PUNICA GRANATUM RIND EXTRACT: ANTIBIOTIC POTENTIATOR AND EFFLUX PUMP INHIBITOR OF MULTIDRUG RESISTANT KLEBSIELLA PNEUMONIAE CLINICAL ISOLATES
Objective: With a rise in multidrug resistant (MDR) bacterial isolates, search for antibiotics or compounds that could act synergistically with them
is a significant area of research. Efflux-mediated resistance, in particular, is a great hurdle that needs to be overcome. In an effort to identify such
synergistic compounds and potential efflux pump inhibitors (EPI), we analyzed the rind of Punica granatum (pomegranate) against MDR clinical
Klebsiella pneumoniae isolates.
Methods: Sequential fractionation of P. granatum rind ethanol (PGR) extract was carried out to obtain hexane, butanol and water fractions.
Antibacterial activity of the plant extracts was confirmed, and synergistic interaction with antibiotics was determined by the checkerboard assay. Gas
chromatography-mass spectrometry (GC-MS) analysis was performed to identify the phytochemical constituents of the hexane extract. To study EPI
activity of the extracts, norfloxacin accumulation assay was carried out.
Results: PGR ethanol extract was found to have synergistic activity with ciprofloxacin, levofloxacin, ceftazidime, cefoxitin, meropenem, and gentamicin
resulting in fold decrease of minimum inhibitory concentration (MIC) ranging from 2 to 32 fold. The hexane fraction was found to have maximum
synergistic activity resulting in a 32-fold reduction of ciprofloxacin MIC followed by butanol and water fractions. The PGR ethanol extract was also
found to have efflux inhibition activity by the norfloxacin accumulation assay. Of the sequential fractions, the butanol fraction had maximum efflux
Conclusion: Therefore, our study shows that PGR extract can potentiate the effect of antibiotics on MDR bacteria, and the mode of action is likely to
be due to EPI.
Keywords: Punica granatum rind, Pomegranate, Synergy with antibiotics, Multidrug resistant, Klebsiella pneumoniae, Efflux pump inhibition.
1. Nikaido H. Multidrug resistance in bacteria. Annu Rev Biochem 2009;78(3):119-46.
Fig. 2: Efflux inhibition activity of Punica granatum rind ethanol extract and sequential fractions
2. Podschun R, Ullmann U. Klebsiella spp. As nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11(4):589-603.
3. Padilla E, Llobet E, DomÃ©nech-SÃ¡nchez A, MartÃnez-MartÃnez L, Bengoechea JA, AlbertÃ S. Klebsiella pneumoniae AcrAB efflux pump contributes to antimicrobial resistance and virulence. Antimicrob Agents Chemother 2010;54(1):177-83.
4. Webber MA, Piddock LJ. The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother 2003;51(1):9-11.
5. Mehta J, Jandaik S. Evaluation of phytochemicals and synergistic interaction between plant extracts and antibiotics for efflux pump inhibitory activity against Salmonella enterica serovar Typhimurium strains. Int J Pharm Pharm Sci 2016;8(10):217-23.
6. Stavri M, Piddock LJ, Gibbons S. Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother 2007;59(6):1247-60.
7. da Silva JA, Rana TS, Narzary D, Verma N, Meshram DT, Ranade S. Pomegranate biology and biotechnology: A review. Sci Hortic Amsterdam 2013;160(8):85-107.
8. Dahham SS, Ali MN, Tabassum H, Khan M. Studies on antibacterial and antifungal activity of pomegranate (Punica granatum L.). Am J Agric Environ Sci 2010;9(3):273-81.
9. Rathinamoorthy R, Udayakumar S, Thilagavathi G. Antibacterial efficacy analysis of Punica granatum L. Leaf, rind and Terminalia chebula fruit extract treated cotton fabric against five most common human pathogenic bacteria. Int J Pharm Life Sci 2011;2(10):1147-53.
10. Chanda S, Rakholiya K. Combination therapy: Synergism between natural plant extracts and antibiotics against infectious diseases. In: MÃ©ndez-Vilas A, editor. Science against Microbial Pathogens: Communicating Current Research and Technological Advances. Spain: Formatex Research Center; 2011. p. 520-9.
11. Shukla N, Kumar M, Akanksha, Ahmad G, Rahuja N, Singh AB, et al. Tectone, a new antihyperglycemic anthraquinone from Tectona grandis leaves. Nat Prod Commun 2010;5(3):427-30.
12. Sarker SD, Nahar L, Kumarasamy Y. Microtitre plate-based antibacterial assay incorporating resazurin as an indicator of cell growth, and its application in the in vitro antibacterial screening of phytochemicals. Methods 2007;42(4):321-4.
13. Stefanovic O, Stankovic MS, Comic L. In vitro antibacterial efficacy of Clinopodium vulgare L. Extracts and their synergistic interaction with antibiotics. J Med Plants Res 2011;5(17):4074-9.
14. Li L, He Z, Pandey GK, Tsuchiya T, Luan S. Functional cloning and characterization of a plant efflux carrier for multidrug and heavy metal detoxification. J Biol Chem 2002;277(7):5360-8.
15. Lautenbach E, Fishman NO, Bilker WB, Castiglioni A, Metlay JP, Edelstein PH, et al. Risk factors for fluoroquinolone resistance in nosocomial Escherichia coli and Klebsiella pneumoniae infections. Arch Intern Med 2002;162(21):2469-77.
16. Poole K. Efflux-mediated resistance to fluoroquinolones in gram-negative bacteria. Antimicrob Agents Chemother 2000;44(9):2233-41.
17. Rafiq Z, Sam N, Vaidyanathan R. Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India. Mem Inst Oswaldo Cruz 2016;111:144-6.
18. Thangavel A, Senthilkumar B, Vijayalakshmi, Duraisamy S. Phytochemical screening, gas chromatography-mass spectrometry (Gc-Ms) analysis of phytochemical constituents and antibacterial activity of coriandrum sativum (l) seeds. Int. J. Pharm. Pharm. Sci. 2015;7(9):153-9.
19. Kabara JJ, Swieczkowski DM, Conley AJ, Truant JP. Fatty acids and derivatives as antimicrobial agents. Antimicrob Agents Chemother 1972;2(1):23-8.
20. Cho H, Oh Y, Park S, Lee Y. Concentration of CCCP Should be optimized to detect the efflux system in quinolone-susceptible Escherichia coli. J Microbiol 2001;39(1):62-6.
21. Aqil F, Khan MS, Owais M, Ahmad I. Effect of certain bioactive plant extracts on clinical isolates of Î²-lactamase producing methicillin resistant Staphylococcus aureus. J Basic Microbiol 2005;45(2):106-14.
22. Braga LC, Leite AA, Xavier KG, Takahashi JA, Bemquerer MP, Chartone-Souza E, et al. Synergic interaction between pomegranate extract and antibiotics against Staphylococcus aureus. Can J Microbiol 2005;51(7):541-7.
23. Reddy BU, Mullick R, Kumar A, Sudha G, Srinivasan N, Das S. Small molecule inhibitors of HCV replication from pomegranate. Sci Rep 2014;4:5411.
The publication is licensed under CC By and is open access. Copyright is with author and allowed to retain publishing rights without restrictions.