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Synergy of aminoglycoside antibiotics by 3-Benzylchroman derivatives from the Chinese drug Caesalpinia sappan against clinical methicillin-resistant Staphylococcus aureus (MRSA).

ABSTRACT

The in vitro antimicrobial activities of three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from Caesalpinia sappan L. (Leguminosae) were assayed, which mainly dealt with synergistic evaluation of aminoglycoside and other type of antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) by the three compounds through the Chequerboard and Time-kill curve methods. The results showed that Compounds 1-3 alone exhibited moderate to weak activity against methicillin-susceptible S. aureus (MSSA) and other standard strains by MICs/MBCs ranged from 32/64 to >1024/>1024 [micro]g/ml, with the order of activity as 1 > 2 > 3. Chequerboard method showed significant anti-MRSA synergy of 1/Aminoglycosides (Gentamicin, Amikacin, Etimicin and Streptomycin) combinations with [(FICIs).sub.50] at 0.375-0.5. The combined [(MICs).sub.50] values ([micro]g/ml) reduced from 32-128/16-64 to 4-8/4-16, respectively. The percent of reduction by MICs ranged from 50% to 87.5%, with a maximum of 93.8% (1/16 of the alone MIC). Combinations of 2 and 3 with Aminoglycosides and the other antibiotics showed less potency of synergy. The dynamic Time-killing experiment further demonstrated that the combinations of 1 /aminoglycoside were synergistically bactericidal against MRSA. The anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of 1/Aminoglycoside combinations was in good consistency, which made the resistance reversed by CLSI guidelines. We concluded that the 3-Benzylchroman derivative Brazilin (1) showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection.

Keywords:

Anti-MRSA activity

Synergy

Brazilin

Aminoglycoside

Bactericidal

Introduction

The clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) is a so called "superbug" which was originated date back to 1961 (Jevons 1961). Nowadays the alarm bell of "Entering a post-antibiotic era" is still ringing (Alanis 2005; Kahrstrom 2013). MRSA is notorious for not only its multi-drug resistant to conventional antibiotics, but also its global epidemiology of healthcare-acquired/associated (HA), community-acquired/associated (CA) and livestock-associated (LA). It is highly prevalent in hospitals worldwide and the highest rates (>50%) were reported in North and South America, Asia and Malta (Stefani et al. 2012; Dubey et al. 2013). The critical shortage of new antibiotics in development against MRSA and other multidrug-resistant bacteria is of great concern, and new targets and modes of action against MRSA are urgently needed (Freire-Moran et al. 2011).

Medicinal plants have been demonstrated as the potential anti-MRSA resources by researchers worldwide and the therapeutic potential of phytochemicals has been increasingly recognized (Gibbons 2004,2008; Mahady 2005; Zuo et al. 2008a, 2008b; Zahin et al. 2010; Radulovic et al. 2013). Moreover, special attentions have been paid to the synergy of phytochemicals with conventional antibiotics in reducing or even reversing the drug-resistance (Hemaiswarya et al. 2008). Synergy research has been emphasized as "Approaching a new generation of phytopharmaceuticals" (Wagner and Ulrich-Merzenich 2009). However, we notice that reports of this field are fewer than those of the abundant screening plant extracts and active compounds (Radulovic et al. 2013). Therefore, we have recently focused on searching for new antibiotics' synergistic phytochemicals from the Chinese medicinal plants (An et al. 2011; Zuo et al" 2011, 2012). We herein report the promising anti-MRSA synergy of aminoglycoside and other types of antibiotics combined with three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) from the Chinese drug Sappan Lignum, the heartwood of Caesalpinia sappan L. (Leguminosae) (NUTCM 2005).

Materials and methods

Antibacterial agents

Eight antibiotics including four Aminoglycosides were purchased from the manufacturers in China, i.e. Gentamicin (GEN) and Ceftazidime (CAZ) (Guangzhou Baiyunshan Tianxin Pharmaceutical Co., Ltd.); Amikacin (AMK) (Jiangsu Wuzhong Pharmaceutical Group Co., Ltd.); Etimicin (ETM) (Wuxi Jimin kexin Shanhe Pharmaceutical Co., Ltd.); Streptomycin (STR) and Penicillin (PEN) (Shandong Lukang Pharmaceutical Co., Ltd.); Cefazolin (CFZ) (Harbin Pharmaceutical Group Co., Ltd.) and Azithromycin (AZM) (Yangtze River Pharmaceutical Group Co., Ltd.). Vancomycin (VAN) (Eli Lilly Japan K. K., Seishin Laboratories) was used as the positive control agent. Cefoxitin disks were purchased from Beijing Tiantan biological products Co., Ltd., China. The three 3-Benzylchroman derivatives Brazilin (1), Brazilein (2) and Sappanone B (3) were isolated from Sappan Lignum, the heartwood of C. sappan L. (Leguminosae) (NUTCM 2005). Their structures were identified mainly by spectral analysis and comparison with the data in the literatures (Saitoh et al. 1986; Kim et al. 1997).

Bacterial strains

Ten MRSA strains with SCCmec III genotype and mecA gene were obtained and characterized from the infectious sputum samples of critically ill patients in Kunming General Hospital as previously reported (An et al. 2011). The control strain was S. aureus (ATCC25923; Methicillin-susceptible S. aureus (MSSA)). MSSA and other standard strains of Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853) and Candida albicans (ATCCY0109) were purchased from the Beijing Tiantan Pharmaceutical and Biological Technology Co., Ltd., China and used in this experiment.

Media

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.

Bioactivity-guided fractionation, isolation and identification of Compounds 1-3

The Sappan Lignum (5.0 kg; Voucher specimen KUN273 in Herbarium of Kunming Institute of Botany, China) was powdered, macerate and extracted with 80% ethanol for three times at the room temperature (7, 3, 2 days x 40, 20 and 151, respectively). The mixtures were filtered and the resulting filtrates were combined. After evaporating the solvent, the crude ethanol extract (685 g, 13.7%) was suspended in 1000 ml deioned water and successively extracted with Petroleum ether, EtOAc and butanol to give four sub-extracts, including the extract from water layer (<1, 400, 42.2, and 24 g, respectively). The 200 g sub-extract from EtOAc which showed the most active against MRSA by disk diffusion method (Zuo et al. 2008b) was subjected to column chromatography with silica gel (200-300 mesh, 4000g; Qingdao Haiyang Chemical Co., Ltd), gradient eluting with Petroleum ether-EtOAc-MeOH (15:10:0-10:15:2.5) to give 19 fractions (SL-1-19). Further activity tracking of the fractions and repeated chromatography of the active SL-7 (14.85g) with silica gel (400 mesh; Petroleum ether-EtOAc-MeOH (25:10:1)) and Sephadex LH-20 (Amersham Pharmacia Biotech.; MeOH) to furnish Compounds 1 (858 mg) and 3 (152.2 mg). The similar treatment of SL-12 (10.05g) with silica gel (400mesh; petroleum ether-EtOAc-MeOH (20:10:1.5); petroleum ether-CHCl3-MeOH (7:3:2)) and polyamide (petroleum ether-chloroform-MeOH (7:3:3)) to furnish Compound 2 (76.3 mg).

Compound 1, Pale yellow needles (MeOH), [C.sub.16][H.sub.14][O.sub.5], ESI-MS m/z: 309 [[M+Na].sup.+]. [sup.1]H NMR: [delta] ppm [400 MHz, [CD.sub.3] OD] [[delta].sub.H] 3.68 (1H, d, J= 11.3, H-2a); 3.92 (1H, d, J= 10.2, H-2b), 4.00 (1H, s, H-4), 7.18 (1H, d, J = 8.4, H-5), 6.46 (1H, dd, J = 8.8, 2.5, H-6), 6.36 (1H, s, H-8), 2.77 (1H, d, J = 15.6, H-9a); 3.02 (1H, d, j= 15.6, H-9b), 6.59 (1H, s, H-2'), 6.70 (1H, s, H-5'). [sup.13]C NMR: [delta] ppm [100 MHz, [CD.sub.3] OD] [[delta].sub.C] 70.8 (C-2), 78.1 (C-3), 51.0 (C-4), 115.5 (C-4a), 132.2 (C-5), 109.9 (C-6), 155.7 (C-7), 104.2 (C-8), 157.9 (C-8a), 42.9 (C-9), 131.3 (C-1'), 112.8 (C-2'), 145.6 (C-3'), 145.3 (C-4'), 112.4 (C-5'), 137.4 (C-6'). The data were in agreement with those of Brazilin (Kim et al. 1997).

Compound 2, Red-brown crystals (MeOH), [C.sub.16][H.sub.14][O.sub.5], ESI-MS m/z: 307 [[M+Na].sup.+].1H NMR: [delta] ppm [400 MHz, [CD.sub.3]OD] [[delta].sub.H] 4.12 (1H, d, J = 9.0, H-2a); 4.47 (1H, d, J= 8.9, H-2b), 8.25 (1H, d, J = 9.9, H-5), 6.77 (1H, dd, J = 8.1, 2.8, H-6), 6.54 (1H, d, J = 2.1, H-8), 3.07 (2H, s, H-9), 6.56 (1H, s, H-2'), 7.49 (1H, s, H-5'). [sup.13]C NMR: [delta] ppm [100 MHz, [CD.sub.3]OD] <5c 70.6 (C-2), 78.4 (C-3), 150.9 (C-4), 108.8 (C-4a), 129.9 (C-5), 108.8 (C-6), 164.4 (C-7), 103.3 (C-8), 158.8 (C-8a), 38.9 (C-9), 159.3 (C-1'), 116.0 (C-2'), 182.2 (C-3'), 152.9 (C-4'), 111.2 (C-5'), 124.8 (C-6'). The data were in agreement with those of Brazilein (Kim et al. 1997).

Compound 3, Yellow needles (MeOH), [C.sub.16][H.sub.14][O.sub.6], EI-MS m/z: 302 ([[M].sup.+], 17), 284 (8), 180 (87), 137 (72), 123 (100). [sup.1]H NMR: [delta] ppm [400 MHz, [CD.sub.3]OD] [[delta].sub.H] 3.98 (1H d, J = 11.2Hz, H-2a); 4.11 (1H, d, J = 11.2, H-2b), 7.70 (1H, d, J = 8.7, H-5), 6.68 (1H, dd, J = 8.0, 3.9, H-6), 6.52 (1H, d, J = 2.2, H-8), 2.74 (1H, d, J= 14.0, H-9a); 2.82 (1H, d, J = 14.0, H-9b), 6.77 (1H, d, J = 2.5, H-2'), 6.69 (1H, d, = 4.7, H-5'). [sup.13]C NMR: [delta] ppm [100 MHz, [CD.sub.3]OD] [[delta].sub.C] 73.3 (C-2), 74.1 (C-3), 195.8 (C-4), 113.2 (C-4a), 130.4 (C-5), 112.2 (C-6), 166.7 (C-7), 103.6 (C-8), 164.9 (C-8a), 40.8 (C-9), 127.7 (C-1'), 115.9 (C-2'), 145.8 (C-3'), 145.3 (C-4'), 118.9 (C-5'), 123.2 (C-6'). The data were in agreement with those of Sappanone B (Saitoh et al. 1986).

Susceptibility testing

MICs/MBCs of Compounds 1-3 were determined by standardized broth microdilution techniques with starting inoculums of 5 x [10.sup.5] CFU/ml according to CLSI guidelines and incubated at 35 [degrees]C for 24 h (CLSI 1999, 2006a,b, 2007). All the experiments were performed in duplicate.

Synergy testing

Potential anti-MRSA interaction type of Compounds 1-3 combined with various antibiotics was evaluated by fractional inhibitory concentration indices (FICIs) and time-kill curves through chequerboard method and dynamic time-killing method, respectively as described previously (An et al. 2011; Zuo et al. 2012). The bacteriostatic interaction type was judged by FICIs as the following: FICI [less than or equal to] 0.5, synergy; 0.5 < FICI [less than or equal to] 1, additivity; and 1 < FICI < 2, indifference (or no effect) and FICI [greater than or equal to] 2, antagonism (Flu et al. 2002; Orhan et al. 2005). The bactericidal interaction type was judged by the [log.sub.10] CFU/ml increase in killing at 24 h ([DELTA][LC.sub.24]) in comparison with the killing by the most active single drug as the following: [DELTA][LC.sub.24] >2 [log.sub.10] CFU/ml, synergy; [DELTA][LC.sub.24] = 1-2 [log.sub.10] CFU/ml, additivity; [DELTA][LC.sub.24] = [+ or -]1 [log.sub.10] CFU/ml, Indifference; Antagonism, [DELTA][LC.sub.24]> -1 [log.sub.10] CFU/ml (bacterial growth in comparison with the least active single agent were considered to represent antagonism) (Chin et al. 2008). The data from time-kill assays are presented as the means [+ or -] standard deviations.

Results and discussion

Three 3-Benzylchroman derivatives, i.e. Brazilin (1), Brazilein (2) and Sappanone B (3) were isolated from Sappan Lignum, the heartwood of C. sappan L. (Leguminosae) (NUTCM 2005) through bioactivity guided fractionation. Their structures (Fig. 1) were identified by spectral analysis and comparison of the data with those reported in the literatures (Saitoh et al. 1986; Kim et al. 1997). We firstly tested their in vitro antimicrobial activities alone against standard clinical pathogens of MSSA (ATCC25923) and MRSA strains, including E. coli, P. aeruginosa and C. albicans by microdilution methods following the CLSI guidelines (Tables 1 and 2) (CLSI 1999,2006a,b, 2007). Then their synergistic anti-MRSA interactions with eight conventional antibiotics through the classical Chequerboard method (Table 3) and Time-kill curve method (Table 4 and Fig. 2) were evaluated (An et al. 2011).

Compounds 1-3 alone showed moderate to weak activity against MSSA by MICs/MBCs ranged 32-128/64-512 ptg/ml, with the order of activity as Brazilin (1)> Brazilein (2) > Sappanone B (3) (Table 1). They all showed much weaker activity against other pathogens with MICs/MBCs from 512 to > 1024 [micro]g/ml (Table 1). The results of alone Anti-MRSA activity of Compounds 1-3, together with the eight antibiotics of Aminoglycosides (GEN, AMK, ETM and STR), (3-Lactams (CAZ, CFZ and PEN) and a Macrolide of AZM are shown in Table 2. The order of activity ([MIC.SUB.50] (n = 10), [micro]g/ml) is: VAN (1)>ETM (16)>(Brazilin (1), GEN and AMK; 32)>Brazilein (2) (64) (STR (64) and PEN (64))>Sappanone B (3) (128)>CFZ (256) > CAZ (512) > AZM (8192). Brazilin (1) showed the most active of the three 3-Benzylchroman derivatives owing to its typical structure, with potency equal to GEN and AMK. Brazilein (2) is the 3'-oxide of Brazilin (1). It caused the anti-MRSA of [MIC.sub.50]/[MBC.sub.50] reducing from 32/64 to 64/256 [micro]g/ml. Sappanone B (3) was considered to be a precursor of Brazilin (1) (Saitoh et al. 1986). It showed the least potency of the three compounds (128/256; Table 2). Compounds 1-3 showed almost the same potencies against both MSSA and MRSA (Tables 1 and 2). Brazilin (1) has been assayed of its bactericidal activity against MRSA and we confirmed it here with the similar potencies against ten clinical MRSA isolates (Xu and Lee 2004).

The results of combined activities in Table 3 assayed by Chequerboard method showed significant anti-MRSA synergies of Brazilin (1)/Aminoglycosides combinations, especially those of 1 combined with GEN, ETM and STR, with (FICI s)50 at 0.375-0.5. There were 6-8 isolates (n = 10) showed synergy and 2-4 showed additivity. The (MICs) 50 values ([micro]g/ml) of Brazilin (l)/Aminoglycosides combinations reduced from 32-128/16-64 (Table 2) to 4-8/4-16 (Table 3), respectively and the percent of reduction ranged from 50% to 87.5%, with a maximum of 93.8%, i.e. the combined MIC reduced to 1/16 in comparison with that of the alone used (Tables 2 and 3). Combinations of Brazilein (2) with the antibiotics AZM, GEN and CAZ showed less frequencies of synergy of 2-7 isolates (n = 10) and more of additivity or indifference. Only three isolates of Sappanone B (3)/CAZ combination were observed synergy, and most of the combinations with AMK, GEN and CFZ exhibited additivity or indifference (Table 3). The synergy of Brazilin (1) and other two 3-Benzylchroman derivatives with different antibiotics is evaluated for the first time so far to the best of our knowledge (Kim et al. 2004; Xu and Lee 2004).

The dynamic Time-killing experiment further demonstrated that the combinations of Brazilin (1)/STR (GEN, AMK and ETM), and 2/AZM were synergistically bactericidal against MRSA (Fig. 2 and Table 4), because the increased killing of Log10CFU/ml at 24 h ([DELTA][LC.sub.24]) were among 2.27-3.18, while combinations of 2/GEN (and CAZ), and 3/GEN (AMK, CAZ and CFZ) showed additively bactericidal against MRSA with [DELTA][LC.sub.24] of 1.24-1.95 (Table 4) (Chin et al. 2008). Meanwhile, Brazilin (1) singly showed equal to (or even a slightly higher than) the killing activity of STR (GEN, AMK and ETM) in the combination at MIC (32 p-g/ml) and 24 h against the isolate of MRSA 144 (Fig. 2), and Brazilin (1) also showed concentrations dependent (from 8 to 64[micro]g/ml or (1/4-4) x MIC) killing effects against other MRSA strains (data not shown) either used alone or in combination with the four antibiotics.

We also noticed the good consistency of anti-MRSA synergy results of the bacteriostatic (Chequerboard method) and bactericidal (time-kill method) efficiencies of Brazilin (1)/Aminoglycosides combinations, which is different from those of our previous reports where the combined killing effects reduced from synergy to additivity or even indifference (An et al. 2011; Zuo et al. 2011).

As the ultimate aim of synergy studies of phytochemicals or the plant secondary metabolites with conventional antibiotics or antibacterial (antifungal) agents is to reduce the MIC/MBC of these agents against the resistant pathogens to their originally susceptible degree, hence, it is necessary to find a criterion for judgment of the antibacterial (antifungal) resistance modifying action. Here we take the newly MIC Interpretive Standards for Staphylococcus spp. in Table 2C of CLSI (CLS1 2012). The related antibiotics are collected in the bottom of Table 2. Judged by this criterion and the results in Tables 2 and 3, we can find the combined [(MICs).sub.50] of Brazilin (1)/GEN and AMK ([micro]g/ml) reduced from 32 to 8 and 16, leading the anti-MRSA activities of GEN and AMK from resistant (R) to their intermediate (I) and susceptible (S) degree, respectively. So we can say that the resistance was reversed. Other combinations only showed more or less the positive resistance modifying effects.

Sappan Lignum is a Chinese crude drug with the function of eliminating blood stasis, swelling and pain in traditional Chinese medicine (TCM) (NUTCM 2005). Its extracts were reported to possess antiinflammatory properties and to treat thrombosis, infectious diseases or tumors. The methanol extract of Sappan Lignum (C. sappan) has been shown of anti-MRSA activity with the potential to restore the effectiveness of [beta]-lactam antibiotics and inhibition of the MRSA invasion to human mucosal fibroblasts (HMFs) (Kim et al. 2004). One of the anti-MRSA principles Brazilin (1) from Sappan Lignum was shown to have ability to inhibit DNA and protein synthesis (Xu and Lee 2004). The mechanisms of synergy of Compounds 1-3 with the antibiotics may be partly attributed to these effects. As the Aminoglycoside antibiotics resistance could be caused by the enzymatic inactivation or efflux pumps overexpression (Nikaido 2009), resistance reversal effects of the 3-Benzylchroman derivative/Aminoglycoside combinations might as well be ascribed to inhibition against the inactivation or overexpression by 3-Benzylchroman derivatives, which should be investigated in the future study.

The aminoglycoside antibiotics are important in the treatment of serious bacterial infections. But they could produce well-known toxicities include nephrotoxicity, ototoxicity, neuromuscular blockade and hypersensitivity reactions (Freeman et al. 1997). The anti-MRSA synergistic interactions of 3-Benzylchroman derivatives could increase the aminoglycoside potency against Gram positive pathogens like MRSA; reduce their toxicities through a smaller clinical dosage against the resistant pathogens. Whether or not the 3-Benzylchroman derivatives could increase the potency against Gram negative pathogens is also interesting for future exploration.

In conclusion, the 3-Benzylchroman derivative Brazilin showed in vitro synergy of bactericidal activities against MRSA when combined with Aminoglycosides, which might be beneficial for combinatory therapy of MRSA infection.

ARTICLE INFO

Article history:

Received 8 October 2013

Received in revised form

11 December 2013

Accepted 2 March 2014

Conflict of interest

There was no conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (NSFC 81073126, 81173504) and the supporting fund ofYunnan Province of China (2008PY001). We are also grateful to Kunming Institute of Botany (CAS) for spectral analysis.

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G.Y. Zuo (a) *, Z.Q. Han (a, b), X.Y. Hao (b), J. Han (c), Z.S. Lid, G.C. Wang (a)

(a) Research Center for Natural Medicines, Kunming General Hospital of Chengdu Military Command, Kunming 650032, China (b) School of Pharmacy, Guiyang Medical University, Guiyang 550004, China

(c) School of Basic Medical Sciences, Yunnan Traditional Chinese Medical College, Kunming 650500, China (d) Kunming Institute of Virology of Chengdu Military Command, Kunming 650032, China

* Corresponding author. Tel.: +86 871 4774941; fax: +86 871 5414186.

E-mail address: zuoguoying@263.net (G.Y. Zuo).

http://dx.doi.org/10.1016/i.phymed.2014.03.004

Table 1
MICs and MBCs of Compounds 1-3 against various pathogens ([micro]g/ml).

Strain                  MSSA (a)   EC (b)   PA (c)   CA (d)

Brazilin (1)      MIC    32          512      512    >1024
                  MBC    64          512      512    >1024

Brazilein (2)     MIC   128         1024    >1024    >1024
                  MBC   256         1024    >1024    >1024

Sappanone B (3)   MIC   128        >1024    >1024    >1024
                  MBC   512        >1024    >1024    >1024

Vancomycin        MIC     1           --       --       --
                  MBC     2           --       --       --

(a) MSSA, methicillin-susceptible Staphylococcus aureus (ATCC25923).

(b) EC, Escherichia coli (ATCC25922).

(c) PA, Pseudomonas aeruginosa (ATCC27853).

(d) CA, Candida albicans (ATCCY0109).

Table 2 MICs (/MBCs) of Compounds 1/3 and antibiotics each used
alone against 10 clinical MRSA strains of SCCmec III type
([micro]g/ml).

Strain             1 (a)          2                3

MRSA 008           32/64          512/512          256/512
MRSA 082           32/64          64/128           128/512
MRSA 092           16/64          64/128           128/256
MRSA 098           32/32          128/256          64/256
MRSA 111           32/64          128/256          128/256
MRSA 135           32/64          128/256          128/512
MRSA 144           32/32          64/128           128/256
MRSA 189           32/128         64/256           64/128
MRSA 321           16/64          64/256           128/128
MRSA 328           32/32          128/512          128/256
Range              16-32/32-128   64-512/128-512   64-256/128-512
50% (n = 10) (b)   32/64          64/256           128/256
90% (n = 10) (c)   32/64          128/512          128/512
MIC            S   --             --               --

Interp.        I   --             --               --
Criteria (d)   R   --             --               --

Strain             ETM    GEN                AMK                STR

MRSA 008           16     32                 64                 64
MRSA 082           16     16                 32                 128
MRSA 092           16     32                 16                 32
MRSA 098           16     32                 16                 64
MRSA 111           16     32                 32                 128
MRSA 135           16     64                 16                 64
MRSA 144           8      32                 64                 64
MRSA 189           16     32                 32                 64
MRSA 321           8      16                 32                 128
MRSA 328           16     32                 32                 64
Range              8-16   16-64              16-64              32-128
50% (n = 10) (b)   16     32                 32                 64
90% (n = 10) (c)   16     32                 64                 128
MIC            S   --     [less than or      [less than or      --
                          equal to] 4        equal to] 16
Interp.        I   --     8                  32                 --
Criteria (d)   R   --     [greater than or   [greater than or   --
                          equal to] 16       equal to] 64

Strain             PEN                CFZ

MRSA 008           128                512
MRSA 082           64                 256
MRSA 092           64                 256
MRSA 098           64                 256
MRSA 111           64                 128
MRSA 135           32                 256
MRSA 144           32                 256
MRSA 189           64                 512
MRSA 321           64                 256
MRSA 328           64                 256
Range              32-128             128-512
50% (n = 10) (b)   64                 256
90% (n = 10) (c)   64                 512
MIC            S   [less than or      [less than or
                   equal to] 0.12     equal to] 8
Interp.        I   --                 16
Criteria (d)   R   [greater than or   [greater than or
                   equal to] 0.25     equal to] 32

Strain             CAZ                AZM

MRSA 008           512                8192
MRSA 082           512                8192
MRSA 092           512                8192
MRSA 098           512                8192
MRSA 111           512                4096
MRSA 135           512                8192
MRSA 144           512                8192
MRSA 189           256                8192
MRSA 321           512                8192
MRSA 328           512                8192
Range              256-512            4096-8192
50% (n = 10) (b)   512                8192
90% (n = 10) (c)   512                8192
MIC            S   [less than or      [less than or
                   equal to] 8        equal to] 2
Interp.        I   16                 4
Criteria (d)   R   [greater than or   [greater than or
                   equal to] 32       equal to] 8

Strain             VAN

MRSA 008           1
MRSA 082           1
MRSA 092           1
MRSA 098           1
MRSA 111           1
MRSA 135           1
MRSA 144           1
MRSA 189           1
MRSA 321           1
MRSA 328           1
Range              1
50% (n = 10) (b)   1
90% (n = 10) (c)   1
MIC            S   [less than
                   or equal to] 4
Interp.        I   8-16
Criteria (d)   R   [greater than
                   or equal to] 32

(a) 1: Brazilin; 2: Brazilein; 3: Sappanone B; GEN: Gentamicin;
ETM: Etimicin; STR: Streptomycin; AMK: Amikacin; AZM: Azithromycin'
CAZ: Ceftazidime' PEN' Penicillin- CFZ: Cefazolin; VAN: Vancomycin.

(b) 50% (n = 10): values against 50% of the 10 MRSA strains.

(c) 90% (n = 10): values against 90% of the 10 MRSA strains.

(d) MIC Interpretive Criteria (2012 CLSI M100-S22); "S":
susceptible; "I": intermediate; "R": resistant.

Table 3
MICs ([micro]g/ml) and FIC indices (FICIs) of Compounds 1-3 and
antibiotics each used in various combinations against 10 clinical
MRSA strains of SCCmec III type.

Combination            Combined activity      Range (n = 10)

Brazilin (1)/GEN (a)   MIC                    2-16/4-16
                       [MIC.sub.Rd] (%) (b)   93.8-50/87.5-50
                       F1C1C                  0.25-1

Brazilin (1)/ETM       MIC                    4-16/2-8
                       [MIC.sub.Rd] (%)       87.5-50/75-50
                       FICI                   0.375-0.75

Brazilin (1)/STR       MIC                    4-16/8-64
                       [MIC.sub.Rd] (%)       87.5-50/87.5-50
                       F1CF                   0.25-1

Brazilin (1)/AMK       MIC                    2-32/2-16
                       [MIC.sub.Rd] (%)       87.5-0/87.5-0
                       FICI                   0.25-2

Brazilein (2)/AZM      MIC                    8-64/128-4096
                       [MIC.sub.Rd] (%)       96.9-50/98.4-50
                       FICI                   047-0.75

Brazilein (2)/GEN      MIC                    8-128/8-16
                       [MIC.sub.Rd] (%)       87.5-50/75-50
                       FICI                   0.375-1

Brazilein (2)/CAZ      MIC                    16-128/8-512
                       [MIC.sub.Rd] (%)       87.5-0/98.4-0
                       FICI                   0.188-2

Brazilein (2)/PEN      MIC                    8-256/16-64
                       [MIC.sub.Rd] (%)       87.5-0/75-0
                       FICI                   0.625-2

Sappanone B (3)/CAZ    MIC                    16-128/64-256
                       [MIC.sub.Rd] (%)       75-50/75-50
                       FICI                   0.5-1

Sappanone B (3)/AMK    MIC                    16-128/4-32
                       [MIC.sub.Rd] (%)       75-50/87.5-50
                       FICI                   0.625-1

Sappanone B (3)/GEN    MIC                    16-128/8-32
                       [MIC.sub.Rd] (%)       75-0/75-50
                       FICI                   0.75-1.5

Sappanone B (3)/CFZ    MIC                    32-128/64-256
                       [MIC.sub.Rd] (%)       75-0/50-0
                       FICI                   0.75-2

Combination            Combined activity      50%        90%
                                              (n = 10)   (n = 10)
                                              (d)        (e)

Brazilin (1)/GEN (a)   MIC                    4/8        8/8
                       [MIC.sub.Rd] (%) (b)   87.5/75    75/50
                       F1C1C                  0.375      0.75

Brazilin (1)/ETM       MIC                    8/4        8/4
                       [MIC.sub.Rd] (%)       75/75      50/75
                       FICI                   0.5        0.75

Brazilin (1)/STR       MIC                    8/16       8/32
                       [MIC.sub.Rd] (%)       75/75      50/50
                       F1CF                   0.5        0.75

Brazilin (1)/AMK       MIC                    8/16       16/16
                       [MIC.sub.Rd] (%)       75/75      50/50
                       FICI                   0.625      1

Brazilein (2)/AZM      MIC                    16/1024    64/2048
                       [MIC.sub.Rd] (%)       87.5/75    50/75
                       FICI                   0.375      0.625

Brazilein (2)/GEN      MIC                    32/8       64/16
                       [MIC.sub.Rd] (%)       75/75      50/50
                       FICI                   0.75       1

Brazilein (2)/CAZ      MIC                    64/256     128/512
                       [MIC.sub.Rd] (%)       50/50      0/0
                       FICI                   1          2

Brazilein (2)/PEN      MIC                    64/32      64/64
                       [MIC.sub.Rd] (%)       50/50      0/0
                       FICI                   1          2

Sappanone B (3)/CAZ    MIC                    32/256     64/256
                       [MIC.sub.Rd] (%)       75/50      50/50
                       FICI                   0.75       1

Sappanone B (3)/AMK    MIC                    32/8       64/32
                       [MIC.sub.Rd] (%)       50/50      50/50
                       FICI                   0.75       1

Sappanone B (3)/GEN    MIC                    64/16      128/16
                       [MIC.sub.Rd] (%)       50/50      0/50
                       FICI                   1          1.25

Sappanone B (3)/CFZ    MIC                    64/128     128/256
                       [MIC.sub.Rd] (%)       50/50      0/0
                       FICI                   1          2

Combination            Combined activity      Interaction (n = 10)

                                              Syn    Add    Ind

Brazilin (1)/GEN (a)   MIC                    8r     2      0
                       [MIC.sub.Rd] (%) (b)
                       F1C1C

Brazilin (1)/ETM       MIC                    7      3      0
                       [MIC.sub.Rd] (%)
                       FICI

Brazilin (1)/STR       MIC                    6      4      0
                       [MIC.sub.Rd] (%)
                       F1CF

Brazilin (1)/AMK       MIC                    4      5      1
                       [MIC.sub.Rd] (%)
                       FICI

Brazilein (2)/AZM      MIC                    7      3      0
                       [MIC.sub.Rd] (%)
                       FICI

Brazilein (2)/GEN      MIC                    3      7      0
                       [MIC.sub.Rd] (%)
                       FICI

Brazilein (2)/CAZ      MIC                    2      5      3
                       [MIC.sub.Rd] (%)
                       FICI

Brazilein (2)/PEN      MIC                    0      5      5
                       [MIC.sub.Rd] (%)
                       FICI

Sappanone B (3)/CAZ    MIC                    3      7      0
                       [MIC.sub.Rd] (%)
                       FICI

Sappanone B (3)/AMK    MIC                    0      10     0
                       [MIC.sub.Rd] (%)
                       FICI

Sappanone B (3)/GEN    MIC                    0      8      2
                       [MIC.sub.Rd] (%)
                       FICI

Sappanone B (3)/CFZ    MIC                    0      7      3
                       [MIC.sub.Rd] (%)
                       FICI

(a) GEN: Gentamicin: ETM: Etimicin; STR: Streptomycin; AMR:
Amikacin; AZM: Azithromycin; CAZ: Ceftazidime; PEN: Penicillin;
CFZ: Cefazolin; All data in the left side of "/" are designated to
Compounds 1-3 and right side to antibiotics.

(b) [MIC.sub.Rd] (%): % of MIC reduced; MICm
(%)=(MICaione--MICcombined) x 100/MICaione.

(c) FICI = ([(MIC of compound).sub.combined]/[(MIC of
compound).sub.alone] + ([(MIC of antibiotic).sub.combined]/[(MIC of
antibiotic).sub.alone]); FICI [less than or equal to] 0.5, synergy
(Syn); 0.5 < FICI [less than or equal to] 1, additivity (Add); 1 <
FICI [less than or equal to] 2, indifference (Ind).

(d) 50% (n = 10): values of inhibition against 50% of the 10 MRSA
strains.

(e) 90% (n = 10): value of inhibition against 90% of the 10 MRSA
strains.

(f) Number of MRSA strains showing the interactions.

Table 4
Time-killing assay results of various combinations
of Compounds 1-3 with antibiotics at 24 h.

Drugs (a)   Brazilin (1)

                           [DELTA]
                           [LC.sub.24]
            Mase (b)       (Int) (c)

STR         Brazilin (1)   3.03 (Syn)
GEN         GEN            2.85 (Syn)
AMK         Brazilin (1)   2.62 (Syn)
ETM         Brazilin (1)   2.27 (Syn)
AZM         --             --
CAZ         --             --
CFZ         --             --

Drugs (a)   Brazilein (2)

                                  [DELTA]
                                  [LC.sub.24]
            Masc                  (Int) (c)

STR         --                    --
GEN         Brazilein (2)         1.37 (Add)
AMK         --                    --
ETM         --                    --
AZM         Brazilein (2) (AZM)   2.7 (Syn) (3.18 (Syn) (d)
CAZ         CAZ                   1.19 (Add)
CFZ         --                    --

Drugs (a)   Sappanone B (3)

                              [DELTA]
                              [LC.sub.24]
            Masc              (Int) (c)

STR         --                --
GEN         GEN               1.29 (Add)
AMK         AMK               1.95 (Add)
ETM         --                --
AZM         --                --
CAZ         CAZ               1.24 (Add)
CFZ         CFZ               1.40 (Add)

(a) GEN: Gentamicin: ETM: Etimicin; STR: Streptomycin; AMK:
Amikacin; AZM: Azithromycin; CAZ: Ceftazidime; PEN: Penicillin;
CFZ: Cefazolin.

(b) Mase: most active single drug.

(c) [DELTA][LC.sub.24]: [DELTA][Log.sub.10] CFU/ml at 24h; Int.:
Interaction; Syn: synergy ([DELTA][LC.sub.24] [greater than or
equal to] 2); Add: additivity (1 < [DELTA][LC.sub.24] <2).

(d) Assayed at 1/2 MIC.
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Title Annotation:Short communication
Author:Zuoa, G.Y.; Hana, Z.Q.; Hao, X.Y.; Han, J.; Lid, Z.S.; Wang, G.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jun 15, 2014
Words:5789
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