Bioactivity of the amikacin: Selenium nanoparticles stabilized by chitosan (AK: CS-SeNPs) on Proteus mirabilis biofilm
Article Main
Abstract
Biofilm-associated diseases have become a challenging issue for the healthcare system due to the aggregation of bacteria within the biofilm, which exhibits increased resistance to broad-spectrum antibiotics at standard or elevated concentrations. Consequently, adopting Chitosan-stabilized selenium nanoparticles (CS-SeNPs) is an efficient way to regulate biofilm creation. Multiple analyses were applied to characterize CS-SeNPs, including ultraviolet-visible absorption, Fourier Transform Infrared Spectroscopy, Zeta potential analysis, Dynamic Light Scattering analysis, Field emission Scanning Electron Microscopy, and Energy Dispersive x-ray. The antibacterial and antibiofilm properties of CS-SeNPs, AK: Cs-SeNPs, and amikacin (AK) were tested using 96-microtiter plates. The resulting data have revealed that CS-SeNPs at a wavelength of 244 nm were stabilized and rounded in shape with an average size of 68±23nm. The minimum inhibitory doses of AK and CS-SeNPs required to prevent the growth of P. mirabilis were 1000±398 and 50±0 µg/mL, respectively. The combination of AK:CS-SeNPs inhibited P. mirabilis strains at MIC of 160±0:12.5±0 µg/mL, which is lower than the MIC of AK and CS-SeNPs applied alone. The lowest concentrations of AK:CS-SeNPs, ranging between 66±23:5±1 μg /mL and 93±23:8±3 μg /Ml, successfully impeded the initial creation of P.mirabilis biofilm .The results demonstrate that The conjugation of AK:CS-SeNPs improves the amikacin's bactericidal efficiency, significantly hinders biofilm's initial development and reduces the viability of established biofilm created by multidrug-resistant P.mirabilis. This therapeutic approach has the potential to serve as a promising strategy for addressing Biofilm-associated diseases caused by resistant strains of P.mirabilis, conferring confidence in the battle against persistent infections.
Article Details
Article Details
Keywords
Antibacterial impact, Antibiofilm impact, Amikacin, Chitosan, Mature biofilm, Proteus mirabilis, Selenium nanoparticles
References
Al Jahdaly, B.A., Al-Radadi, N.S., Eldin, G.M.G., Almahri, A., Ahmed, M.K., Shoueir, K. & Janowska, I. ( 2021) . Selenium nanoparticles synthesized using an eco-friendly method: Dye decolorization from aqueous solutions, cell viability, antioxidant, and antibacterial effectiveness. J. Mater. Res. Technol. 11, 85–97. https://doi.org/10.1016/j.jmrt.2020.12.098
Bardbari, A.M., Arabestani, M.R., Karami, M., Keramat, F., Aghazadeh, H., Alikhani, M.Y. & Bagheri, K.P. (2018). Highly synergistic activity of melittin with imipenem and colistin in biofilm inhibition against multidrug-resistant strong biofilm producer strains of Acinetobacter baumannii. Eur. J. Clin. Microbial. Infect. Dis. 37, 443–454. https://doi.org/10.1007/s10096-018-3189-7
Bhattacharjee, S. (2016). DLS and zeta potential - What they are and what they are not? J. Control. Release 235, 337–351. https://doi.org/10.1016/j.jconrel.2016.06.017
Chen, W., Li, Y., Yang, S., Yue, L., Jiang, Q. & Xia, W. (2015). Synthesis and antioxidant properties of chitosan and carboxymethyl chitosan-stabilized selenium nanoparticles. Carbohydr. Polym. 132, 574–581. https://doi.org/10.1016/j.carbpol.2015.06.064
Fang, Ferric C. & R.T.S.(2020). Antimicrobial resistance—The glass Is half full. N. Engl. J. Med. 382, 1361–1363. https://doi.org/10.1056/nejme2002121
Geoffrion, L.D., Hesabizadeh, T., Medina-Cruz, D., Kusper, M., Taylor, P., Vernet-Crua, A., Chen, J., Ajo, A., Webster, T.J. & Guisbiers, G., (2020). Naked Selenium Nanoparticles for Antibacterial and Anticancer Treatments. ACS Omega. https://doi.org/10.1021/acsomega.9b03172
Haji, S.H., Ali, F.A., & Aka, S.T.H. (2022). Synergistic antibacterial activity of silver nanoparticles biosynthesized by carbapenem-resistant Gram-negative bacilli. Sci. Rep. 12, 1–13. https://doi.org/10.1038/s41598-022-19698-0
Huang, T., Holden, J.A., Heath, D.E., O’Brien-Simpson, N.M. & O’Connor, A.J. (2019). Engineering highly effective antimicrobial selenium nanoparticles through control of particle size. Nanoscale 11, 14937–14951. https://doi.org/10.1039/c9nr04424h
Huang, X., Chen, X., Chen, Q., Yu, Q., Sun, D. & Liu, J. (2016). Investigation of functional selenium nanoparticles as potent antimicrobial agents against superbugs. Acta Biomater. 30, 397–407. https://doi.org/10.1016/j.actbio.2015.10.041
Jacobsen, S.M., Stickler, D.J., Mobley, H.L.T. & Shirtliff, M.E. (2008). Complicated catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin. Microbial. Rev. 21, 26–59. https://doi.org/10.1128/CMR.00019-07
Javed, R., Sajjad, A., Naz, S., Sajjad, H. & Ao, Q. (2022). Significance of Capping Agents of Colloidal Nanoparticles from the Perspective of Drug and Gene Delivery, Bioimaging, and Biosensing: An Insight. Int. J. Mol. Sci. 23. https://doi.org/10.3390/ijms231810521
Jernigan, J.A., Hatfield, K.M., Wolford, H., Nelson, R.E., Olubajo, B., Reddy, S.C., McCarthy, N., Paul, P., McDonald, L.C., Kallen, A., Fiore, A., Craig, M. & Baggs, J. (2020). Multidrug-Resistant Bacterial Infections in U.S. Hospitalized Patients, 2012–2017. N. Engl. J. Med. 382, 1309–1319. https://doi.org/10.1056/nejmoa1914433
Kaiser, T.D.L., Pereira, E.M., dos Santos, K.R.N., Maciel, E.L.N., Schuenck, R.P. &Nunes, A.P.F.(2013). Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis. Diagn. Microbial. Infect. Dis. 75, 235–239. https://doi.org/10.1016/j.diagmicrobio.2012.11.014
Kamer, A.M.A., El Maghraby, G.M., Shafik, M.M. & Al-Madboly, L.A. (2024). Silver nanoparticle with potential antimicrobial and antibiofilm efficiency against multiple drug resistant, extensive drug resistant Pseudomonas aeruginosa clinical isolates. BMC Microbial. 24, 1–16. https://doi.org/10.1186/s12866-024-03397-z
Karthik, K.K., Cheriyan, B.V., Rajeshkumar, S. & Gopalakrishnan, M. (2024). A review on selenium nanoparticles and their biomedical applications. Biomed. Technol. 6, 61–74. https://doi.org/10.1016/j.bmt.2023.12.001
Lin, W., Zhang, J., Xu, J.F. & Pi, J. (2021). The Advancing of Selenium Nanoparticles Against Infectious Diseases. Front. Pharmacol. 12, 1–16. https://doi.org/10.3389/fphar.2021.682284
Medina, Eva & D.H.P. (2016). " Tackling threats and future problems of multidrug-resistant bacteria. Curr. Top. Microbiol. Immunol. 33. https://doi.org/10.1007/82
Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T. &Yacaman, M.J. (2005). The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346–2353. https://doi.org/10.1088/0957-4484/16/10/059
Munir, M.U. & Ahmad, M.M. (2022). Nanomaterials Aiming to Tackle Antibiotic-Resistant Bacteria. Pharmaceutics 14, 1–17. https://doi.org/10.3390/pharmaceutics14030582
Pachouri, C., Tripathi, S., Shukla, S. & Pandey, A. (2021). Development and Evaluation of Chitosan Nanoparticles Based Dry Powder Inhalation Formulations of Isoniazid and Its Release Kinetics. Oxid. Commun. 44, 755–768.
Ridha, D.M., AL-Rafyai, H.M. & Najii, N.S. (2021). Bactericidal Potency of Green Synthesized Silver Nanoparticles against Waterborne Escherichia coli Isolates. Nano Biomed. Eng. 13, 372–379. https://doi.org/10.5101/nbe.v13i4.p372-379
Ridha, D.M., AL-Rafyai, H.M. & Saleh, M.A.A.-J.M. (2024). Evaluation of the Antibacterial Impact of the Conjugated Gentamycin-biosynthesized Silver Nanoparticles on Resistant Pseudomonas aeruginosa Isolates In Vitro. Nano Biomed. Eng. 0–10. https://doi.org/10.26599/nbe.2024.9290067
Roy, N., Nivedya, T., Paira, P. & Chakrabarty, R. (2025). Selenium-based nanomaterials: Green and conventional synthesis methods, applications, and advances in dye degradation. RSC Adv. 15, 3008–3025. https://doi.org/10.1039/d4ra07604d
San Keskin, N.O., Akbal Vural, O. & Abaci, S. (2020). Biosynthesis of Noble Selenium Nanoparticles from Lysinibacillus sp. NOSK for Antimicrobial, Antibiofilm Activity, and Biocompatibility. Geomicrobiol. J. 37, 919–928. https://doi.org/10.1080/01490451.2020.1799264
Schaffer, J.N. & Pearson, M.M. (2016). Proteus mirabilis and urinary tract infections. Urin. Tract Infect. Mol. Pathog. Clin. Manag. 383–433. https://doi.org/10.1128/9781555817404.ch17
Sentkowska, A. & Pyrzyńska, K. (2022). The Influence of Synthesis Conditions on the Antioxidant Activity of Selenium Nanoparticles. Molecules 27, 1–14. https://doi.org/10.3390/molecules27082486
Shahmoradi, S., Shariati, A., Zargar, N., Yadegari, Z., Asnaashari, M., Amini, S.M. & Darban-Sarokhalil, D. (2021). Antimicrobial effects of selenium nanoparticles in combination with photodynamic therapy against Enterococcus faecalis biofilm. Photodiagnosis Photodyn. Ther. 35, 102398. https://doi.org/10.1016/j.pdpdt.2021.102398
Shakibaie, M., Forootanfar, H., Golkari, Y., Mohammadi-Khorsand, T. & Shakibaie, M.R. (2015). Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. J. Trace Elem. Med. Biol. 29, 235–241. https://doi.org/10.1016/j.jtemb.2014.07.020
Sharma, D., Maheshwari, D., Philip, G., Rana, R., Bhatia, S., Singh, M., Gabrani, R., Sharma, S.K., Ali, J., Sharma, R.K. & Dang, S. (2014). Formulation and optimization of polymeric nanoparticles for intranasal delivery of lorazepam using Box-Behnken design: In vitro and in vivo evaluation. Biomed Res. Int. 2014. https://doi.org/10.1155/2014/156010
Tumbarello, M., Trecarichi, E.M., Fiori, B., Losito, A.R., D’Inzeo, T., Campana, L., Ruggeri, A., Di Meco, E., Liberto, E., Fadda, G., Cauda, R. & Spanu, T. (2012). Multidrug-resistant Proteus mirabilis bloodstream infections: Risk factors and outcomes. Antimicrob. Agents Chemother. 56, 3224–3231. https://doi.org/10.1128/AAC.05966-11
Wadhwani, S.A., Gorain, M., Banerjee, P., Shedbalkar, U.U., Singh, R., Kundu, G.C. & Chopade, B.A. (2017). Green synthesis of selenium nanoparticles using Acinetobacter sp. SW30: Optimization, characterization and its anticancer activity in breast cancer cells. Int. J. Nanomedicine 12, 6841–6855. https://doi.org/10.2147/IJN.S139212
33. WHO. (2017). GLASS | Global antimicrobial resistance surveillance system (GLASS) report 2016-17, Who.
Yuan, Q., Xiao, R., Afolabi, M., Bomma, M. & Xiao, Z. (2023). Evaluation of Antibacterial Activity of Selenium Nanoparticles against Food-Borne Pathogens. Microorganisms 11. https://doi.org/10.3390/microorganisms 11061519
Zhang, C., Zhai, X., Zhao, G., Ren, F. & Leng, X. (2015). Synthesis, characterization, and controlled release of selenium nanoparticles stabilized by chitosan of different molecular weights. Carbohydr. Polym. 134, 158–166. https://doi.org/10.1016/j.carbpol.2015.07.065
Zhang, H., Li, Z., Dai, C., Wang, P., Fan, S., Yu, B. & Qu, Y. (2021). Antibacterial properties and mechanism of selenium nanoparticles synthesized by Providencia sp. DCX. Environ. Res. 194, 110630. https://doi.org/10.1016/j.envres.2020.110630
Zou, L., Wang, J., Gao, Y., Ren, X., Rottenberg, M.E., Lu, J. & Holmgren, A. (2018). Synergistic antibacterial activity of silver with antibiotics correlating with the upregulation of the ROS production. Sci. Rep. 8, 1–11. https://doi.org/10.1038/s41598-018-29313-w
Bardbari, A.M., Arabestani, M.R., Karami, M., Keramat, F., Aghazadeh, H., Alikhani, M.Y. & Bagheri, K.P. (2018). Highly synergistic activity of melittin with imipenem and colistin in biofilm inhibition against multidrug-resistant strong biofilm producer strains of Acinetobacter baumannii. Eur. J. Clin. Microbial. Infect. Dis. 37, 443–454. https://doi.org/10.1007/s10096-018-3189-7
Bhattacharjee, S. (2016). DLS and zeta potential - What they are and what they are not? J. Control. Release 235, 337–351. https://doi.org/10.1016/j.jconrel.2016.06.017
Chen, W., Li, Y., Yang, S., Yue, L., Jiang, Q. & Xia, W. (2015). Synthesis and antioxidant properties of chitosan and carboxymethyl chitosan-stabilized selenium nanoparticles. Carbohydr. Polym. 132, 574–581. https://doi.org/10.1016/j.carbpol.2015.06.064
Fang, Ferric C. & R.T.S.(2020). Antimicrobial resistance—The glass Is half full. N. Engl. J. Med. 382, 1361–1363. https://doi.org/10.1056/nejme2002121
Geoffrion, L.D., Hesabizadeh, T., Medina-Cruz, D., Kusper, M., Taylor, P., Vernet-Crua, A., Chen, J., Ajo, A., Webster, T.J. & Guisbiers, G., (2020). Naked Selenium Nanoparticles for Antibacterial and Anticancer Treatments. ACS Omega. https://doi.org/10.1021/acsomega.9b03172
Haji, S.H., Ali, F.A., & Aka, S.T.H. (2022). Synergistic antibacterial activity of silver nanoparticles biosynthesized by carbapenem-resistant Gram-negative bacilli. Sci. Rep. 12, 1–13. https://doi.org/10.1038/s41598-022-19698-0
Huang, T., Holden, J.A., Heath, D.E., O’Brien-Simpson, N.M. & O’Connor, A.J. (2019). Engineering highly effective antimicrobial selenium nanoparticles through control of particle size. Nanoscale 11, 14937–14951. https://doi.org/10.1039/c9nr04424h
Huang, X., Chen, X., Chen, Q., Yu, Q., Sun, D. & Liu, J. (2016). Investigation of functional selenium nanoparticles as potent antimicrobial agents against superbugs. Acta Biomater. 30, 397–407. https://doi.org/10.1016/j.actbio.2015.10.041
Jacobsen, S.M., Stickler, D.J., Mobley, H.L.T. & Shirtliff, M.E. (2008). Complicated catheter-associated urinary tract infections due to Escherichia coli and Proteus mirabilis. Clin. Microbial. Rev. 21, 26–59. https://doi.org/10.1128/CMR.00019-07
Javed, R., Sajjad, A., Naz, S., Sajjad, H. & Ao, Q. (2022). Significance of Capping Agents of Colloidal Nanoparticles from the Perspective of Drug and Gene Delivery, Bioimaging, and Biosensing: An Insight. Int. J. Mol. Sci. 23. https://doi.org/10.3390/ijms231810521
Jernigan, J.A., Hatfield, K.M., Wolford, H., Nelson, R.E., Olubajo, B., Reddy, S.C., McCarthy, N., Paul, P., McDonald, L.C., Kallen, A., Fiore, A., Craig, M. & Baggs, J. (2020). Multidrug-Resistant Bacterial Infections in U.S. Hospitalized Patients, 2012–2017. N. Engl. J. Med. 382, 1309–1319. https://doi.org/10.1056/nejmoa1914433
Kaiser, T.D.L., Pereira, E.M., dos Santos, K.R.N., Maciel, E.L.N., Schuenck, R.P. &Nunes, A.P.F.(2013). Modification of the Congo red agar method to detect biofilm production by Staphylococcus epidermidis. Diagn. Microbial. Infect. Dis. 75, 235–239. https://doi.org/10.1016/j.diagmicrobio.2012.11.014
Kamer, A.M.A., El Maghraby, G.M., Shafik, M.M. & Al-Madboly, L.A. (2024). Silver nanoparticle with potential antimicrobial and antibiofilm efficiency against multiple drug resistant, extensive drug resistant Pseudomonas aeruginosa clinical isolates. BMC Microbial. 24, 1–16. https://doi.org/10.1186/s12866-024-03397-z
Karthik, K.K., Cheriyan, B.V., Rajeshkumar, S. & Gopalakrishnan, M. (2024). A review on selenium nanoparticles and their biomedical applications. Biomed. Technol. 6, 61–74. https://doi.org/10.1016/j.bmt.2023.12.001
Lin, W., Zhang, J., Xu, J.F. & Pi, J. (2021). The Advancing of Selenium Nanoparticles Against Infectious Diseases. Front. Pharmacol. 12, 1–16. https://doi.org/10.3389/fphar.2021.682284
Medina, Eva & D.H.P. (2016). " Tackling threats and future problems of multidrug-resistant bacteria. Curr. Top. Microbiol. Immunol. 33. https://doi.org/10.1007/82
Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramírez, J.T. &Yacaman, M.J. (2005). The bactericidal effect of silver nanoparticles. Nanotechnology 16, 2346–2353. https://doi.org/10.1088/0957-4484/16/10/059
Munir, M.U. & Ahmad, M.M. (2022). Nanomaterials Aiming to Tackle Antibiotic-Resistant Bacteria. Pharmaceutics 14, 1–17. https://doi.org/10.3390/pharmaceutics14030582
Pachouri, C., Tripathi, S., Shukla, S. & Pandey, A. (2021). Development and Evaluation of Chitosan Nanoparticles Based Dry Powder Inhalation Formulations of Isoniazid and Its Release Kinetics. Oxid. Commun. 44, 755–768.
Ridha, D.M., AL-Rafyai, H.M. & Najii, N.S. (2021). Bactericidal Potency of Green Synthesized Silver Nanoparticles against Waterborne Escherichia coli Isolates. Nano Biomed. Eng. 13, 372–379. https://doi.org/10.5101/nbe.v13i4.p372-379
Ridha, D.M., AL-Rafyai, H.M. & Saleh, M.A.A.-J.M. (2024). Evaluation of the Antibacterial Impact of the Conjugated Gentamycin-biosynthesized Silver Nanoparticles on Resistant Pseudomonas aeruginosa Isolates In Vitro. Nano Biomed. Eng. 0–10. https://doi.org/10.26599/nbe.2024.9290067
Roy, N., Nivedya, T., Paira, P. & Chakrabarty, R. (2025). Selenium-based nanomaterials: Green and conventional synthesis methods, applications, and advances in dye degradation. RSC Adv. 15, 3008–3025. https://doi.org/10.1039/d4ra07604d
San Keskin, N.O., Akbal Vural, O. & Abaci, S. (2020). Biosynthesis of Noble Selenium Nanoparticles from Lysinibacillus sp. NOSK for Antimicrobial, Antibiofilm Activity, and Biocompatibility. Geomicrobiol. J. 37, 919–928. https://doi.org/10.1080/01490451.2020.1799264
Schaffer, J.N. & Pearson, M.M. (2016). Proteus mirabilis and urinary tract infections. Urin. Tract Infect. Mol. Pathog. Clin. Manag. 383–433. https://doi.org/10.1128/9781555817404.ch17
Sentkowska, A. & Pyrzyńska, K. (2022). The Influence of Synthesis Conditions on the Antioxidant Activity of Selenium Nanoparticles. Molecules 27, 1–14. https://doi.org/10.3390/molecules27082486
Shahmoradi, S., Shariati, A., Zargar, N., Yadegari, Z., Asnaashari, M., Amini, S.M. & Darban-Sarokhalil, D. (2021). Antimicrobial effects of selenium nanoparticles in combination with photodynamic therapy against Enterococcus faecalis biofilm. Photodiagnosis Photodyn. Ther. 35, 102398. https://doi.org/10.1016/j.pdpdt.2021.102398
Shakibaie, M., Forootanfar, H., Golkari, Y., Mohammadi-Khorsand, T. & Shakibaie, M.R. (2015). Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis. J. Trace Elem. Med. Biol. 29, 235–241. https://doi.org/10.1016/j.jtemb.2014.07.020
Sharma, D., Maheshwari, D., Philip, G., Rana, R., Bhatia, S., Singh, M., Gabrani, R., Sharma, S.K., Ali, J., Sharma, R.K. & Dang, S. (2014). Formulation and optimization of polymeric nanoparticles for intranasal delivery of lorazepam using Box-Behnken design: In vitro and in vivo evaluation. Biomed Res. Int. 2014. https://doi.org/10.1155/2014/156010
Tumbarello, M., Trecarichi, E.M., Fiori, B., Losito, A.R., D’Inzeo, T., Campana, L., Ruggeri, A., Di Meco, E., Liberto, E., Fadda, G., Cauda, R. & Spanu, T. (2012). Multidrug-resistant Proteus mirabilis bloodstream infections: Risk factors and outcomes. Antimicrob. Agents Chemother. 56, 3224–3231. https://doi.org/10.1128/AAC.05966-11
Wadhwani, S.A., Gorain, M., Banerjee, P., Shedbalkar, U.U., Singh, R., Kundu, G.C. & Chopade, B.A. (2017). Green synthesis of selenium nanoparticles using Acinetobacter sp. SW30: Optimization, characterization and its anticancer activity in breast cancer cells. Int. J. Nanomedicine 12, 6841–6855. https://doi.org/10.2147/IJN.S139212
33. WHO. (2017). GLASS | Global antimicrobial resistance surveillance system (GLASS) report 2016-17, Who.
Yuan, Q., Xiao, R., Afolabi, M., Bomma, M. & Xiao, Z. (2023). Evaluation of Antibacterial Activity of Selenium Nanoparticles against Food-Borne Pathogens. Microorganisms 11. https://doi.org/10.3390/microorganisms 11061519
Zhang, C., Zhai, X., Zhao, G., Ren, F. & Leng, X. (2015). Synthesis, characterization, and controlled release of selenium nanoparticles stabilized by chitosan of different molecular weights. Carbohydr. Polym. 134, 158–166. https://doi.org/10.1016/j.carbpol.2015.07.065
Zhang, H., Li, Z., Dai, C., Wang, P., Fan, S., Yu, B. & Qu, Y. (2021). Antibacterial properties and mechanism of selenium nanoparticles synthesized by Providencia sp. DCX. Environ. Res. 194, 110630. https://doi.org/10.1016/j.envres.2020.110630
Zou, L., Wang, J., Gao, Y., Ren, X., Rottenberg, M.E., Lu, J. & Holmgren, A. (2018). Synergistic antibacterial activity of silver with antibiotics correlating with the upregulation of the ROS production. Sci. Rep. 8, 1–11. https://doi.org/10.1038/s41598-018-29313-w
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Bioactivity of the amikacin: Selenium nanoparticles stabilized by chitosan (AK: CS-SeNPs) on Proteus mirabilis biofilm. (2025). Journal of Applied and Natural Science, 17(2), 810-820. https://doi.org/10.31018/jans.v17i2.6563
