SYNERGISTIC EFFECT OF RHAMNOLIPID IN NANO-CHITOSAN WITH ANTIBIOTICS AGAINST MULTIDRUG RESISTANCE SKIN PATHOGENS
DOI:
https://doi.org/10.36103/vpe69p76Keywords:
antibacterial activity, chitosan, mic,nanoparticles, rhamnolipid, synergistic effectAbstract
This study was aimed to purify and characterize the rhamnolipid biosurfactant produced by an environmental isolate of P.aeruginosa (PO10), and to evaluate its antibacterial activity alone in combination with chitosan as nanoparticles. The isolate showed strong hemolytic activity (36.6mm clear zone), high emulsification index (E24%=66). Rhamnolipid was extracted and purified using chloroform: methanol (2:1) solvent system and silica gel column chromatography (3.5 x 30 cm), and was characterized by FTIR and GC-MS. The MIC values of rhamnolipid were 12.5mg/ml for both P.aeruginosa and S.aureus. Chitosan/rhamnolipid nanoparticles (C/RL-NPs) were synthesized via ionic gelation and characterized by UV-Vis, FTIR, AFM, Zeta potential (+39mV), and FESEM (44.78-60.71 nm, spherical shape).These analyses confirmed the successful formation and high stability of the C/RL-NPs. The MIC of C/RL-NPs was reduced to 1.25mg/ml. This study is the first to investigate the synergistic effect of C/RL-NPs in combination with antibiotics, resulted in significant increases in inhibition zones against both pathogenic isolate of P.aeruginosa and S.aureus compared to antibiotics or nanoparticles alone.
References
Abouseoud, M., Yataghene, A., Amrane, A., & Maachi, R. (2008). Biosurfactant production by free and alginate entrapped cells of Pseudomonas fluorescens. Journal of Industrial Microbiology & Biotechnology, 35(11), 1303–1308. https://doi.org/10.1007/s10295-008-0411-0 DOI: https://doi.org/10.1007/s10295-008-0411-0
Ahmad, A., Ahmad, I., Ramzan, S., Kiyani, M., Dubal, D., & Mubarak, N. M. (2021). Nanomaterial synthesis protocols. In F. Verpoort, I. Ahmad, A. Ahmad, A. Khan, & C. Y. Chee (Eds.), Nanomedicine Manufacturing and Applications (pp. 37–85). Elsevier.
https://doi.org/10.1016/B978-0-12-820773-4.00010-X DOI: https://doi.org/10.1016/B978-0-12-820773-4.00010-X
Ali, A., & Ahmed, S. (2018). A review on chitosan and its nanocomposites in drug delivery. International Journal of Biological Macromolecules,109,273–286.
https://doi.org/10.1016/j.ijbiomac.2017.12.078 DOI: https://doi.org/10.1016/j.ijbiomac.2017.12.078
.Balouiri, M., Sadiki, M., & Ibnsouda, S. K.(2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis, 6(2), 71–79.
https://doi.org/10.1016/j.jpha.2015.11.005 DOI: https://doi.org/10.1016/j.jpha.2015.11.005
Bettencourt, A. F., Tome, C., Oliveira, T., Martin, V., Santos, C., Goncalves, L., ... & Ribeiro, I. A. (2021). Exploring the potential of chitosan-based particles as delivery carriers for promising antimicrobial glycolipid biosurfactants. Carbohydrate polymers, 254, 117433.
https://doi.org/10.1016/j.carbpol.2020.117433 DOI: https://doi.org/10.1016/j.carbpol.2020.117433
Bharali, P., Saikia, J., Ray, A., & Konwar, B. (2013). Rhamnolipid (RL) from Pseudomonas aeruginosa OBP1: A novel chemotaxis and antibacterial agent. Colloids and Surfaces B: Biointerfaces, 103, 502–509. https://doi.org/10.1016/j.colsurfb.2012.10.064 DOI: https://doi.org/10.1016/j.colsurfb.2012.10.064
Bustelo, M., Pinazo, A., Manresa, M., Mitjans, M., Vinardell, M., & Pérez, L. (2017). Monocatenary histidine-based surfactants: Role of the alkyl chain length in antimicrobial activity and their selectivity over red blood cells. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 532,501–509.
https://doi.org/10.1016/j.colsurfa.2017.04.017 DOI: https://doi.org/10.1016/j.colsurfa.2017.04.017
Corbett, J. C., McNeil-Watson, F., Jack, R. O., & Howarth, M. (2012). Measuring surface zeta potential using phase analysis light scattering in a simple dip cell arrangement. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 396, 169–176.
https://doi.org/10.1016/j.colsurfa.2011.12.065 DOI: https://doi.org/10.1016/j.colsurfa.2011.12.065
Costa, E. M., Silva, S., Veiga, M., Baptista, P., Tavaria, F. K., & Pintado, M. E. (2021). Textile dyes loaded chitosan nanoparticles: Characterization, biocompatibility and staining capacity. Carbohydrate Polymers, 251, Article117120.
https://doi.org/10.1016/j.carbpol.2020.117120 DOI: https://doi.org/10.1016/j.carbpol.2020.117120
De la Fuente- 10. De la Fuente-Núñez, C., Reffuveille, F. L., Fernández, L., & Hancock, R. E. W. (2013). Bacterial biofilm development as a multicellular adaptation: Antibiotic resistance and new therapeutic strategies. Current Opinion in Microbiology, 16(5), 580–589.
https://doi.org/10.1016/j.mib.2013.06.013 DOI: https://doi.org/10.1016/j.mib.2013.06.013
Fontes, G. C., Amaral, P. F. F., Nele, M., & Coelho, M. A. Z. (2010). Factorial design to optimize biosurfactant production by Yarrowia lipolytica. BioMed Research International, 2010, Article 821306. https://doi.org/10.1155/2010/821306 DOI: https://doi.org/10.1155/2010/821306
Grobelny, J., DelRio, F. W., Pradeep, N., Kim, D. I., Hackley, V. A., & Cook, R. F. (2011). Size measurement of nanoparticles using atomic force microscopy. In Characterization of nanoparticles intended for drug delivery (pp. 71-82). Totowa, NJ: Humana Press.
https://doi.org/10.1007/978-1-60327-198-1_7 DOI: https://doi.org/10.1007/978-1-60327-198-1_7
Hussain, S., Joo, J., Kang, J., Kim, B., Braun, G. B., She, Z. G., ... & Ruoslahti, E. (2018). Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy. Nature biomedical engineering, 2(2), 95-103.
https://doi.org/10.1038/s41551-017-0187-5 DOI: https://doi.org/10.1038/s41551-017-0187-5
Irfan, M., Shahi, S. K., & Sharma, P. K. (2015). In vitro synergistic effect of biosurfactant produced by Bacillus subtilis MTCC 441 against drug-resistant Staphylococcus aureus. Journal of Applied Pharmaceutical Science, 5(3), 113–116.
https://doi.org/10.7324/JAPS.2015.50320 DOI: https://doi.org/10.7324/JAPS.2015.50320
Karamchandani, B. M., Maurya, P. A., Dalvi, S. G., Waghmode, S., Sharma, D., Rahman, P. K., ... & Satpute, S. K. (2022). Synergistic activity of rhamnolipid biosurfactant and nanoparticles synthesized using fungal origin chitosan against phytopathogens. Frontiers in bioengineering and biotechnology, 10, 917105.
https://doi.org/10.3389/fbioe.2022.917105 DOI: https://doi.org/10.3389/fbioe.2022.917105
King, A. T., Davey, M. R., Mellor, I. R., Mulligan, B. J., & Lowe, K. C. (1991). Surfactant effects on yeast cells. Enzyme and Microbial Technology, 13(2), 148–153.
https://doi.org/10.1016/0141-0229(91)90171-6 DOI: https://doi.org/10.1016/0141-0229(91)90171-6
Kucukoglu, V., Uzuner, H., Kenar, H., & Karadenizli, A. (2019). In vitro antibacterial activity of ciprofloxacin-loaded chitosan microparticles and their effects on human lung epithelial cells. International Journal of Pharmaceutics, 569, Article 118578.
https://doi.org/10.1016/j.ijpharm.2019.118578 DOI: https://doi.org/10.1016/j.ijpharm.2019.118578
Lovaglio, R. B., dos Santos, F. J., Junior, M. J., & Contiero, J. (2011). Rhamnolipid emulsifying activity and emulsion stability: pH rules. Colloids and Surfaces B: Biointerfaces, 85(2),301–305.
https://doi.org/10.1016/j.colsurfb.2011.03.001 DOI: https://doi.org/10.1016/j.colsurfb.2011.03.001
Liu, Y. Y., Wang, Y., Walsh, T. R., Yi, L. X., Zhang, R., Spencer, J., ... & Shen, J. (2016). Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. The Lancet infectious diseases, 16(2), 161-168.
https://doi.org/10.1016/S1473-3099(15)00424-7 DOI: https://doi.org/10.1016/S1473-3099(15)00424-7
Maier, R. M., & Soberón-Chávez, G. (2000). Pseudomonas aeruginosa rhamnolipids: Biosynthesis and potential applications. Applied Microbiology and Biotechnology, 54(5),625–633.
https://doi.org/10.1007/s002530000443 DOI: https://doi.org/10.1007/s002530000443
Manna, M. S., Tamer, Y. T., Gaszek, I., Poulides, N., Ahmed, A., Wang, X., ... & Toprak, E. (2021). A trimethoprim derivative impedes antibiotic resistance evolution. Nature communications, 12(1), 2949.
https://doi.org/10.1038/s41467-021-23191-z DOI: https://doi.org/10.1038/s41467-021-23191-z
Marangon, C. A., Martins, V. C., Ling, M. H., Melo, C. C., Plepis, A. M. G., Meyer, R. L., & Nitschke, M. (2020). Combination of rhamnolipid and chitosan in nanoparticles boosts their antimicrobial efficacy. ACS applied materials & interfaces, 12(5), 5488-5499.
https://doi.org/10.1021/acsami.9b19253 DOI: https://doi.org/10.1021/acsami.9b19253
Mnif, I., Ellouz-Chaabouni, S., & Ghribi, D. (2018). Glycolipid biosurfactants: Main classes, functional properties, and related potential applications in environmental biotechnology. Journal of Polymers and the Environment, 26(6), 2192–2206.
https://doi.org/10.1007/s10924-017-1076-4 DOI: https://doi.org/10.1007/s10924-017-1076-4
Pasieczna-Patkowska, S., Cichy, M., & Flieger, J. (2025). Application of Fourier Transform Infrared (FTIR) Spectroscopy in Characterization of Green Synthesized Nanoparticles. Molecules, 30(3), 684. https://doi.org/10.3390/molecules30030684 DOI: https://doi.org/10.3390/molecules30030684
Niladevi, K. N., & Prema, P. (2005). Mangrove actinomycetes as the source of ligninolytic enzymes. Actinomycetologica, 19(2),40–47.
https://doi.org/10.3209/saj.19.40 DOI: https://doi.org/10.3209/saj.19.40
Ohikhena, F. U., Wintola, O. A., & Afolayan, A. J. (2017). Evaluation of the antibacterial and antifungal properties of Phragmanthera capitata (Sprengel) Balle (Loranthaceae), a mistletoe growing on rubber tree, using the dilution techniques. The Scientific World Journal, 2017, Article 9658598.
https://doi.org/10.1155/2017/9658598 DOI: https://doi.org/10.1155/2017/9658598
Pan, C., Qian, J., Zhao, C., Yang, H., Zhao, X., & Guo, H. (2020). Study on the relationship between crosslinking degree and properties of TPP crosslinked chitosan nanoparticles. Carbohydrate Polymers, 241, Article116349.
https://doi.org/10.1016/j.carbpol.2020.116349 DOI: https://doi.org/10.1016/j.carbpol.2020.116349
Paulino, B. N., Pessôa, M. G., Mano, M. C. R., Molina, G., Neri-Numa, I. A., & Pastore, G. M. (2016). Current status in biotechnological production and applications of glycolipid biosurfactants. Applied Microbiology and Biotechnology, 100, 10265–10293.
https://doi.org/10.1007/s00253-016-7980-z DOI: https://doi.org/10.1007/s00253-016-7980-z
Pontes, C., Alves, M., Santos, C., Ribeiro, M. H., Gonçalves, L., Bettencourt, A. F., & Ribeiro, I. A. (2016). Can Sophorolipids prevent biofilm formation on silicone catheter tubes?. International Journal of Pharmaceutics, 513(1-2), 697-708.
https://doi.org/10.1016/j.ijpharm.2016.09.074 DOI: https://doi.org/10.1016/j.ijpharm.2016.09.074
Prabaharan, M., & Mano, J. F. (2005). Chitosan-based particles as controlled drug delivery systems. Drug Delivery, 12(1), 41–57.
https://doi.org/10.1080/10717540590889781 DOI: https://doi.org/10.1080/10717540590889781
Qazi, M. A., Malik, Z. A., Qureshi, G. D., Hameed, A., & Ahmed, S. (2013). Yeast extract as the most preferable substrate for optimized biosurfactant production by rhlB gene-positive Pseudomonas putida SOL-10 isolate. Journal of Bioremediation & Biodegradation,4(2).
. Raafat, D., von Bargen, K., Haas, A., & Sahl, H.-G. (2008). Insights into the mode of action of chitosan as an antibacterial compound. Applied and Environmental Microbiology, 74(12), 3764–3773. https://doi.org/10.1128/AEM.00453-08 DOI: https://doi.org/10.1128/AEM.00453-08
Sana, S., Datta, S., Biswas, D., & Sengupta, D. (2018). Assessment of synergistic antibacterial activity of combined biosurfactants revealed by bacterial cell envelope damage. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1860(2), 579–585.
https://doi.org/10.1016/j.bbamem.2017.09.027 DOI: https://doi.org/10.1016/j.bbamem.2017.09.027
Sánchez, M., Aranda, F. J., Espuny, M. J., Marqués, A., Teruel, J. A., Manresa, Á., & Ortiz, A. (2007). Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in aqueous media. Journal of colloid and interface science, 307(1), 246-253.
https://doi.org/10.1016/j.jcis.2006.11.041 DOI: https://doi.org/10.1016/j.jcis.2006.11.041
Santa Anna, L. M., Sebastian, G. V., Menezes, E. P., Alves, T. L. M., Santos, A. S., Pereira Jr., N., & Freire, D. M. G. F. (2002). Production of biosurfactants from Pseudomonas aeruginosa PA1 isolated in oil environments. Brazilian Journal of Chemical Engineering,19(2),159–166.
https://doi.org/10.1590/S0104-66322002000200011 DOI: https://doi.org/10.1590/S0104-66322002000200011
Shaban, S. M. (2016). Studying the effect of newly synthesized cationic surfactant on silver nanoparticles formation and their biological activity. Journal of Molecular Liquids, 216,137–145.
https://doi.org/10.1016/j.molliq.2015.12.098 DOI: https://doi.org/10.1016/j.molliq.2015.12.098
Shah, M., Fawcett, D., Sharma, S., Tripathy, S. K., & Poinern, G. E. J. (2015). Green synthesis of metallic nanoparticles via biological entities. Materials, 8(11), 7278–7308.
https://doi.org/10.3390/ma8115377 DOI: https://doi.org/10.3390/ma8115377
Sundar, S., Kundu, J., & Kundu, S. C. (2010). Biopolymeric nanoparticles. Science and Technology of Advanced Materials, 11(1).
https://doi.org/10.1088/1468-6996/11/1/014104 DOI: https://doi.org/10.1088/1468-6996/11/1/014104
Shekhar, S., Sundaramanickam, A., & Balasubramanian, T. (2015). Biosurfactant producing microbes and their potential applications: A review. Critical Reviews in Environmental Science and Technology, 45(14),1522–1554. https://doi.org/10.1080/10643389.2014.955631 DOI: https://doi.org/10.1080/10643389.2014.955631
Sifour, M., Al-Jilawi, M. H., & Aziz, G. M. (2007). Emulsification properties of biosurfactant produced from Pseudomonas aeruginosa RB28. Pakistan Journal of Biological Sciences, 10(8), 1331–1335. https://doi.org/10.3923/pjbs.2007.1331.1335 DOI: https://doi.org/10.3923/pjbs.2007.1331.1335
Singh, N., Pemmaraju, S. C., Pruthi, P. A., Cameotra, S. S., & Pruthi, V. (2013). Candida biofilm disrupting ability of di-rhamnolipid (RL-2) produced from Pseudomonas aeruginosa DSVP20. Applied Biochemistry and Biotechnology, 169(8), 2374–2391.
https://doi.org/10.1007/s12010-013-0149-7 DOI: https://doi.org/10.1007/s12010-013-0149-7
Sudarshan, N., Hoover, D. G., & Knorr, D. (1992). Antibacterial action of chitosan. Food Biotechnology,6(3),257–272.
https://doi.org/10.1080/08905439209549838 DOI: https://doi.org/10.1080/08905439209549838
Thanomsub, B., Pumeechockchai, W., Limtrakul, A., Arunrattiyakorn, P., Petchleelaha, W., Nitoda, T., & Kanzaki, H. (2006). Chemical structures and biological activities of rhamnolipids produced by Pseudomonas aeruginosa B189 isolated from milk factory waste. Bioresource Technology, 97(18),2457–2461.
https://doi.org/10.1016/j.biortech.2005.10.029 DOI: https://doi.org/10.1016/j.biortech.2005.10.029
Varvaresou, A., & Iakovou, K. (2015). Biosurfactants in cosmetics and biopharmaceuticals. Letters in Applied Microbiology,61(3),214–223.
https://doi.org/10.1111/lam.12440 DOI: https://doi.org/10.1111/lam.12440
Vatanparast, H., Shahabi, F., Bahramian, A., Javadi, A., & Miller, R. (2018). The role of electrostatic repulsion on increasing surface activity of anionic surfactants in the presence of hydrophilic silica nanoparticles. Scientific Reports, 8(1), 7251. https://doi.org/10.1038/s41598-018-25493-7 DOI: https://doi.org/10.1038/s41598-018-25493-7
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