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Synergistic effects of diosmetin with erythromycin against ABC transporter over-expressed methicillin-resistant Staphylococcus aureus (MRSA) RN4220/pUL5054 and inhibition of MRSA pyruvate kinase.

Introduction

The evolution of antimicrobial resistance among nosocomial and community-acquired pathogens such as the well-known multidrug-resistant (MDR) organism Staphylococcus aureus is of great concern, and it has compromised the treatment of serious bacterial infections (Castanheira et al., 2012). Flavonoids from natural products have been shown to possess antibacterial actions against methicillin-resistant S. aureus (MRSA) by antagonizing its resistance mechanisms (Shibata et al., 2003; Wagner and Ulrich-Merzenich, 2009). For example, our previous study (Chan et at, 2011) demonstrated that baicalein could significantly reverse the ciprofloxacin resistance of MRSA possibly by inhibiting the NorA efflux pump in vitro and inhibition of a newly discovered MRSA specific pyruvate kinase (PK) (Zoraghi et al., 2010). Diosmin (3',5,7-trihydroxy-4'-methoxyflavone-7-rutinoside) (Fig. 1a), a natural flavonoid found in a variety of citrus fruits is considered to be a vascular protecting agent used to treat chronic venous insufficiency, hemorrhoids, lymphedema and varicose veins (Hitzenberger, 1997). Diosmetin (3',5,7-trihydroxy-4'-methoxyflavone) (Fig. 1a), the aglycone of diosmin, has also been shown to possess antibacterial activities against Helicobacter pylon (Bae et al., 1999) and Bacillus subtilis (Meng et al., 2000) with MICs > 80 and 50 [mu]g/ml, respectively. However, the antibacterial activities and the mechanisms of action of diosmin and diosmetin against MRSA have not been studied. Since the chemical structures of diosmin and diosmetin are similar to baicalein, it is interesting to see whether diosmin and diosmetin also exhibit synergistic effects with antibiotics against MRSA, and their potential inhibitory actions on MRSA PK.

[FIGURE 1 OMITTED]

Materials and methods

Antimicrobial agents and culture medium

Diosmetin and diosmin were obtained from INDOFINE Chemical Company (Hillsborough, USA). Mueller-Hinton (MH) broth was obtained from Becton, Dickinson and company (USA). MH agar plates were used for plating of samples for determination of bacterial density. All other chemicals were purchased from Sigma Chemical Co. (St. Louis, USA).

Bacterial strains

Five laboratory S. aureus strains were used for the susceptibility tests. S. aureus SA-1199B (harboring resistance to fluoroquinolones through overexpression of the NorA efflux pump is ciprofloxacin resistant (Kerns et al., 2003). SA-RN4220-pUL5054 is resistant to macrolides including erythromycin and contains the multicopies plasmid pUL5054 coding for MsrA, an efflux pump (Ross et al., 1990). Three aminoglycosides resistant strains were also included in this study: (a) SA-APH2"-AAC6' is resistant to gentamicin, (b) SA-APH3' is resistant to kanamycin and (c) SA-ANT4' is resistant to fusidic acid. SA-ATCC25293 is a methicillin sensitive strain which was used as quality control strain. Cultures were performed at 37 C in Mueller-Hinton (MH) liquid broth or on MH agar plates.

Determination of minimum inhibitory concentration (MIC), checkerboard assay and time - kill curves

For antimicrobial susceptibility tests, cells ([10.sub.6] CFU/ml) were inoculated into MH broth and dispensed at 200 [mu]1/well in 96-well microtiter plates. MlCs were determined by serial 2-fold dilution of the test compound in MH broth. The bacterial broth was incubated for 18 h at 37[degrees]C and inhibition of bacterial growth was examined visually. Bacterial growth in each well was assayed by absorption at 620 nm using a spectrophotometer (Multimode Detector DTX 880, Beckman Coulter, USA). The percentage of growth in each well was calculated as the OD of each well divided by the OD of the drug-free well after subtracting the background OD obtained from microorganism-free microtiter plates. For the tested compounds, MIC was defined as the lowest concentration of antibacterial, which resulted in [greater than or equal to]90% inhibition of growth compared with that of the drug-free control. MIC determination against S. aureus (ATCC 25923), a sensitive strain, was performed in parallel to each test against resistant strain as a positive control.

The checkerboard assay was conducted to measure the synergy between diosmetin/diosmin and antibiotics against tested strains (Chan et al., 2011). The concentrations of antibiotics and diosmin/diosmetin used were 0, 2, 4, 8, 16, 32, 64 and 128 [mu]g/ml. MIC values obtained for a given combination were used to evaluate the effects of combination between diosmin/diosmetin and antibiotics by calculating the Fractional Inhibitory Concentration Index (FICI) using fractional inhibitory concentration (FIC) according to the following formulae: AC of diosmin/diosmetin = MIC diosmin/diosmetin in combination divided by MIC of diosmin/diosmetin alone; hence FICI = FIC of diosmin/diosmetin + FIC of antibiotics. "Synergy" was defined when FIC index was less than or equal to 0.5; while "additive" in which the FIC index was greater than 0.5 and less than or equal to 1.0; whereas "indifferent" when the FIC index was greater than 1.0 and less than or equal to 2.0; and "antagonistic" in cases which the FIC index was greater than 2.0 (Climo et al., 1999). For the checkerboard assay using SA-RN4220-pUL5054, verapamil which is known to inhibit efflux pumps of bacteria (Kristiansen et al., 2003) was included as a positive control.

Time-kill curve methods as previously described (Chan et al., 2011) were used to evaluate the synergistic activities of diosmetin alone or in combination with erythromycin against SA-RN4220/pUL5054.

MRSA PK activity assay

MRSA-252 and E. coli constructs in pET-28a(+) vector were used to generate relevant recombinant His-tagged PK proteins in E coli BL-21(DE3) as previously described (Zoraghi et al., 2010). The effects of diosmin/diosmetin on MRSA PK were determined using a continuous assay coupled to lactate dehydrogenase (LDH) in which the change in absorbance at 340 nm owing to oxidation of NADH was measured using a Benchmark Plus microplate spectrophotometer (Bio-Rad, Canada). PK activity proportional to the rate of change at 340 nm was expressed as specific activity ([mu]mol/min/mg), which is defined as the amount of PK that catalyzes the formation of 1 [mu]mol of either product/min. Results for diosmin/diosmetin were expressed as percentage inhibition of MRSA PK enzymatic activity relative to a DMS0 negative control.

Statistical analysis

Data were expressed as mean [+ or -] standard deviation. Statistical analyses and significance, as measured by the Student's t-test for paired samples were performed using Prism software version 5.0 (GraphPad Software, CA, USA). In all comparisons, p < 0.05 was considered statistically significant.

Results and discussion

The growth inhibitory effect of diosmetin alone was moderate even at 128 [mu]g/ml, but significant synergistic activities against SA-RN4220/pUL5054 were observed when combinations of diosmetin (8-64[mu]g/ml, 1/128-1/4 x MIC) with erythromycin (32-64 [mu]g/ml, 1/8-1/4 x MIC) were used. The FICI value of diosmetin and erythromycin was 0.28 (0.16-0.31) which was smaller than 0.5, suggesting that they acted synergistically to inhibit SA-RN4220/pUL5054 growth (Table 1(a)). Moreover, the synergism of diosmetin with erythromycin against SA-RN4220/pUL5054 was stronger than the positive control verapamil that the MIC of diosmetin is five-fold less than verapamil. An additive effect was observed when diosmetin (64 [mu]g/ml) was combined with ciprofloxacin (8[mu]g/ml) against the NorA efflux pump overexpressed SA-1199B. Diosmetin and diosmin alone were ineffective in inhibiting the growth of all tested strains as the MICs were all greater than 128 [mu]g/ml (Table 1(a) and (b)). To further confirm the synergistic activity of disometin with erythromycin, an in vitro time - kill curve for SA-RN4220-pUL5054 was performed (Fig. 1b). After 24 h incubation, disometin (8 [mu]g/ml) or erythromycin (32 [mu]g/ml) alone did not inhibit the growth of SA-11998. In contrast, when disometin was combined with erythromycin, the antibacterial activity of erythromycin was significantly enhanced from 12 h to 24 h, with over 4 log reduction in CFU from 6.236 [+ or -] 0.47 to 2.41 [+ or -] 0.97 loglo CFU/ml after 24 h incubation.
Table 1 Minimum inhibitory concentrations (MIC) and fractional
inhibitory concentration indices (FICI) of (a) diosmin and (b)
diosmetin with antibiotics against different MRSA strains (n = 3).
FICI [less than or equal to]0.5 is defined as synergistic; >
0.5-[less than or equal to]1.0 as additive; >1.0-[less than or
equal to]2.0as indifferent; and >2.0 as antagonistic.

S. aureus Agent MIC FIC
strains ([mu]g/
 ml)

 Alone Combination Median (range)

(a)

1199B Diosmin >128 32 0.13(0.13-0.50)

 Ciprofloxacin 8 8 1.00(1.00-1.00)

APH2"AAC6 Diosmin >128 >128 1.00(1.00-1.00)

 Gentamicin >128 >128 1.00(1.00-1.00)

APH3' Diosmin >128 >128 1.00(1.00-1.00)

 Kanamycin >256 >256 1.00(1.00-1.00)

ANT4' Diosmin >128 8 0.01(0.01-0.03)

 Fusidicacid 128 64 0.50(0.50-0.50)

ATCC25293 Diosmin >128 32 0.13(0.02-0.13)

 Oxacillin 0.25 0.125 1.00(1.00-1.00)

RN4220/ Diosmin >128 128 0.50(0.50-0.50)
pUL5054

 Erythromycin 256 128 0.50(0.50-1.00)

S. aureus FICI
strains

 Median
 (range)

(a)

1199B 1.13
 (1.13-1.50)

APH2"AAC6 2.00
 (2.00-2.00)

APH3' 2.00
 (2.00-2.00)

ANT4' 0.53
 (0.51-0.53)

ATCC25293 2.00
 (1.02-1.13)

RN4220/ 1.00
pUL5054 (1.00-1.50)

S. aureus Agent MIC([mu]g/ FIC FICI
strains ml)

 Alone Combination Median (range)

(b)

1199B Diosmetin >128 64 0.13(0.13-0.50)

 Ciprofloxacin 8 4 0.25(0.25-0.50)

APH2"AAC6 Diosmetin >128 16 0.01(0.01-0.13)

 Gentamicin >128 256 0.50(0.50-0.50)

APH3' Diosmetin >128 >128 1.00(1.00-1.00)

 Kanamycin >256 >256 1.00(1.00-1.00)

ANT4' Diosmetin >128 32 0.13(0.13-0.25)

 Fusidicacid 128 32 0.25(0.25-0.50)

ATCC25293 Diosmetin >128 >128 0.02(0.01-0.25)

 Oxacillin 0.25 0.125 1.00(1.00-1.00)

RN4220/ Diosmetin >128 8 0.03(0.03-0.06)
pUL5054

 Erythromycin 256 32 0.25(0.13-0.25)

 Verapamil 1024 256 0.25(0.25-0.25)

 Erythromycin 256 32 0.13(0.13-0.13)

S. aureus
strains

 Median
 (range)

(b)

1199B 0.63
 (0.50-1.00)

APH2"AAC6 0.56
 (0.51-0.63)

APH3' 2.00
 (2.00-2.00)

ANT4' 0.50
 (0.38-0.63)

ATCC25293 1.01
 (1.00-1.25)

RN4220/ 0.28
pUL5054 (0.16-0.31)

 0.38
 (0.38-0.38)


For three of the aminoglycoside-resistant strains, significant growth inhibitory effects were observed in one of the strains SA-ANT4. Diosmin (8 pg/ml) and diosmetin (32 [mu]g/ml) could reduced the MIC of fusidic acid from 128 [mu]g/ml to 64 and 32 p,g/ml, respectively (Table 1a and b). Additive growth inhibitory effect were also observed when diosmetin (16 [mu]g/ml) with high concentration of gentamicin (256 [mu]g/ml) against SA-APH2"AAC6 (Table 1 b) and both tested flavonoids were not active against SA-APH3' with kanamycin (Table 1 a and b).

The effects of diosmetin and diosmin on MRSA PK activity were shown in Fig. 1 c. Diosmetin could inhibit MRSA PK enzymatic activity in a dose-dependent manner (Fig.1c(i)) with inhibitory activity of 48% observed at 10 WM diosmetin. To determine whether diosmetin could selectively inhibit MRSA PK without an effect on the human ortholog, 2 human isoforms, M1 and M2, were tested. Compared with MRSA PK, the inhibitory action of diosmetin on M1 and M2 were significantly weaker at 5 to 10[mu]M (Fig.1c(ii)). In contrast, diosmin did not show inhibitory activity against MRSA PK enzymatic activity (less than 5% inhibitory activity even at 10[mu]M diosmin) (Fig. 1c(i)).

Examples of efflux-related resistance mechanisms that have been described for S. aureus include those conferred by QacA and NorA, which are multidrug-resistant transporters, and the more specific MsrA and TetK transport proteins (Gibbons et al., 2003). By using 5 laboratory MRSA strains with known resistant mechanisms, we found that diosmetin could synergistically inhibit the growth of SA-RN4220-pUL5054 with erythromycin, which contains multicopies of plasmid pUL5054 coding for the efflux pump MsrA and resistant to 14-and 15-membered macrolides. MsrA is a 488-amino-acid protein with two ATP-binding motifs and function independently when cloned in SA-RN4220 (Ross et al., 1990). Besides, diosmetin in combination with ciprofloxacin, inhibited the growth of the NorA overexpressed strain SA-1199B in an additive manner. Unlike MsrA, NorA is an ATP-independent efflux pump, suggesting that the inhibitory activities of diosmetin against MRSA were more related to the ATP dependent process of the bacteria. By studying the architecture of the MRSA interactome, MRSA strain 252 pyruvate kinase (PK) was identified as a potential novel drug target based upon it being a highly-connected, essential hub protein in the MRSA interactome (Zoraghi et al., 2010). In the present study, we showed that diosmetin could selectively inhibit the enzymatic activity of MRSA PK over the human PK Ml and M2 isoforms. It is likely that interfering with ATP production by diosmetin may stop the function of ATP-dependent MsrA pump and contribute to the mode of action of the synergism with erythromycin against SA-RN4220-pUL5054. However, diosmetin does not inhibit the H. pylori urease activity (Bae et al., 1999) and its effect on PK appear to be specific towards this enzyme. Interestingly, diosmetin was shown to induce osteoblastic maturation and differentiation by increasing the activities of different protein kinases in vitro (Hsu and Kuo, 2008). Taken together, the inhibitory activity of diosmetin against the MRSA PK is specific and the interference of the ATP generation process by diosmetin might inhibit the MsrA pump in SA-RN4220-pUL5054 and ultimately enhanced the bactericidal activities of erythromycin.

Similar to the structure of diosmetin, diosmin has been shown to partially inhibit the ATP synthase purified from E coli (Chinnam et al., 2010) but we found that diosmin was not active against MRSA PK and did not demonstrate any direct or synergistic inhibitory activities against tested MRSA strains. In another antibacterial study (Bae et al., 1999), only diosmetin but not diosmin was active against H. pylon. As the only difference between diosmin and the diosmetin is the rutinose moiety present on diosmin (Fig. 1 a), it is therefore suggested that the presence of rutinose in the chemical structure of diosmin may hamper its antibacterial activities in vitro. However, it is interesting to note that diosmin is hydrolyzed by enzymes of the intestinal microflora before absorption of its aglycone diosmetin in vivo (Campanero et al., 2010). Apart from antibacterial activities, differential effects of these two flavones were also reported that only diosmetin but not diosmin could inhibit cytochrome P450 1A1 activity in MCF-7 human breast epithelial cancer cells (Ciolino et al., 1998).

In conclusion, our results demonstrated that diosmetin together with erythromycin, could synergistically inhibit the growth of ABC-pump overexpressed MRSA-RN4220/pUL5054 in vitro. The inhibition of MRSA PK by diosmetin could lead to a deficiency of ATP and affect the bacterial efflux pump which might contribute to the antibacterial actions of diosmetin against MRSA.

Acknowledgements

We acknowledge the support from the CUHK Scheme D grant for 2010/2.

0944-71135--see front matter[c]2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.02.007

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Ben C.L. Chan (a), (d), Margaret Ip (b), H. Gong (e), S.L. Lui (a), (d), Raymond H. See (e), Claude Jolivalt (f), (g), K.P. Fung (a), (c), (d), P.C. Leung (a), (d), Neil E. Reiner (e), Clara B.S. Lau* (a), (d).

(a.) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(b.) Department of Microbiology, The Chinese University of Hong Kong Shatin, New Territories, Hong Kong

(c.) School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(d.) State Key Laboratory of Phytochemistry Er Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(e.) Division of Infectious Diseases, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

(f.) ENSCP ChimieParisTech, Laboratoire Charles Friedel, 75005 Paris, France

(g.) CNRS, UMR 7223, 75005 Paris, France

* Corresponding author at: The Institute of Chinese Medicine, E305, Science Centre East Block, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. Tel.: +852 3943 6109; fax: +852 2603 5248.

E-mail address: claralau@cuhk.edu.hk (C.B.S. Lau).
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Title Annotation:Short communication
Author:Chan, Ben C.L.; Ip, Margaret; Gonge, H.; Lui, S.L.; See, Raymond H.; Jolivalt, Claude; Fung, K.P.; L
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:May 15, 2013
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