Synergistic effects between silibinin and antibiotics on methicillin-resistant Staphylococcus aureus isolated from clinical specimens

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1 Biotechnol. J. 2011, 6, DOI /biot Research Article Synergistic effects between silibinin and antibiotics on methicillin-resistant Staphylococcus aureus isolated from clinical specimens Hyun-Kyung Kang 1, Hye-Young Kim 2 and Jeong-Dan Cha 2 1 Department of Dental Hygiene, College of Medical and Life Sciences, Silla University, Busan, South Korea 2 Department of Dental Hygiene, College of Natural Sciences, Dongeui University, Busan, South Korea Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous microorganism, and creates serious medical problems. It causes many types of infections in humans and often acquires multidrug resistance. In this study, silibinin was evaluated against 20 clinical isolates of MRSA, either alone or in combination with ampicillin or oxacillin, using a checkerboard assay. The silibinin exhibited good activity against isolates of MRSA, and MRSA ATCC33952 and MSSA ATCC25923, with minimum inhibitory concentrations/minimum bactericidal concentrations (MICs/MBCs) ranging between 2 8/4 16 μg/ml, for ampicillin / μg/ml, and for oxacillin / μg/ml. The range of MIC 50 and MIC 90 were μg/ml and 2 8 μg/ml, respectively. The MICs/MBCs for the combination of silibinin plus oxacillin or ampicillin were reduced by 4-fold against the MRSA isolates tested, demonstrating a synergistic effect, as defined by a fractional inhibitory concentration index (FICI) of 0.5. Furthermore, a time-kill study evaluating the growth of the tested bacteria showed that growth was completely attenuated after 2 5 h of treatment with the 1/2 MIC of silibinin, regardless of whether it was administered alone or with oxacillin (1/2 MIC) or ampicillin (1/2 MIC). In conclusion, silibinin exerted synergistic effects when administered with oxacillin or ampicillin and the antibacterial activity and resistant regulation of silibinin against clinical isolates of MRSA might be useful in controlling MRSA infections. Received 22 November 2010 Revised 17 January 2011 Accepted 31 January 2011 Keywords: Fractional inhibitory concentration MRSA Synergistic effects Minimum bactericidal concentrations Minimum inhibitory concentrations 1 Introduction Staphylococcus aureus is an important human pathogen, causing many serious infections such as wound infections, furuncles and carbuncles, and Correspondence: Professor Jeong-Dan Cha, Department of Dental Hygiene, College of Natural Sciences, Dongeui University, 995 Eomgwangno, Busan jin-gu, Busan, South Korea joungdan@deu.ac.kr Abbreviations: FIC(I), fractional inhibitory concentration (index); MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive S. aureus bullous impetigo, through locally invasive diseases such as cellulitis, osteomyelitis, sinusitis, and pneumonia, to major life-threatening septicemia and meningitis [1, 2]. Furthermore, S. aureus can spread easily, and has been found in the nasal passages of approximately 40 50% of healthy people. It is the etiological agent commonly associated with many diseases, and is normally related to subclinical or chronic infections. [3]. Methicillin-resistant S. aureus (MRSA) has become a major problem in many countries, resulting in significant morbidity, mortality, and health care costs. It has gained much attention in the past decade, as a major cause of hospital-acquired infections [4, 5].The β-lactam resistance of MRSA is caused by the production of a novel penicillin-binding protein (PBP) designated 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1397

2 Biotechnology Journal Biotechnol. J. 2011, 6, PBP 2 (or PBP 2a), which, unlike the intrinsic set of PBPs (PBP 1 4) of S. aureus, has remarkably reduced binding affinities to β-lactam antibiotics [6, 7]. PBP 2 is encoded by a meca gene located on the chromosome of MRSA. Resistance to methicillin is caused by this meca gene, which is located on a mobile genetic element, the Staphylococcal Cassette Chromosome mec (SCCmec) [7].The emergence of drug-resistant S. aureus, along with the undesirable side effects and poor systemic absorption of methicillin and vancomycin, has led to research on new anti-mrsa or vacomycin-resistant S. aureus (VRSA) agents from medicinal plants [8 10]. One of the most promising natural drugs is silymarin/silibinin. Silymarin is a standardized extract obtained from the seeds of milk thistle (Silybum marianum), which contains approximately 65 80% of the silymarin flavonolignans [11, 12]. Silymarin is a complex mixture of polyphenolic molecules, including seven closely related flavonolignans (silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin) and one flavonoid [12]. Silibinin is a major bioactive component of silymarin flavonolignans [12, 13]. It has been found to provide cytoprotection and, above all, hepato- and cirrhosis protection, and to protect the liver against poisoning from exposure to chemical and environmental toxins, including insect stings, mushroom poisoning, and alcohol [12, 14, 15]. The antiviral mechanism of silibinin action is still unclear because its chemical and pharmacological properties may change when it is administered orally [16]. In a previous study, silibinin showed antibacterial activity against gram-positive bacteria Bacillus subtilis, S. aureus, and Staphylococcus epidermidis and gramnegative bacteria [17]. Silibinin significantly inhibited macromolecule synthesis such as RNA and proteins in gram-positive bacteria [17, 18]. In the present study, the antimicrobial activities of silibinin against MRSA isolated in a clinic were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of a combination with antibiotics. 2 Materials and methods 2.1 Preparation of bacterial strains Twenty isolates of MRSA isolated from the Wonkwang University Hospital, as well as standard strains of methicillin-sensitive S. aureus (MSSA) ATCC and of MRSA ATCC were used. Antibiotic susceptibility was determined in testing the inhibition zones (inoculums 0.5 McFarland suspension, CFU/mL) and MIC/MBC (minimum inhibitory concentration/minimum bactericidal concentration) (inoculums CFU/mL) for strains, measured as described in the National Committee for Clinical Laboratory Standards (NCCLS, 1999). To rapidly identify methicillin resistance, the presence of the meca gene in MRSA isolates was detected using a PCR method [19]. 2.2 MIC/MBC assay The antimicrobial activity of silibinin against the 20 clinical MRSA isolates and reference strains (MSSA and MRSA) was determined using the broth dilution method [9]. The MIC was recorded as the lowest concentration of test samples resulting in the complete inhibition of visible growth. For clinical strains, MIC 50 s and MIC 90 s, defined as MICs at which, 50 and 90%, respectively of the isolates were inhibited, were determined. The MBC was determined based on the lowest concentration of the extracts required to kill 99.9% of bacteria from the initial inoculum as determined by plating on agar. 2.3 Checkerboard dilution test The synergistic combinations were investigated in the preliminary checkerboard method performed using the MRSA, MSSA, and one clinical isolate strains via MIC determination [9, 20].The fractional inhibitory concentration index (FICI) is the sum of the FICs of each of the drugs, which were defined as the MIC of each drug when used in combination divided by the MIC of each drug when used alone, i.e., FICI = (MIC of drug A in combination/mic of drug A alone) + (MIC of drug B in combination/mic of drug B alone). FICI were graded as: 0.5, synergy; > , additive; > , indifference; and >2.0, antagonism [20]. The synergic effect is shown graphically by applying published isobole methods [21]. 2.4 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 CFU/mL were exposed to the MIC of silibinin alone, or silibinin (1/2 MIC) plus oxacillin (1/2 MIC) or silibinin (1/2 MIC) plus ampicillin (1/2 MIC).Viable counts were measured 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 h at 37 C. All experiments were repeated several times and colony counts were conducted in duplicate, after which the means were determined Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

3 Biotechnol. J. 2011, 6, Results and discussion Many plants have been evaluated not only for direct antimicrobial activity, but also as a resistancemodifying agent [8, 19, 22]. Flavonoid compounds constitute an important class of phytochemicals, which possess diverse biological activities against MRSA [9, 23].The results of the antibacterial activity showed that the silibinin exhibited inhibitory activities against MRSA isolates and reference stains, MRSA and MSSA. In Table 1, the MICs/MBCs for silibinin against reference strains and MRSA isolates 1 20 ranged between 2 8/4 16 µg/ml, for ampicillin / µg/ml, and for oxacillin / µg/ml.the range of MIC 50 and MIC 90 were µg/ml and 2 8 µg/ml, respectively.the MIC 50 and MIC 90 determinations for all the clinical isolates confirmed the high bacteriostatic activity of silibinin against reference strains and MRSA 1-20 isolates.the range of MIC 50 and MIC 90 were µg/ml and 2 8 µg/ml, respectively. 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 [20, 24]. The synergistic effect of oxacillin and silibinin resulted in a reduction of the MICs/MBCs for all bacteria, with the MICs/MBCs of / µg/ml for oxacillin becoming / µg/ml and reduced by 4-fold in most of S. aureus tested, evidencing a synergistic effect as defined by a FICI of 0.5, except for isolates MRSA 5, 7, and 10 (Table 2 and Fig. 1). Considering the synergistic effect between silibinin and ampicillin, the MICs/MBCs were reduced by = 4-fold in most of S. aureus tested, with a FICI of 0.5, except for isolates MRSA 5, and 16 (Table 3 and Fig. 2). The effects of silibinin administered in combination with oxacillin or ampicillin against standard (MSSA and MRSA) and clinical isolates of MRSA (MRSA 1 20) were confirmed by time-kill curve experiments (Figs. 3 6). Cultures of each strain of bacteria with a cell density of CFU/ ml were exposed to the MIC of silibinin alone or silibinin (1/2 MIC) with oxacillin (1/2 MIC) or ampicillin (1/2 MIC). We observed that after 1 3 h of silibinin with ampicillin or oxacillin an increased rate of killing was seen as compared to that observed with silibinin (MIC) alone (Figs. 3 and 4). A profound bactericidal effect was exerted Table 1. Antibacterial activity of silibinin and antibiotics in isolated MRSA and some of reference bacteria Samples Silibinin (μg/ml) Ampicillin Oxacillin MIC 50< MIC 90< MIC/MBC MIC/MBC (μg/ml) MSSA ATCC a) 2 8 8/16 2/2 0.25/0.5 MRSA ATCC b) 1 4 4/8 1024/2048 8/16 MRSA 1 c) 1 4 4/4 1024/2048 8/16 MRSA /8 128/256 8/16 MRSA /4 1024/1024 8/8 MRSA /16 128/256 16/16 MRSA /16 128/256 16/32 MRSA /4 128/256 8/16 MRSA /4 128/512 16/32 MRSA /8 128/128 8/8 MRSA /16 128/512 16/32 MRSA /16 64/128 8/16 MRSA /8 128/128 16/16 MRSA /8 128/128 32/32 MRSA /16 64/128 32/64 MRSA /16 128/256 16/32 MRSA /16 64/128 8/16 MRSA /4 64/128 16/32 MRSA /4 128/128 8/16 MRSA /4 64/128 4/16 MRSA /16 64/128 4/8 MRSA /8 128/512 16/16 a) MSSA (ATCC 25923): reference strain methicillin-sensitive S. aureus. b) MRSA (ATCC 33591): reference strain methicillin-resistant S. aureus. c) MRSA 1 20: methicillin-resistant S. aureus isolated at clinic Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1399

4 Biotechnology Journal Biotechnol. J. 2011, 6, Figure 1. Isobologram curve revealing the synergistic effect of silibinin with oxacillin against methicillin-sensitive S. aureus (MSSA) ATCC and methicillin-resistant S. aureus (MRSA) ATCC strains and clinical isolates of MRSA. Mean FICs of drug oxacillin and silibinin were taken on y and x axis, respectively. Data points are from four experiments Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

5 Biotechnol. J. 2011, 6, Figure 2. Isobologram curve revealing the synergistic effect of silibinin with ampicillin against methicillin-sensitive S. aureus (MSSA) ATCC and methicillin-resistant S. aureus (MRSA) ATCC strains and clinical isolates of MRSA. Mean FICs of drug ampicillin and silibinin were taken on y and x axis, respectively. Data points are from four experiments Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1401

6 Biotechnology Journal Biotechnol. J. 2011, 6, Table 2. Synergistic effects of the silibinin with oxacillin in isolated MRSA and some of reference bacteria Samples Agent MIC/MBC (μg/ml) FIC FICI b) Outcome Alone Combination a) MSSA ATCC c) Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Oxacillin 0.25/ / /0.25 MRSA ATCC d) Silibinin 4/8 1/4 0.25/ /0.75 Synergistic/Additive Oxacillin 8/16 2/4 0.25/0.25 MRSA 1 e) Silibinin 4/4 1/2 0.25/ /0.75 Synergistic/Additive Oxacillin 8/16 2/4 0.25/0.25 MRSA 2 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Oxacillin 8/16 2/4 0.25/0.25 MRSA 3 Silibinin 4/4 1/2 0.25/ /1.0 Synergistic/Additive Oxacillin 8/8 2/4 0.25/0.5 MRSA 4 Silibinin 8/16 2/8 0.25/ /1.0 Synergistic/Additive Oxacillin 16/16 4/8 0.25/0.5 MRSA 5 Silibinin 4/16 2/8 0.5/ /0.75 Additive/Additive Oxacillin 16/32 4/8 0.25/0.25 MRSA 6 Silibinin 4/4 1/2 0.25/ /0.75 Synergistic/Additive Oxacillin 8/16 2/4 0.25/0.25 MRSA 7 Silibinin 2/4 0.5/2 0.25/ /0.75 Additive/Additive Oxacillin 16/32 8/8 0.5/0.25 MRSA 8 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Oxacillin 8/8 1/ /0.25 MRSA 9 Silibinin 8/16 2/4 0.25/ /0.375 Synergistic/Synergistic Oxacillin 16/32 4/4 0.25/0.125 MRSA 10 Silibinin 8/16 2/8 0.25/ /0.5 Additive/Synergistic Oxacillin 8/16 4/4 0.5/0.25 MRSA 11 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Oxacillin 16/16 4/4 0.25/0.25 MRSA 12 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Oxacillin 32/32 8/8 0.25/0.25 MRSA 13 Silibinin 8/16 2/8 0.25/ /0.75 Synergistic/Additive Oxacillin 32/64 8/ /0.25 MRSA 14 Silibinin 8/16 2/8 0.25/ /0.75 Synergistic/Additive Oxacillin 16/32 4/8 0.25/0.25 MRSA 15 Silibinin 4/16 1/4 0.25/ /0.375 Synergistic/Synergistic Oxacillin 8/16 2/2 0.25/0.125 MRSA 16 Silibinin 2/4 0.5/2 0.25/ /0.375 Synergistic/Synergistic Oxacillin 16/32 4/4 0.25/0.125 MRSA 17 Silibinin 4/4 1/2 0.25/ /0.5 Synergistic/Synergistic Oxacillin 8/16 2/4 0.25/0.25 MRSA 18 Silibinin 2/4 0.5/1 0.25/ /0.5 Synergistic/Synergistic Oxacillin 4/16 1/4 0.25/0.25 MRSA 19 Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Oxacillin 4/8 1/2 0.25/0.25 MRSA 20 Silibinin 8/8 2/4 0.25/ /0.75 Synergistic/Additive Oxacillin 16/16 4/4 0.25/0.25 a) The MIC and MBC of the silibinin with oxacillin. b) The FIC index. c) MSSA (ATCC 25923): reference strain methicillin-sensitive S. aureus. d) MRSA (ATCC 33591): reference strain methicillin-resistant S. aureus. e) MRSA 1 20: methicillin-resistant S. aureus isolated at clinic Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

7 Biotechnol. J. 2011, 6, Table 3. Synergistic effects of silibinin with ampicillin in isolated MRSA and some of reference bacteria Samples Agent MIC/MBC (μg/ml) FIC FICI b) Outcome Alone Combination a) MSSA ATCC c) Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 2/2 0.5/ /0.25 MRSA ATCC d) Silibinin 4/8 1/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 1024/ / /0.25 MRSA 1 e) Silibinin 4/4 1/2 0.25/ /0.75 Synergistic/Additive Ampicillin 1024/ / /0.25 MRSA 2 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Ampicillin 128/256 32/ /0.25 MRSA 3 Silibinin 4/4 1/2 0.25/ /1.0 Synergistic/Additive Ampicillin 1024/ / /0.5 MRSA 4 Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 128/256 32/ /0.25 MRSA 5 Silibinin 4/16 2/4 0.5/ /0.5 Additive/Synergistic Ampicillin 128/256 32/ /0.25 MRSA 6 Silibinin 4/4 1/2 0.25/ /0.375 Synergistic/Synergistic Ampicillin 128/256 32/ /0.125 MRSA 7 Silibinin 2/4 0.5/1 0.25/ /0.5 Synergistic/Synergistic Ampicillin 128/512 32/ /0.25 MRSA 8 Silibinin 4/8 1/4 0.25/ /1.0 Synergistic/Additive Ampicillin 128/128 32/ /0.5 MRSA 9 Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 128/512 32/ /0.25 MRSA 10 Silibinin 8/16 1/ / /0.375 Synergistic/Synergistic Ampicillin 64/128 16/ /0.125 MRSA 11 Silibinin 4/8 1/4 0.25/ /0.75 Synergistic/Additive Ampicillin 128/128 32/ /0.25 MRSA 12 Silibinin 4/8 1/2 0.25/ /0.5 Synergistic/Synergistic Ampicillin 128/128 32/ /0.25 MRSA 13 Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 64/128 16/ /0.25 MRSA 14 Silibinin 8/16 2/8 0.25/ /0.75 Synergistic/Additive Ampicillin 128/256 32/ /0.25 MRSA 15 Silibinin 4/16 1/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 64/128 16/ /0.25 MRSA 16 Silibinin 2/4 1/2 0.5/ /0.75 Additive/Additive Ampicillin 64/128 16/ /0.25 MRSA 17 Silibinin 4/4 1/2 0.25/ /0.75 Synergistic/Additive Ampicillin 128/128 16/ /0.25 MRSA 18 Silibinin 2/4 0.5/1 0.25/ /0.5 Synergistic/Synergistic Ampicillin 64/128 16/ /0.25 MRSA 19 Silibinin 8/16 2/4 0.25/ /0.5 Synergistic/Synergistic Ampicillin 64/128 16/ /0.25 MRSA 20 Silibinin 8/8 1/ / /0.75 Synergistic/Additive Ampicillin 128/512 32/ /0.25 a) The MIC and MBC of the silibinin with ampicillin. b) The FIC index. c) MSSA (ATCC 25923): reference strain methicillin-sensitive S. aureus. d) MRSA (ATCC 33591): reference strain methicillin-resistant S. aureus. e) MRSA 1 20: methicillin-resistant S. aureus isolated a clinic Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1403

8 Biotechnology Journal Biotechnol. J. 2011, 6, 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 silibinin, regardless of whether it was administered alone or with oxacillin (1/2 MIC) or ampicillin (1/2 MIC). Silibinin also shows susceptibility on gram-positive bacteria and gram-negative bacteria, except Figure 3. Time-kill curves of MIC of the silibinin alone and 1/2 MIC of silibinin with 1/2 MIC of oxacillin or ampicillin against MRSA isolates 1 4 and methicillin-sensitive S. aureus (MSSA) ATCC and methicillin-resistant S. aureus (MRSA) ATCC strains. Bacteria were incubated with the silibinin alone ( ) and with ampicillin ( ) or with oxacillin (f) over time. Data points are the mean values ± SEM of four experiments. CFU, colony-forming units Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

9 Biotechnol. J. 2011, 6, against gram-negative bacteria Escherichia coli and Proteus vulgaris [17, 18]. Tests of antifungal activities showed that neither silibinin nor silymarin II had antifungal activity against yeast [25]. The gram-positive bacteria-specific properties of silibinin are caused by the inhibition of RNA and protein synthesis, rather than by attacking the bacterial membrane [17]. Figure 4. Time-kill curves of MIC of the silibinin alone and 1/2 MIC of silibinin with 1/2 MIC of oxacillin or ampicillin against MRSA isolates Bacteria were incubated with the silibinin alone ( ) and with ampicillin ( ) or with oxacillin (f) over time. Data points are the mean values ± SEM of four experiments Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1405

10 Biotechnology Journal Biotechnol. J. 2011, 6, Conclusion In conclusion, silibinin exerted synergistic effects when administered with oxacillin or ampicillin, and the antimicrobial effect and resistant regulation of silibinin against clinical isolates of MRSA might be useful for potential application as a natural product agent. Figure 5. Time-kill curves of MIC of the silibinin alone and 1/2 MIC of silibinin with 1/2 MIC of oxacillin or ampicillin against MRSA isolates Bacteria were incubated with the silibinin alone ( ) and with ampicillin ( ) or with oxacillin (f) over time. Data points are the mean values ± SEM of four experiments Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

11 Biotechnol. J. 2011, 6, Figure 6. Time-kill curves of MIC of the silibinin alone and 1/2 MIC of silibinin with 1/2 MIC of oxacillin or ampicillin against MRSA isolates Bacteria were incubated with the silibinin alone ( ) and with ampicillin ( ) or with oxacillin (f) over time. Data points are the mean values ± SEM of four experiments. We thank Prof. S. M. Kim, Department of Clinical Laboratory Science, Wonkwang Health Science University for the confirmation and isolation of MRSA. The authors have declared no conflict of interest. 5 References [1] Corey, G. R., Staphylococcus aureus bloodstream infections: Definitions and treatment. Clin. Infect. Dis. 2009, 15, S [2] Petti, C.A., Fowler,V. G. Jr., Staphylococcus aureus bacteremia and endocarditis. Cardiol. Clin. 2003, 21, [3] Malm, A., Biernasiuk, A., Los, R., Kosikowska, U. et al., Slime production and cell surface hydrophobicity of nasopharygeal and skin staphylococci isolated from healthy people. Pol. J. Microbiol. 2005, 54, [4] Defres, S., Marwick, C., Nathwani, D., MRSA as a cause of long infection including airway infection, community-acquired pneumonia and hospital-acquired pneumonia. Eur. Respir. J. 2009, 34, [5] Maltezou, H. C., Giamarellou, H., Community-acquired methicillin-resistant Staphylococcus aureus infections. Int. J. Antimicrob. Agents 2006, 27, [6] Soltan-Dallal, M. M., Salehipour, Z., Mehrabadi, J. F., Molecular epidemiology of Staphylococcus aureus in food samples based on the protein A gene polymorphic region DNA sequence. Can. J. Microbiol. 2010, 56, [7] Chen, L., Mediavilla, J. R., Oliveira, D. C., Willey, B. M. et al., Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing. J. Clin. Microbiol. 2009, 47, [8] Aqil, F., Khan, M. S., Osais, M., Ahmad, I., Effect of certain bioactive plant extracts on clinical isolates of beta-lactamase producing methicillin-resistant Staphylococcus aureus. J. Basic Microbiol. 2005, 45, [9] Cha, J. D., Moon, S. E., Kim, J. Y., Jung, E. K. et al., Antibacterial activity of sophoraflavanone G isolated from the roots of Sophora flavescens against methicillin-resistant Staphylococcus aureus. Phytother. Res. 2009, 17, [10] Shiota, S., Shimizu, M., Sugiyama, J., Morita, Y. et al., Mechanisms of action of corilain and tellimagrandin 1 that remarkably potentiate the activity of beta-lactams agninst methicillin-resistant Staphylococcus aureus. Microbiol. Immunol. 2004, 48, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1407

12 Biotechnology Journal Biotechnol. J. 2011, 6, [11] Saller, R., Melzer, J., Reichling, J., Brignoli, R. et al., An updated systematic review of the pharmacology of silymarin. Forsch Komplementmed. 2007, 14, [12] Kren, V., Walterova, D., Silybin and silymarin-new effects and applications. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc. Czech Repub. 2005, 149, [13] Tyagi, A., Raina, K., Singh, R. P., Gu, M. et al., Chemopreventive effects of silymarin and silibinin on N-butyl-N-(4-hydroxybutyl) nitrosamine induced urinary bladder carcinogenesis in male ICR mice. Mol. Cancer Ther. 2007, 6, [14] Singh, R. P., Deep, G., Chittezhath, M., Kaur, M. et al., Effect of silibinin on the growth and progression of primary lung tumors in mice. J. Natl. Cancer Inst. 2006, 98, [15] Pradhan, S. C., Girish, C., Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical medicine. Indian J. Med. Res. 2006, 124, [16] Gazak, R., Purchartova, K., Marhol, P., Zivna, L. et al., Antioxidant and antiviral activities of silybin fatty acid conjugates. Eur. J. Med. Chem. 2010, 45, [17] Lee, D. G., Kim, H. K., Park, Y., Park, S. C. et al., Gram-positive bacteria specific properties of silybin derived from Silybum marianum. Arch. Pharm. Res. 2003, 26, [18] Jung, H. J., Lee, D. G., Synergistic antibacterial effect between silybin and N,N -dicyclohexylcarbodiimide in clinical Pseudomonas aeruginosa isolates. J. Microbiol. 2008, 46, [19] Kim, K. J.,Yu, H. H., Jeong, S. I., Cha, J. D. et al., Inhibitory effects of Caesalpinia sappan on growth and invasion of methicillin-resistant Staphylococcus aureus. J. Ethnopharmacol. 2004, 91, [20] Climo, M. W., Patron, R. L., Archer, G. L., Combinations of vancomycin and beta-lactams are synergistic against staphylococci with reduced susceptibilities to vancomycin. Antimicrob. Agents Chemother. 1999, 43, [21] Wagner, H., Ulrich-Merzenich, G., Synergy research: Approaching a new generation of phytopharmaceuticals. Phytomedicine 2009, 16, [22] Hatano,T., Shintani,Y.,Aga,Y., Shiota, S. et al., Phenolic constituents of licorice. VIII. Structures of glicophenone and glicoisoflavanone, and effects of licorice phenolics on methicillin-resistant Staphylococcus aureus. Chem. Pharm. Bull. 2000, 48, [23] Fukai, T., Marumo, A., Kaitou, K., Kanda, T. et al., Antimicrobial activity of licorice flavonoids against methicillin-resistant Staphylococcus aureus. Fitoterapia 2000, 73, [24] Cudic, M., Lockatell, C. V., Johnson, D. E., Otvos, L. Jr., In vitro and in vivo activity of an antibacterial peptide analog against uropathogens. Peptides 2003, 24, [25] Maser, P., Vogel, D., Schmid, C., Raz, B. et al., Identification and characterization of trypanocides by functional expression of an adenosine transporter from Trypanosoma brucei in yeast. J. Mol. Med. 2001, 79, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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