Green synthesis of Iron nanoparticles from Pseudomonas aeruginosa isolated from soil and their antibacterial and antibiofilm activity against pathogenic bacteria
DOI:
https://doi.org/10.31210/spi2026.29.01.38Keywords:
Pseudomonas aeruginosa, Iron nanoparticles, Green synthesis, antibiofilm, antibacterial activityAbstract
Fifty-four soil samples were collected from various sites (agricultural, oil-polluted, riverbank, and animal dung-contaminated soils) in northern Basrah, southern Iraq, between September 18 and November 28, 2022. The specimens were transported to the laboratory within 3 hours, serially diluted, and cultivated on selective media. The screening indicated that 28 samples (51.9 %) exhibited positive bacterial growth. Phenotypic and molecular identification via PCR amplification of the 16S rRNA gene (~1400 bp) revealed the presence of Pseudomonas aeruginosa (8 isolates), Escherichia coli (7 isolates), Klebsiella pneumoniae (4 isolates), Staphylococcus aureus (4 isolates), Bacillus cereus (3 isolates), and Bacillus subtilis (2 isolates). Among these, a dominant P. aeruginosa isolate was successfully utilized for the green biosynthesis of iron nanoparticles (FeNPs). The successful formation of biogenic FeNPs was initially indicated by a rapid color transition from yellow to dark brown. UV-Vis spectroscopy confirmed nanoparticle synthesis with a sharp surface plasmon resonance peak at 304 nm, while FT-IR spectroscopy verified the definitive metal-oxygen (Fe–O) stretching bond at 589.97 cm⁻¹ along with capping bacterial functional groups. Scanning electron microscopy (SEM) demonstrated a predominantly spherical nanoparticle morphology with a well-defined representative particle size of 22.83 nm. The quantitative microtiter plate and Congo red agar assays confirmed that the pathogenic isolates possessed a robust baseline capacity for biofilm formation. The biosynthesized FeNPs exhibited exceptional, concentration-dependent antibiofilm efficacy, achieving characteristically high biofilm inhibition rates of up to 91.64 % at concentrations of 50 and 100 µg/mL. Furthermore, the nanoparticles displayed potent antibacterial activity via the agar well diffusion method, with inhibition zones expanding proportionally with concentration and reaching a maximum diameter of 21.0 mm against P. aeruginosa. These collective findings suggest that biosynthesized iron nanoparticles could serve as highly effective, biocompatible, and environmentally friendly nano-antimicrobials for mitigating multidrug-resistant pathogens and persistent biofilm-associated infections.
References
1. Jin, S. (2024). Pseudomonas aeruginosa. In Y.-W. Tang, M. Sussman, D. Liu, I. Poxton, & J. Schwartzman (Eds.), Molecular medical microbiology (3rd ed., pp. 811–825). Academic Press. https://doi.org/10.1016/B978-0-12-818619-0.00064-2
2. Kuraimsh, K. K. (2025). Prevalence of resistance to antimicrobial agents by Pseudomonas aeruginosa and Acinetobacter baumannii isolated from Iraqi patients with burns at Al-Nasiriya Hospital. Journal of the Faculty of Medicine Baghdad, 67 (4), 447–456. https://doi.org/10.32007/jfacmedbaghdad3204
3. Khan, F., Jeong, G.-J., Singh, P., Tabassum, N., Mijakovic, I., & Kim, Y.-M. (2022). Retrospective analysis of the key molecules involved in the green synthesis of nanoparticles. Nanoscale, 14 (40), 14824–14857. https://doi.org/10.1039/d2nr03632k
4. Dutt, Y., Pandey, R. P., Dutt, M., Gupta, A., Vibhuti, A., Vidic, J., Raj, V. S., Chang, C.-M., & Priyadarshini, A. (2023). Therapeutic applications of nanobiotechnology. Journal of Nanobiotechnology, 21 (1). https://doi.org/10.1186/s12951-023-01909-z
5. Tabassum, N., Khan, F., Kang, M.-G., Jo, D.-M., Cho, K.-J., & Kim, Y.-M. (2023). Inhibition of polymicrobial biofilms of Candida albicans – Staphylococcus aureus/Streptococcus mutans by fucoidan–gold nanoparticles. Marine Drugs, 21 (2), 123. https://doi.org/10.3390/md21020123
6. Singh, P., Pandit, S., Beshay, M., Mokkapati, V. R. S. S., Garnaes, J., Olsson, M. E., Sultan, A., Mackevica, A., Mateiu, R. V., Lütken, H., Daugaard, A. E., Baun, A., & Mijakovic, I. (2018). Anti-biofilm effects of gold and silver nanoparticles synthesized by the Rhodiola rosea rhizome extracts. Artificial Cells, Nanomedicine, and Biotechnology, 46 (sup3), 886–899. https://doi.org/10.1080/21691401.2018.1518909
7. Khan, A. A., Khan, S., Khan, S., Rentschler, S., Laufer, S., & Deigner, H.-P. (2021). Biosynthesis of iron oxide magnetic nanoparticles using clinically isolated Pseudomonas aeruginosa. Scientific Reports, 11 (1). https://doi.org/10.1038/s41598-021-99814-8
8. Asoso, O. S., Idris, O. O., Ayodele, O. S., & Laoye, B. J. (2022). Isolation and identification of bacteria and fungi associated with tomatoes. Medical & Clinical Research, 7 (6), 1–11. https://doi.org/10.33140/mcr.07.06.02
9. Sambrook, J., & Russell, D. W. (2006). Purification of nucleic acids by extraction with phenol: Chloroform. Cold Spring Harbor Protocols, 2006 (1), pdb. https://doi.org/10.1101/pdb.prot4455
10. Leszczewicz, M., Broncel, N., Frączak, O., Kapela, T., & Makowski, K. (2025). Screening of moderately halophilic bacteria producing ectoine resulting in selection of Virgibacillus salarius BHTA19. Food Technology and Biotechnology, 63 (3), 310. https://doi.org/10.17113/ftb.63.03.25.8727
11. Shenoy, V., Harika, K., Narasimhaswamy, N., & Chawla, K. (2020). Detection of biofilm production and its impact on antibiotic resistance profile of bacterial isolates from chronic wound infections. Journal of Global Infectious Diseases, 12 (3), 129–134. https://doi.org/10.4103/jgid.jgid_150_19
12. Almeida, C., Azevedo, N. F., Santos, S., Keevil, C. W., & Vieira, M. J. (2011). Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS ONE, 6 (3), e14786. https://doi.org/10.1371/journal.pone.0014786
13. Tang, J., Kang, M., Chen, H., Shi, X., Zhou, R., Chen, J., & Du, Y. (2011). The staphylococcal nuclease prevents biofilm formation in Staphylococcus aureus and other biofilm-forming bacteria. Science China Life Sciences, 54 (9), 863–869. https://doi.org/10.1007/s11427-011-4195-5
14. Kamnev, A. A., Dyatlova, Y. A., Kenzhegulov, O. A., Vladimirova, A. A., Mamchenkova, P. V., & Tugarova, A. V. (2021). Fourier Transform Infrared (FTIR) spectroscopic analyses of microbiological samples and biogenic selenium nanoparticles of microbial origin: Sample preparation effects. Molecules, 26 (4), 1146. https://doi.org/10.3390/molecules26041146
15. Ashrafi-Saiedlou, S., Rasouli-Sadaghiani, M., Fattahi, M., & Ghosta, Y. (2025). Biosynthesis and characterization of iron oxide nanoparticles fabricated using cell-free supernatant of Pseudomonas fluorescens for antibacterial, antifungal, antioxidant, and photocatalytic applications. Scientific Reports, 15 (1). https://doi.org/10.1038/s41598-024-84974-0
16. Mathur, T., Singhal, S., Khan, S., Upadhyay, D., Fatma, T., & Rattan, A. (2006). Detection of biofilm formation among the clinical isolates of Staphylococci: An evaluation of three different screening methods. Indian Journal of Medical Microbiology, 24 (1), 25. https://doi.org/10.4103/0255-0857.19890
17. Majumdar, M., Khan, S. A., Nandi, N. B., Roy, S., Panja, A. S., Roy, D. N., & Misra, T. K. (2020). Green synthesis of iron nanoparticles for investigation of biofilm inhibition property. ChemistrySelect, 5 (43), 13575–13583. https://doi.org/10.1002/slct.202003033
18. Kamble, S., Bhosale, K., Mohite, M., & Navale, S. (2023). Methods of preparation of nanoparticles. International Journal of Advanced Research in Science, Communication and Technology, 3 (7), 121–126. https://doi.org/10.48175/IJARSCT-9485
19. Huseen, R., Taha, A., & Abdulhusein, O. (2021). Study of biological activities of magnetic iron oxide nanoparticles prepared by co-precipitation method. Journal of Applied Sciences and Nanotechnology, 1 (2), 37–48. https://doi.org/10.53293/jasn.2021.11635
20. Aghamollaei, H., Moghaddam, M. M., Kooshki, H., Heiat, M., Mirnejad, R., & Barzi, N. S. (2015). Detection of Pseudomonas aeruginosa by a triplex polymerase chain reaction assay based on lasI/R and gyrB genes. Journal of Infection and Public Health, 8 (4), 314–322. https://doi.org/10.1016/j.jiph.2015.03.003
21. Kridi, N., Al-Shater, M. S., & Al Zoubi, M. M. (2021). Isolation and identification of some bacterial isolates from soil contaminated with crude oil and Testing Their Effectiveness. Baghdad Science Journal, 18 (4). https://doi.org/10.21123/bsj.2021.18.4(suppl.).1476
22. Ali, A. A., Hameed, K. W., & Nadder, M. I. (2022). Isolation of Pseudomonas aeruginosa from soil and production of lipase enzyme. IOP Conference Series: Earth and Environmental Science, 961 (1), 012087. https://doi.org/10.1088/1755-1315/961/1/012087
23. Podschun, R., & Ullmann, U. (1998). Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clinical Microbiology Reviews, 11 (4), 589–603. https://doi.org/10.1128/cmr.11.4.589
24. Wyres, K. L., & Holt, K. E. (2016). Klebsiella pneumoniae population genomics and antimicrobial-resistant clones. Trends in Microbiology, 24 (12), 944–956. https://doi.org/10.1016/j.tim.2016.09.007
25. Yang, F., Deng, B., Liao, W., Wang, P., Chen, P., & Wei, J. (2019). High rate of multiresistant Klebsiella pneumoniae from human and animal origin. Infection and Drug Resistance, 12, 2729–2737. https://doi.org/10.2147/idr.s219155
26. Chi, X., Berglund, B., Zou, H., Zheng, B., Börjesson, S., Ji, X., Ottoson, J., Lundborg, C. S., Li, X., & Nilsson, L. E. (2019). Characterization of clinically relevant strains of extended-spectrum β-lactamase-producing Klebsiella pneumoniae occurring in environmental sources in a rural area of China by using whole-genome sequencing. Frontiers in Microbiology, 10, 211. https://doi.org/10.3389/fmicb.2019.00211
27. Kämpfer, P., & Glaeser, S. P. (2013). Prokaryote characterization and identification. In E. Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt, & F. Thompson (Eds.), The Prokaryotes: Prokaryotic biology and symbiotic associations (4th ed., pp. 123–147). Springer. https://doi.org/10.1007/978-3-642-30194-0_6
28. Rocha, J., Henriques, I., Gomila, M., & Manaia, C. M. (2022). Common and distinctive genomic features of Klebsiella pneumoniae thriving in the natural environment or in clinical settings. Scientific Reports, 12 (1). https://doi.org/10.1038/s41598-022-14547-6
29. Verheijen, F., Jelinčić, A., Jeffery, S., Domingos, T., Khodaparast, Z., & Bastos, A. C. (2025). Correction: Escherichia coli thrives in soil 24 months after grazing exclusion in a rainfed Mediterranean biodiverse pasture. Frontiers in Environmental Science, 13. https://doi.org/10.3389/fenvs.2025.1708183
30. Murphy, C. M., Weller, D. L., Bardsley, C. A., Ingram, D. T., Chen, Y., Oryang, D., Rideout, S. L., & Strawn, L. K. (2024). Survival of twelve pathogenic and generic Escherichia coli strains in agricultural soils as influenced by strain, soil type, irrigation regimen, and soil amendment. Journal of Food Protection, 87 (10), 100343. https://doi.org/10.1016/j.jfp.2024.100343
31. Gerken, T., Wiegner, T. N., & Economy, L. M. (2022). A comparison of soil Staphylococcus aureus and fecal indicator bacteria concentrations across land uses in a Hawaiian watershed. Journal of Environmental Quality, 51 (5), 916–929. https://doi.org/10.1002/jeq2.20380
32. Mahapatra, S., Yadav, R., & Ramakrishna, W. (2022). Bacillus subtilis impact on plant growth, soil health and environment: Dr. Jekyll and Mr. Hyde. Journal of Applied Microbiology, 132 (5), 3543–3562. https://doi.org/10.1111/jam.15480
33. Foysal, M. J., & Lisa, A. K. (2018). Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria. Journal of Genetic Engineering and Biotechnology, 16 (2), 387–392. https://doi.org/10.1016/j.jgeb.2018.01.005
34. Al-Mojamaee, N. A. H., & Altaii, H. A. J. (2023). Comparison of two methods for the detection of Pseudomonas aeruginosa biofilm formation isolated from different clinical samples. Iraqi Journal of Humanities and Social Science Research, 11 (3), 651–668.
35. Basavaraju, M., & Gunashree, B. S. (2022). Escherichia coli: An overview of main characteristics. In M. S. Erjavec (Ed.), Escherichia coli: Old and new insights (pp. 1–18). IntechOpen. https://doi.org/10.5772/intechopen.105508
36. Abbas, R., Chakkour, M., Zein El Dine, H., Obaseki, E. F., Obeid, S. T., Jezzini, A., Ghssein, G., & Ezzeddine, Z. (2024). General overview of Klebsiella pneumoniae: Epidemiology and the role of siderophores in its pathogenicity. Biology, 13 (2), 78. https://doi.org/10.3390/biology13020078
37. Todorova, S., & Kozhuharova, L. (2009). Characteristics and antimicrobial activity of Bacillus subtilis strains isolated from soil. World Journal of Microbiology and Biotechnology, 26 (7), 1207–1216. https://doi.org/10.1007/s11274-009-0290-1
38. Stecchini, M. L., Spaziani, M., Torre, M. D., & Pacor, S. (2009). Bacillus cereus cell and spore properties as influenced by the micro-structure of the medium. Journal of Applied Microbiology, 106 (6), 1838–1848. https://doi.org/10.1111/j.1365-2672.2009.04162.x
39. Silva-Santana, G., Cabral-Oliveira, G. G., Oliveira, D. R., Nogueira, B. A., Pereira-Ribeiro, P. M. A., & Mattos-Guaraldi, A. L. (2020). Staphylococcus aureus biofilms: an opportunistic pathogen with multidrug resistance. Reviews in Medical Microbiology, 32 (1), 12–21. https://doi.org/10.1097/mrm.0000000000000223
40. Hajiali, S., Daneshjou, S., & Daneshjoo, S. (2022). Biomimetic synthesis of iron oxide nanoparticles from Bacillus megaterium to be used in hyperthermia therapy. AMB Express, 12 (1). https://doi.org/10.1186/s13568-022-01490-y
41. Qurban, A., & Ameen, A. (2020). Bacterial identification by 16S ribotyping: A review. Biotechnology: An Indian Journal, 16 (2), 204.
42. Prashanthi, R., Shreevatsa, G. K., Krupalini, S., & Manoj, L. (2021). Isolation, characterization, and molecular identification of soil bacteria showing antibacterial activity against human pathogenic bacteria. Journal of Genetic Engineering and Biotechnology, 19 (1), 120. https://doi.org/10.1186/s43141-021-00219-x
43. Al-Dhabaan, F. A. (2019). Morphological, biochemical and molecular identification of petroleum hydrocarbons biodegradation bacteria isolated from oil polluted soil in Dhahran, Saud Arabia. Saudi Journal of Biological Sciences, 26 (6), 1247–1252. https://doi.org/10.1016/j.sjbs.2018.05.029
44. Singh, B., Dahiya, M., Kumar, V., Ayyagari, A., Chaudhari, D. N., & Ahire, J. J. (2025). Biofilm and antimicrobial resistance: Mechanisms, implications, and emerging solutions. Microbiology Research, 16 (8), 183. https://doi.org/10.3390/microbiolres16080183
45. Shkodenko, L., Kassirov, I., & Koshel, E. (2020). Metal oxide nanoparticles against bacterial biofilms: Perspectives and limitations. Microorganisms, 8 (10), 1545. https://doi.org/10.3390/microorganisms8101545
46. Dawan, J., & Ahn, J. (2022). Bacterial stress responses as potential targets in overcoming antibiotic resistance. Microorganisms, 10 (7), 1385. https://doi.org/10.3390/microorganisms10071385
47. Carnero Canales, C. S., Marquez Cazorla, J. I., Marquez Cazorla, R. M., Sábio, R. M., Santos, H. A., & Pavan, F. R. (2025). Combating Gram-negative infections: The role of antimicrobial peptides and nanotechnology in overcoming antibiotic resistance. Materials Today Bio, 35, 102381. https://doi.org/10.1016/j.mtbio.2025.102381
48. Zhang, T.-G., & Miao, C.-Y. (2024). Iron oxide nanoparticles as promising antibacterial agents of new generation. Nanomaterials, 14 (15), 1311. https://doi.org/10.3390/nano14151311
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 І. Дж. Аббасс, Е. А. Аль-Імара, Д. А. Альхасан
This work is licensed under a Creative Commons Attribution 4.0 International License.
