community-acquired/associated (CA) MRSA]. The livestock-associated MRSA [(LA) MRSA] has also occurred [3]. MRSA is able to produce resistance to nearly all common classes of antibiotics including βlactams, aminoglycosides, macrolides, tetracyclines and quinolones, and even the vancomycin-resistant S. aureus (VRSA) has also been reported [4]. The decreasing effectiveness of conventional drugs is continuously haunting both clinicians and drug researchers, and the critical shortage of new antibiotics in development against MRSA and other multidrug-resistant bacteria is of great concern worldwide. New targets with novel strategy of therapy and mechanism of action for development of antibacterial agents against MRSA are urgently needed [5].
Plant natural resources have been demonstrated to possess great chemical and biological diversities and promising findings of antibacterial phytochemicals which showed not only anti-MRSA activity alone, but also synergistic potentials when they were used in combination with conventional antibacterial agents against MRSA [6][7][8][9][10]. In recent years, we are devoting efforts to search for novel phytochemicals that showed synergy with conventional antibacterial agents against MRSA from medicinal plant sources, especially from the traditional Chinese medicines (TCM) [11][12][13]. We found two diallylbiphenols, i.e. magnolol (ML) and honokiol (HL) are such phytochemicals contained in the Chinese crude drug Hou-po, the stem or root bark of Magnolia officinalis Rehd. et Wils. (Magnoliaceae) [14].
Hou-po is an important species in TCM [14]. It has been traditionally used for respiration, digestion and infection related ailments like cough, diarrhea, and allergic rhinitis [14]. Modern pharmacological reports also demonstrated its antimicrobial, anti-inflammatory and analgesic, antianxiety and antidepressant, antitumor and anticoagulant effects as well as myocardial/cerebral ischemia protections [15]. ML and HL are two main phenolic constituents primarily isolated from Hou-po and also found in other Magnolia sp., together with other nonphenolic constituents such as alkaloids and essential oils [16][17][18]. The antimicrobial effects of Hou-po extracts on Bacillus anthracis, S. aureus and other pathogens were found as early as six decades ago [19][20][21]. Previous reports on antimicrobial activities of ML and HL and their synthetic derivatives include antibacterial [22][23][24][25][26][27][28][29][30][31], antifungal [32][33][34], antiviral [35] and nemicidal [36] activities. Although their antibacterial activities against MRSA and vancomycin-resistant enterococci (VRE) have been reported [37,38], their potential for combined action on conventional antibacterial agents against MRSA has not been studied. Only the synergy of honokiol with fluconazole against clinical isolates of azole-resistant Candida albicans [39], and synergistic effect of lysozyme on bactericidal activity of magnolol and honokiol against a cariogenic bacterium of Streptococcus mutans OMZ 176 [40] including their potentiation of the antitumor agents [41][42][43][44][45][46] were reported. In this paper, we will show the potential synergistic effects of ML and HL in combination with conventional antibacterial agents against clinical MRSA strains through the checkerboard and time-kill curve methods.

Bacterial strains and media
Ten MRSA strains (MRSA 01-10) were isolated and characterized from the infectious sputum sample of critically ill patients in KGH as previously reported [11][12][13]. The strains were determined with zone diameter (ZD) ≤ 21 mm against cefoxitin disk and the properties of susceptible (S), intermediate (I) and resistant (R) to antibacterial agents were determined according to the ZD Interpretive Criteria of Table 2C in 2012 CLSI by comparison with the ZD of corresponding antibacterial agents (Table 1) [47]. The presence of mecA gene and SCCmec genotypes were determined by multiplex PCR methods in Kunming Institute of Virology, PLA, China, as previously reported [48]. The control strain for MRSA was S. aureus (ATCC 25923; methicillin-susceptible S. aureus (MSSA)) which was purchased from the Beijing Tiantan Pharmaceutical and Biological Technology Co., Ltd., China. Standard Mueller-Hinton agar and broth (MHA and MHB, Tianhe Microbial Agents Co., Hangzhou, China) were used as bacterial culture media. Colony counts were determined using MHA plates. MHB was used for quantitative susceptibility testing and dynamic time-kill experiments.

Susceptibility testing
The test of susceptibility spectrum of the ten clinical MRSA strains to conventional antibacterial agents was performed by disk diffusion test following the CLSI guideline [47,49,50]. MICs/MBCs of ML and HL were determined by standardized broth microdilution techniques with inoculums of 5 × 10 5 CFU/mL according to CLSI guidelines and incubated at 35°C for 24 h [51][52][53]. The solvent used for dissolving the compounds and the antibiotics was MHB (or containing the final concentration of less than 5 % of dimethyl sulphoxide).

Statistical analysis
All the experiments were performed in triplicate. Data are expressed as the mean ± standard error. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS 20.0) software (SPSS Inc., Chicago, IL, USA). Data were analysed by Kruskal-Wallis test and the significant differences between groups were analysed by Dunnett's test. Statistical significance was accepted at a level of p < 0.01.

Antimicrobial effects of ML and HL
The MICs/MBCs of ML, HL and eight conventional antibacterial agents alone against MSSA and MRSA are shown in Table 2. As a whole, ML and HL appeared as two moderate bactericidal agents against both MSSA   Table 2). Table 3 shows the different degree of synergistic interactions of ML and HL in combination with the eight antibacterial agents against the ten clinical MRSA isolates.

Synergy of ML and HL in combination with antibacterial agents against MRSA
There are three to ten MRSA strains that showed synergy with FICIs between 0.25~0.5, leading to the combined MICs decreasing to as low as 1~2 and 1~16 mg/L for ML (HL) and the agents, respectively. MIC 50 of the combinations decreased from 16 mg/L to 1~4 mg/L for ML (HL) and 8~128 mg/L to 2~64 mg/L for the antibacterial agents, which exhibited a broad spectrum of synergistic action with aminoglycosides (amikacin, etilmicin and gentamicin), floroquinolones (levofloxacin, ciprofloxacin and norfloxacin), fosfomycin and piperacillin. The times of dilution (TOD, the extent of decreasing in MIC value) were determined up to 16 for the combined MIC. The more significant synergy after combining was determined as ML (HL) with AMK, ETM, GEN and FOS. Therefore, ML and HL showed generally the same synergistic bacteriostatic effects on the tested antibacterial agents. Moreover, ML (HL) combined with antibacterial agents did not show antagonistic effects on any of the ten MRSA strains. There were only 1~2 strains that showed indifference.
To further evaluate the dynamic bactericidal effects of ML (HL) in combination with the antibacterial agents, the time-kill curve experiments were performed and the results are shown in Table 4 (Table 4). All the combinations showed better bactericidal activity against MRSA than any of single ML (HL) or the agents at 24 h incubation. The bactericidal efficiency of the combinations generally lasted longer than that of the single agents (Figs. 2  and 3). The more significant combinations were determined as HL + ETM, HL + LEV and ML + ETM, with △LC 24 of 2.08~2.25 (Table 4). The combinations of antibacterial agents with HL showed more significant killing effects than those with ML for a same antibacterial agent. For example, the combination of HL + LEV showed synergy but the combination of ML + LEV showed only additively. Therefore, HL is a more optimistic agent for bactericidal potentiation of the effect of conventional antibacterial agents ( Table 4).

Reversal of MRSA resistance to amikacin and gentamicin by ML and HL
Besides the synergy effectiveness, the combination of ML (HL) with amikacin (AMK) led MICs (mg/L) of AMK to decrease markedly even to reverse the MRSA resistance to AMK by the MIC Interpretive Criteria of CLSI Performance Standards, i.e. MIC ≤16 mg/L (susceptible), MIC =32 mg/L (intermediate), MIC ≥64 mg/L (resistant) [47]. There is an equivalent eight strains of MRSA (n = 10) that showed MICs ≤16 mg/L against AMK when it was used in combination with ML and HL, respectively (p <0.01) ( Table 3 and Fig. 4). Similarly, three strains of MRSA against GEN (MIC ≤4 mg/L (susceptible), MIC =8 mg/L (intermediate), MIC ≥16 mg/L (resistant)) also showed reversal interaction in combination with ML (HL) ( Table 3) [47].

Discussion
In the present report, we performed the antibacterial evaluations of ML and HL against MRSA both used alone and in combination with clinical conventional antibacterial agents of broad classes. The synergism of ML and HL on MRSA is found for the first time so far to the best of our knowledge [37,39]. We determined the antibacterial activity of ML and HL against MRSA alone with MIC 50 /MBC 50 (n = 10) of 16~64 mg/L which are similar to the results of the previous report which showed MIC/MBC of 12.5/25 mg/L [37]. The difference is reasonable for the varied MRSA strains used. From the results of combinatory effect on MRSA and the reported antifungal synergism [39], the two compounds are demonstrated to possess a wide spectrum of antimicrobial potentiation of conventional chemotherapy. It would be beneficial for the treatment of mixed infections and even the co-infection of tumor diseases, considering ML (HL) also potentiating antitumor agents [41][42][43][44][45][46]. It is important and valuable of ML and HL that potentiate the antimicrobial activity of aminoglycosides and other antibacterial drugs against MRSA, which could prevent the drugs from development of MDR to the troublesome germ. As aminoglycosides are one class of the important antimicrobials for the treatment of infectious diseases, the MRSA resistance reversal effects of ML and HL to the aminoglycosides agents (AMK, ETM and GEN) are especially significant for their application with largely reducing toxic reactions of the hosts' liver, kidney and neural system by a substantially lowered dosage. Hence, greater sample scales are needed in order to draw a more reliable significance of the effectiveness on MRSA from clinical specimens and antibiotics.
The mechanism of the action of ML and HL against MRSA together with their synergism with antibacterial agents is still an unmet question. Two previous reviews summarized four main resistance mechanisms from bacteria: (i) receptor or active site modification, (ii) enzymatic degradation or modification of antibiotic, (iii) decreased penetration, or (iv) increased efflux [9,10]. It was reported that the antimicrobial mechanisms of Magnolia officinalis extract resulted mainly in cell membrane and wall damage, causing increased permeability of cell membranes or lysis of cell walls and loss of cellular constituents, impairment of structural components and changes in bacterial cell morphology [21], which could ascribed to ML and HL, the two main constituents of the plant. Some studies also have demonstrated that increased permeability of   the bacterial plasma membrane plays an important role in modulating resistance to aminoglycoside [57,58].
Another study showed a phenolic diterpene totarol inhibits multidrug efflux pump activity in Staphylococcus aureus [59]. Therefore, the agents in the present report which shows the resistance reversal effects on the aminoglycosides (AMK and GEN) or synergistic potentiation of other conventional drugs could be through these mechanisms to a certain degree, though the real mechanism is remained to be clarified. There are additional evidences revealing ML and HL as the modulators of the microbial membrane permeability. The two compounds showed active to extremely broad pathogenic microbial species. Apart from MSSA and MRSA, they showed as well antimicrobial activities against many other bacteria [19][20][21][22][23][24][25][26][27][28][29][30][31] and fungi [32][33][34] species and even exhibited antiviral [35] and nemicidal [36] activities. This antimicrobial mode is suggested like the antibacterial compounds of surface-active types which share the characters of usually nonselective to bacteria and very close MIC/MBC concentrations [30]. Therefore, ML and HL very likely target the extra cytoplasmic region as a nonionic surfactant and thus does not need to enter the cell, thereby avoiding most cellular pump-based resistance mechanisms as previously proposed [30].
The effects of ML (HL) on MRSA present here will expand the knowledge of their antimicrobial action and the future direction of anti-MDR investigations for drug development.

Conclusions
ML and HL showed a broad spectrum of synergistic potentiation of conventional antibacterial agents, especially the resistance reversal of AMK and GEN against clinical MRSA isolates. These in vitro activities of ML and HL might partly ascribe to modulate the bacterial cell membrane penetration and warrant further pharmacological investigation.

Ethics statement
The study was conducted in compliance with the ethics principles of the Declaration of Helsinki and Good Clinical Practice and China regulatory requirements. The study protocol (RCNM0116) was approved 10 June 2011 by the Ethics Committee and health authorities of Kunming General Hospital (KGH). Written informed consent was obtained from all subjects prior to sample commencement.