Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora

BMC Complementary and Alternative Medicine

Kang Nee Ting¹  Hwei-San Loh¹  Kuan-Hon Lim¹  Ee Leen Pang¹  Carolina Santiago¹ 

[1] Faculty of Science, University of Nottingham Malaysia Campus, Jalan Broga, Semenyih, 43500, Selangor, Malaysia

关键词: Antimicrobial;    Synergistic activity;    PBP2a;    MRSA;    Duabanga grandiflora;   

Others  :  1217077
DOI  :  10.1186/s12906-015-0699-z

 received in 2014-06-02, accepted in 2015-05-29,  发布年份 2015

【 摘 要 】

Background

The inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present.

Methods

Fractionation of the ethyl acetate leaf extract was achieved by successive column chromatography which eventually led to isolation of an active fraction, F-10. Both extract and F-10 were analyzed for the presence of major classes of phytochemicals in addition to obtaining a high performance liquid chromatography (HPLC) profile to reveal the complexity of the fraction F-10. Broth microdilution method was employed to determine minimum inhibitory concentration (MIC) of the extract and fractions against MRSA. Evaluation of synergistic activity of the active fraction with ampicillin was determined using checkerboard methodand kinetic growth experiments. Effect of combination treatments on expression of PBP2a, a protein that confers resistance to beta-lactam antibiotics, was elucidated with the Western blot assay.

Results

MIC of F-10 against MRSA was 750 mg/L which showed an improved activity by 4-fold compared to its crude extract (MIC = 3000 mg/L). Phytochemical analysis revealed occurrence of tannins, saponin, flavonoids, sterols, and glycosides in F10 fraction. In FIC index interpretation, the most synergistic activity was achieved for combinations of 1/64 × MIC ampicillin + 1/4 × MIC F-10. The combination also evidently inhibited MRSA growth in kinetic growth curve assay. As a result of this synergistic interaction, MIC of ampicillin against MRSA was reduced to 0.78 mg/L (64-fold) from initial value of 50 mg/L. Western blot analysis suggested inhibition of PBP2a in MRSA cultures grown in synergistic combination treatment in which no PBP2a band was expressed.

Conclusions

The results demonstrated synergism between fraction F-10 of D. grandiflora with ampicillin in suppressing MRSA growth via PBP2a inhibition.

【 授权许可】

   
2015 Santiago et al.

【 图 表 】

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【 参考文献 】

• [1]David MZ, Daum RS. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev. 2010; 23:616-87.
• [2]Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H et al.. Meticillin-resistant Staphylococcus aureus (MRSA): global epidemiology and harmonisation of typing methods. Int J Antimicrob Agents. 2012; 39:273-82.
• [3]Hartman BJ, Tomasz A. Expression of methicillin resistance in heterogeneous strains of Staphylococcus aureus. Antimicrob Agents Chemother. 1986; 29:85-92.
• [4]Berger-Bächi B. Expression of resistance to methicillin. Trends Microbiol. 1994; 2:389-93.
• [5]Berger-Bächi B, Rohrer S. Factors influencing methicillin resistance in staphylococci. Arch Microbiol. 2002; 178:165-71.
• [6]Hemaiswarya S, Kruthiventi AK, Doble M. Synergism between natural products and antibiotics against infectious diseases. Phytomedicine. 2008; 15:639-52.
• [7]Wagner H, Ulrich-Merzenich G. Synergy research: approaching a new generation of phytopharmaceuticals. Phytomedicine. 2009; 16:97-110.
• [8]Schulz V. Incidence and clinical relevance of the interactions and side effects of Hypericum preparations. Phytomedicine. 2001; 8:152-60.
• [9]Stauss-Grabo M, Atiye S, Warnke A, Wedemeyer RS, Donath F, Blume HH. Observational study on the tolerability and safety of film-coated tablets containing ivy extract (Prospan® Cough Tablets) in the treatment of colds accompanied by coughing. Phytomedicine. 2011; 18:433-6.
• [10]Graham SA, Hall J, Sytsma K, Shi S. Phylogenetic Analysis of the Lythraceae Based on Four Gene Regions and Morphology. Int J Plant Sci. 2005; 166:995-1017.
• [11]Auamcharoen W, Chandrapatya A, Kijjoa A, Silva AMS, Herz W. Chemical constituents of Duabanga grandiflora (Lythraceae). Biochem Syst Ecol. 2009; 37:535-7.
• [12]Shankar R, Devalla RB. Conservation of folk healing practices and commercial medicinal plants with special reference to Nagaland. Int J Biodivers Conserv. 2012; 4:155-63.
• [13]Rudikoff D, Lebwohl M. Atopic dermatitis. Lancet. 1998; 351:1715-21.
• [14]Nakamura Y, Oscherwitz J, Cease KB, Chan SM, Muñoz-Planillo R, Hasegawa M et al.. Staphylococcus δ-toxin induces allergic skin disease by activating mast cells. Nature. 2013; 503:397-401.
• [15]Othman M, Genapathy S, Liew PS, Ch’ng QT, Loh HS, Khoo TJ et al.. Search for antibacterial agents from Malaysian rainforest and tropical plants. Nat Prod Res. 2011; 25:1857-64.
• [16]Othman M, Loh HS, Wiart C, Khoo TJ, Lim KH, Ting KN. Optimal methods for evaluating antimicrobial activities from plant extracts. J Microbiol Methods. 2011; 84:161-6.
• [17]Spigno G, De Faveri DM. Antioxidants from grape stalks and marc: Influence of extraction procedure on yield, purity and antioxidant power of the extracts. J Food Eng. 2007; 78:793-801.
• [18]Clinical and Laboratory Standards Institute (CLSI): Laboratory Methods for Detection of Methicillin-Resistant Staphylococcus Aureus (MRSA). Supplement. Volume M100-S170 2007, 36–41.
• [19]Cos P, Vlietinck AJ, Vanden D, Maes L. Anti-infective potential of natural products?: How to develop a stronger in vitro “proof-of-concept”. J Ethnopharmacol. 2006; 106:290-302.
• [20]White R, Burgess D, Manduru M, Bosso J. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother. 1996; 40:1914-8.
• [21]Williamson EM. Synergy and other interactions in phytomedicines. Phytomedicine. 2001; 8:401-9.
• [22]Jones WP, Kinghorn AD. Extraction of Plant Metabolites. In: Natural Product Isolation. Sarker SD, Latif AI, Gray AI, editors. Humana Press, New Jersey; 2005: p.323-51.
• [23]Leung DYM. Why is eczema herpeticum unexpectedly rare? Antiviral Res. 2013; 98:153-7.
• [24]Shimizu M, Shiota S, Mizushima T, Ito H, Hatano T, Yoshida T et al.. Marked potentiation of activity of beta-lactams against methicillin-resistant Staphylococcus aureus by corilagin. Antimicrob Agents Chemother. 2001; 45:3198-201.
• [25]Song MD, Wachi M, Doi M, Ishino F, Matsuhashi M. Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion. FEBS Lett. 1987; 221:167-71.
• [26]Archer GL, Bosilevac JM. Signaling antibiotic resistance in staphylococci. Science. 2001; 291:1915-6.
• [27]Zhang HZ, Hackbarth CJ, Chansky KM, Chambers HF. A proteolytic transmembrane signaling pathway and resistance to beta-lactams in staphylococci. Science. 2001; 291:1962-5.
• [28]Arêde P, Milheiriço C, de Lencastre H, Oliveira DC. The anti-repressor MecR2 promotes the proteolysis of the mecA repressor and enables optimal expression of β-lactam resistance in MRSA. PLoS Pathog. 2012; 8: Article ID e1002816
• [29]Geissman TA. Flavonoid compounds, tannins, lignins and related compounds. In: Pyrrole Pigments, Isoprenoid Compounds and Phenolic Plant Constituents. Florkin M, Stotz EH, editors. Elsevier, New York; 1963: p.265.
• [30]Mason TL, Bruce PW. Inactivation of red beet β-glucan synthase by native and oxidized phenolic compounds. Phytochemistry. 1987; 26:2197-202.
• [31]Fuda C, Suvorov M, Vakulenko SB, Mobashery S. The basis for resistance to beta-lactam antibiotics by penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus. J Biol Chem. 2004; 279:40802-6.
• [32]Singh G, Kumar P, Jindal A. Antibacterial potential of sterols of some medicinal plant. Int J Pharm Pharm. 2012; 4:15-8.
• [33]Nazemiyeh H, Rahman MM, Gibbons S, Nahar L, Delazar A, Ghahramani M-A et al.. Assessment of the antibacterial activity of phenylethanoid glycosides from Phlomis lanceolata against multiple-drug-resistant strains of Staphylococcus aureus. J Nat Med. 2008; 62:91-5.