Inhibitory activity of clove oil and cinnamic acid on Klebsiella pneumonia biofilm
Main Article Content
Keywords
Klebsiella pneumonia, MTT, FBLC, FIC, CFU
Abstract
New approaches must be developed to treat illnesses brought on by drug-resistant bacteria because of the rise in antibiotic-resistant bacteria and the insufficiency of new antibiotics on the market. Combining current antibiotics with phytochemicals (PCs) may be a method to boost antibiotic efficacy. Lab studies have linked essential oils (EOs) and their constituents to a set of phytochemicals
that have these effects. As an illustration, we chose cinnamic acid (CAc) and clove oil (COs) as phytochemicals that can work in concert with antibiotics (cefepime) to combat an isolate of Klebsiella pneumonia. In the present study, initial evaluation of the biofilm forming abilities, haem agglutination, yeast agglutination & string test of 84 isolates of K. pneumoniae were performed. The strongest
biofilm forming & positive virulence factors isolates was selected for further experimentation. The COs and CAc's impact on early cell attachment was dosage dependent, according to the crystal violet (CV) assay (p less than 0.05). The inhibition of biofilm formation was highest at 4 MIC of clove oil then 0.25 MIC of CAc. When the CAc & COs were tested against a preformed biofilm, the percentage inhibition was increased with increasing with incubation time. As determined by the CV assay.
The methyl-thiazolyl blue tetrazolium bromide (MTT) assay results showed that the COs considerably decreased the metabolic activity of K. pneumonia biofilms at 4 MIC (P˂ 0.05). The addition of COS and CAC to prevent initial cell attachment lowered both the biomass of the cells (as shown by the CV assay) and their metabolic activity, resulting in 95.03% inhibitor of COs and 93.5% inhibitor of CAc.
The K. pneumonia was grown on Foley balloon latex catheter (FBLC) and then calculated sessile cells. The number of cells shows statistically significant differences between adherent cells and free cells (P˂0.05), and the median of growth for sessile cells treated with CAc & COs were reduced to almost half compared to untreated bacterial biofilm.
By using sonicating water bath (SWB), Foley catheter was put into SWB to dislodge the sessile cells in the solution to made suspension of bacteria, with measuring colony-forming unit (CFU), the suitable time was 6 min among (6, 10 & 15) min.
Synergistic interaction by combination between cefepime and phytochemicals (Cos & CAc), in cefipime-resistant strains of K. pneumonia, synergistic effects measured as metabolic activity decrease and restoration of cefipime sensitivity. AMN3 “Human murine mammary adenocarcinoma” cell attachment phenotypes phenotypes appeared by K. pneumonia strains. The results showed the dissolution of the biofilm adhesions when treated with phytochemicals compared to the control.
Finally, based on K. pneumoniae's reported genome, genes (magA, Aerobactin, mrkJ, mrkA, AcrAB, blaKPC, blaCTX-M and entB) were found. To further confirm the inhibitory effects of the cinnamic acid and clove oil on the K. pneumoniae, which is strong biofilm-producing and their biofilmassociated genes (mrkA and mrkJ) were determined by quantitative RT- PCR. This study showed that
the down-regulation of type III fimbria (mrkA) biosynthesis gene and Phosphodiesterase (mrkJ ) responsible for biofilm development after treatment with cinnamic acid and clove oil.
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Klebsiella pneumoniae harboring several virulence and β-lactamase encoding genes in a Brazilian intensive care unit. Front Microbiol.
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9. Bayles KW. The biological role of death and lysis in biofilm development. Nat Rev Microbiol. 2007;5(9):721–6.
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Pharmaceuticals. 2010;3(5):1374–93.
11. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu E, Sintim HO. Biofilm formation mechanisms and targets for developing
antibiofilm agents. Future Med Chem.2015;7(4):493–512.
12. Tamayo R, Pratt JT, Camilli A. Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annu Rev Microbiol.
2007;61:131–48.
13. Li Y, Li X, Hao Y, Liu Y, Dong Z, Li K. Biological and physiochemical methods of biofilm adhesion resistance control of medicalcontext surface. Int J Biol Sci. 2021;17(7):1769.
14. Steinberg D, Feldman M, Ofek I, Weiss EI. Cranberry high molecular weight constituents promote Streptococcus sobrinus desorption from
artificial biofilm. Int J Antimicrob Agents.2005;25(3):247–51.
15. Dreeszen PH. Biofilm: The key to understanding and controlling bacterial growth in automated drinking water systems. Edstrom Ind Waterford.2003;
16. Kumar CG, Anand SK. Significance of microbial biofilms in food industry: a review. Int J Food Microbiol. 1998;42(1–2):9–27.
17. Darbandi A, Asadi A, Mahdizade Ari M, Ohadi E, Talebi M, Halaj Zadeh M, et al. Bacteriocins:Properties and potential use as antimicrobials. J
Clin Lab Anal. 2022;36(1):e24093.
18. Chikindas ML, Weeks R, Drider D, Chistyakov VA, Dicks LMT. Functions and emerging applications of bacteriocins. Curr Opin
Biotechnol. 2018;49:23–8.
19. Sandasi M, Leonard CM, Van Vuuren SF, Viljoen AM. Peppermint (Mentha piperita) inhibits microbial biofilms in vitro. South African
J Bot. 2011;77(1):80–5.
20. Rehman R, Hanif MA, Mushtaq Z, Al-Sadi AM. Biosynthesis of essential oils in aromatic plants:A review. Food Rev Int. 2016;32(2):117–60.
21. Vidhani SI, Vyas VG, Parmar HJ, Bhalani VM,Hassan MM, Gaber A, et al. Evaluation of some chemical composition, minerals fatty acid
profiles, antioxidant and antimicrobial activities of Tulsi (Ocimum sanctum) from India. Am J Food Sci Technol. 2016;4(2):52–7.
22. Hussein, A. Q., and Al-Meani SAL. Assessment the Relationship of Antibiotic Profile and Virulence Factors in Klebsiella pneumoniae. Int J
Health Sci (Qassim). 2022;6(56):9024–36.
23. CLSI 30th Edition. M100 Performance Standards for Antimicrobial. 30th ed. 2020.
24. Wu M-C, Lin T-L, Hsieh P-F, Yang H-C, Wang J-T. Isolation of genes involved in biofilm formation of a Klebsiella pneumoniae strain
causing pyogenic liver abscess. PLoS One.2011;6(8):e23500.
25. Häkkinen SH, Kärenlampi SO, Mykkänen HM, Heinonen IM, Törrönen AR. Ellagic acid content in berries: Influence of domestic processing and storage. Eur Food Res Technol. 2000;212(1):75–80.
26. Rodríguez Luis O, Sánchez Casas RM, Verde Star MJ, Núñez A, Ríos R, Chávez A. Obtaining the essential oil of Syzygium aromaticum,
identification of eugenol and its effect on Streptococcus mutans. J oral Res. 2014;3(4):218–24.
27. Jafari-Sales A, Hossein-Nezhad P, Bolouri P. Identification of chemical composition of essential oil and evaluation of antimicrobial
effects of ethanolic extract of Mentha pulegium on Staphylococcus aureus and Escherichia coli. Heal Biotechnol Biopharma. 2019;3(1):29–38.
28. Khalil N, Ashour M, Fikry S, Singab AN, Salama O. Chemical composition and antimicrobial activity of the essential oils of selected Apiaceous fruits. Futur J Pharm Sci. 2018;4(1):88–92.
29. 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.
30. Kolarević S, Milovanović D, Avdović M, Oalde M, Kostić J, Sunjog K, et al. Optimisation of the microdilution method for detection of minimum inhibitory concentration values in selected bacteria. Bot Serbica. 2016;40(1):29–36.
31. Reffuveille F, Haney EF, Straus SK, Hancock REW. Broad-spectrum anti-biofilm peptide that targets a cellular stress responses. PLoS Pathog.
2014;10:e1004152.
32. Sandasi M, Leonard CM, Viljoen AM. The in vitro antibiofilm activity of selected culinary herbs and medicinal plants against Listeria
monocytogenes. Lett Appl Microbiol. 2010;50(1):30–5.
33. Djordjevic D, Wiedmann M, McLandsborough LA. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl
Environ Microbiol. 2002;68(6):2950–8.
34. Schillaci D, Arizza V, Dayton T, Camarda L, Stefano V Di. In vitro anti‐biofilm activity of Boswellia spp. oleogum resin essential oils. Lett
Appl Microbiol. 2008;47(5):433–8.
35. Kırmusaoğlu S. The methods for detection of biofilm and screening antibiofilm activity of agents. Antimicrob Antibiot Resist antibiofilm
Strateg Act methods. 2019;1–17.
36. Pericolini E, Colombari B, Ferretti G, Iseppi R, Ardizzoni A, Girardis M, et al. Real-time monitoring of Pseudomonas aeruginosa biofilm
formation on endotracheal tubes in vitro. BMC Microbiol. 2018;18(1):1–10.
37. Al-Qaysi A-MK, Al-Ouqaili MTS, Al-Meani SAL. Ciprofloxacin-and gentamicin-mediated inhibition of Pseudomonas aeruginosa biofilms is
enhanced when combined the volatile oil from Eucalyptus camaldulensis. Syst Rev Pharm. 2020;11:98–105.
38. Saldana Z, Erdem AL, Schuller S, Okeke IN, Lucas M, Sivananthan A, et al. The Escherichia coli common pilus and the bundle-forming pilus
act in concert during the formation of localized adherence by enteropathogenic E. coli. J Bacteriol. 2009;191(11):3451–61.
39. Taweechaisupapong S, Ngaonee P, Patsuk P, Pitiphat W, Khunkitti W. Antibiofilm activity and post antifungal effect of lemongrass oil on clinical Candida dubliniensis isolate. South African J Bot. 2012;78:37–43.
40. Cruz-Córdova A, Esteban-Kenel V, EspinosaMazariego K, Ochoa SA, Moreno Espinosa S, de la Garza Elhain A, et al. Pathogenic determinants
of clinical Klebsiella pneumoniae strains associated with their persistence in the hospital environment. Bol Med Hosp Infant Mex.
2014;71(1):15–24.
41. Purkrtova S, Turoňová H, Pilchova T, Demnerová K, Pazlarova J. Resistance of Listeria monocytogenes biofilms to disinfectants 326.
Czech J Food Sci. 2010;28(4):326–32.
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Mar 17, 2023
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Aseel Qassim Hussein, & Safaa A.L. Al-Meani. (2023). Inhibitory activity of clove oil and cinnamic acid on Klebsiella pneumonia biofilm. Journal of Population Therapeutics and Clinical Pharmacology, 30(2), 375–392. https://doi.org/10.47750/jptcp.2023.1121
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