Anti-virulence potential of eugenol-rich fraction of Syzygium aromaticum against multidrug resistant uropathogens isolated from catheterized patients

Document Type: Original Research Article

Authors

Renal Research Lab, Centre for Bio Medical Research, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, India

Abstract

Objective: Considering the emergence of biofilm-associated pathogens with multidrug resistance, the objective(s) of the present study was to evaluate the anti-virulence property of Syzygium aromaticum extracts/fractions against 2 multidrug-resistant catheter isolates.
Materials and Methods: Pulverized clove buds were subjected to bioactivity-guided isolation to identify the bioactive extract/fraction(s) with significant anti-virulence property. The clove bud powder was subjected to Soxhlet extraction and all these extracts were investigated in terms of anti-virulent efficacy using initial readout assays. Bioassay-guided partial purification was performed through column and size exclusion chromatography. Detailed testing of the anti-virulence and anti-biofilm activity of sub-minimum inhibitory concentration (sub-MIC) levels of the active fraction, was performed besides the cytotoxicity profiling in RAW 264.7 cells.
Results: Gas chromatography-mass spectrometry (GC-MS) analysis of the clove bioactive fraction-2 (CBF-2), done after the bioassay-guided fractionation, revealed eugenol as the major bioactive ingredient present in CBF-2. Reduced mRNA levels of virulence factor genes after CBF-2 (700 μg/ml) treatment correlated well with the respective phenotypic assays (p<0.001). Similarly, CBF-2 (700 μg/ml) treatment exhibited significantly low mRNA levels of quorum sensing (QS) receptor genes compared to their cognate synthase genes (p<0.001). Crystal violet staining and scanning electron micrographs of CBF-2-treated biofilms showed lesser macrocolonies with remarkably simplified architecture. Cytotoxic evaluation of CBF-2 suggested a minute reduction in viability even at the highest tested concentration (1600 μg/ml, p<0.05).
Conclusion: The present study indicated that eugenol-rich CBF-2 has potent anti-virulence and anti-biofilm activity against urinary catheter isolates and can be regarded as an alternative for treatment of catheter-associated urinary tract infections.

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Abraham SVPI, Palani A, Ramaswamy BR, Shunmugiah KP, Arumugam VR. 2011. Antiquorum sensing and antibiofilm potential of Capparis spinosa. Arch Med Res, 42:658-668.

Adonizio A, Kong KF, Mathee K. 2008. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother, 52:198-203.

Annapoorani A, Kalpana B, Musthafa KS, Pandian SK, Ravi AV. 2013. Antipathogenic potential of Rhizophora spp. against the quorum sensing mediated virulence factors production in drug resistant Pseudomonas aeruginosa. Phytomedicine, 20:956-963.

Bhattacharjee S. 2014. Membrane lipid peroxidation and its conflict of interest: the two faces of oxidative stress. Curr Sci, 107:1811-23.

Cortés-Rojas DF, de Souza CRF, Oliveira WP. 2014. Clove (Syzygium aromaticum): a precious spice. Asian Pac J Trop Biomed, 4:90-96.

Ćurčić MG, Stanković MS, Mrkalić EM, Matović ZD, Banković DD, Cvetković DM, Đačić DS, Marković SD.2012. Antiproliferative and proapoptotic activities of methanolic extracts from Ligustrum vulgare L. as an individual treatment and in combination with palladium complex. Int J Mol Sci,13:2521-2534.

Galati G, O'Brien PJ. 2004. Potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. Free Radic Biol Med. 37:287-303.

García-Contreras R, Maeda T, Wood TK.2013.Resistance to quorum-quenching compounds.ApplEnviron Microbiol.79:6840-6846.

García-Contreras R, Peréz-Eretza B, Jasso-Chávez R, Lira-Silva E, Roldán-Sánchez JA, González-Valdez A, Soberón-Chávez G, Coria-Jiménez R, Martínez-Vázquez M, Alcaraz LD. 2015. High variability in quorum quenching and growth inhibition by furanone C-30 in Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients. Pathog Dis.73:ftv040.

Gibot S. 2004. Fighting the enemy properly? Crit Care Med.32:1223-1224.

Husain FM, Ahmad I, Asif M, Tahseen Q. 2013. Influence of clove oil on certain quorum-sensing-regulated functions and biofilm of Pseudomonas aeruginosa and Aeromonas hydrophila. J Biosci.38:835-844.

Jakobsen TH, Bragason SK, Phipps RK, Christensen LD, van Gennip M, Alhede M, Skindersoe M, Larsen TO, Høiby N, Bjarnsholt T. 2012. Food as a source for QS inhibitors: iberin from horseradish revealed as a quorum sensing inhibitor of Pseudomonas aeruginosa.Appl Environ Microbiol. AEM: 05992-11.

Janero DR. 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med. 9:515-540.

Kalia VC. 2013. Quorum sensing inhibitors: an overview. Biotechnol Adv. 31:224-245.

Kampa M, Nifli AP, Notas G, Castanas E.2007. Polyphenols and cancer cell growth. In Reviews of physiology, biochemistry and pharmacology, Springer: 79-113.

Khan MSA, Zahin M, Hasan S, Husain FM, Ahmad I. 2009. Inhibition of quorum sensing regulated bacterial functions by plant essential oils with special reference to clove oil. Lett Appl Microbiol.49:354-360.

Kong X, Liu X, Li J, Yang Y. 2014. Advances in pharmacological research of eugenol. Curr Opin Complement Alternat Med.1:8-11.

Krishnan T, Yin WF, Chan KG. 2012. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa PAO1 by Ayurveda spice clove (Syzygium aromaticum) bud extract. Sensors. 12:4016-4030.

Kumar L, Chhibber S, Kumar R, Kumar M, Harjai K.2015. Zingerone silences quorum sensing and attenuates virulence of Pseudomonas aeruginosa. Fitoterapia. 102:84-95.

Magiorakos AP, Srinivasan A, Carey R, Carmeli Y, Falagas M, Giske C, Harbarth S, Hindler J, Kahlmeter G, Olsson‐Liljequist B. 2012. Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol Infect. 18:268-281.

Mittal R, Aggarwal S, Sharma S, Chhibber S, Harjai K. 2009. Urinary tract infections caused by Pseudomonas aeruginosa: a minireview. J Infect Public Health.2:101-111.

Ni N, Li M, Wang J, Wang B. 2009. Inhibitors and antagonists of bacterial quorum sensing. Med Res Rev. 29:65-124.

Packiavathy IASV, Agilandeswari P, Musthafa KS, Pandian SK, Ravi AV. 2012. Antibiofilm and quorum sensing inhibitory potential of Cuminum cyminum and its secondary metabolite methyl eugenol against Gram negative bacterial pathogens. Food Res Int. 45:85-92.

Rathinam P, Viswanathan P. 2014. Effects of antibiotics upon quorum sensing regulated characters: a propitious scheme against device associated infections. Int J Pharm Pharm Sci. 6:85-90.

Salini R. Sindhulakshmi M, Poongothai T, Pandian SK. 2015. Inhibition of quorum sensing mediated biofilm development and virulence in uropathogens by Hyptis suaveolens. A Van Leeuw J Microb. 107: 1095-1106.

Sarabhai S, Sharma P, Capalash N. 2013. Ellagic acid derivatives from Terminalia chebula Retz. Downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa PAO1 virulence.PLoS One.8:e53441.

Thompson DC, Barhoumi R, Burghardt RC. 1998. Comparative toxicity of eugenol and its quinone methide metabolite in cultured liver cells using kinetic fluorescence bioassays. Toxicol Appl Pharmaco.149:55-63.

Unnikrishnan P, Suthindhiran K, Jayasri M.2014. Inhibitory potential of Turbinaria ornata against key metabolic enzymes linked to diabetes.Bio Med Res Int.:2014.

Van Delden C, Iglewski BH. 1998. Cell-to-cell signaling and Pseudomonas aeruginosa infections. Emerg Infect Diseases.4:551.

Viswanathan P, Rathinam P, Suneeva S. 2015. Plant Quorum Sensing Inhibitors: Food, Medicinal Plants, and Others. In Quorum Sensing vs Quorum Quenching: A Battle with No End in Sight, Springer: 269-281.

Wikler M. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard. 2009.

Yates EA, Philipp B, Buckley C, Atkinson S, Chhabra SR, Sockett RE, Goldner M, Dessaux Y, Cámara M, Smith H. 2002. N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa. Infect immun. 70:5635-5646.