IcaA Gene in Environmental Isolates of Biofilm Producing Staphylococcus aureus
DOI:
https://doi.org/10.31674/mjmr.2026.v010i02.003Abstract
Background: Biofilm-forming (BF) Staphylococcus aureus bacteria are a major environmental and health concern due to their role in antibiotic resistance and chronic infections. The presence of biofilm-associated genes, such as the IcaA gene, plays a significant role in biofilm formation and increases its virulence in the environment. Objectives: This study aims to isolate and identify biofilm-producing Staphylococcus aureus from environmental samples, detect the presence of the IcaA gene using polymerase chain reaction, compare different biofilm detection methods, and detect antibiotic resistance and its patterns in the isolates. Methods: One hundred and forty environmental samples were collected from various locations, including soil and wastewater. Staphylococcus aureus was isolated and identified by culturing on different media and then characterized using biochemical methods. Biofilm formation was detected using Congo Red agar and tube-based methods. The IcaA gene was detected molecularly using Polymerase Chain Reaction (PCR). Antibiotic susceptibility testing was performed using disk diffusion. Results: A total of 140 environmental samples, 55 isolates exhibited biofilm-forming potential. Thirteen of these isolates were identified as Staphylococcus aureus. Biofilm detection showed that 76.92% of the isolates were positive using the Congo Red agar method, and 23.07% were positive using the tube method. Polymerase chain reaction (PCR) results indicated that 4 of the 13 isolates (30.76%) carried the IcaA gene. Antibiotic susceptibility testing revealed high resistance to vancomycin, amoxicillin, and oxacillin, while high susceptibility was observed to gentamicin and amikacin. Conclusion: Environmental Staphylococcus aureus isolates exhibited a marked capacity for biofilm formation and antibiotic resistance. The presence of the IcaA gene confirmed the genetic basis for biofilm formation in some isolates. The tube method showed greater agreement with molecular detection compared to the Congo red agar method. Continued monitoring of environmental Staphylococcus aureus and biofilm-associated genes is recommended due to their role in antibiotic resistance and public health risks.
Keywords:
Antibiotic Resistant; , BF Gene;, Congo Red;, S. aureus;, Virulence FactorDownloads
References
Afzal, M., Vijay, A. K., Stapleton, F., & Willcox, M. D. (2021). Susceptibility of ocular Staphylococcus aureus to antibiotics and multipurpose disinfecting solutions. Antibiotics, 10(10), 1203. https://doi.org/10.3390/antibiotics10101203
Ahmad, S., Rahman, H., Qasim, M., Nawab, J., Alzahrani, K. J., Alsharif, K. F., & Alzahrani, F. M. (2022). Staphylococcus epidermidis pathogenesis: Interplay of IcaAdbc operon and mscramms in biofilm formation of isolates from pediatric bacteremia in Peshawar, Pakistan. Medicina, 58(11), 1510. https://doi.org/10.3390/medicina58111510
Ajmal, A. W., Saroosh, S., Mulk, S., Hassan, M. N., Yasmin, H., Jabeen, Z., ... & Mumtaz, S. (2021). Bacteria isolated from wastewater irrigated agricultural soils adapt to heavy metal toxicity while maintaining their plant growth promoting traits. Sustainability, 13(14), 7792. https://doi.org/10.3390/su13147792
Alharbi, O. S., Alhazmi, K. A., Gazzaz, M., Almuhayya, S., Aldehalan, F. A., Sharif, A. T., ... & Ibrahem, K. (2025). A review vancomycin role in Gram Positive biofilm-associated infections: challenges and emerging solutions. Therapeutics and Clinical Risk Management, 1569-1578. https://doi.org/10.2147/TCRM.S541780
Alibegli, M., Bay, A., Fazelnejad, A., Ghezelghaye, P. N., Soghondikolaei, H. J., & Goli, H. R. (2025). Contribution of IcaADBC genes in biofilm production ability of Staphylococcus aureus clinical isolates collected from hospitalized patients at a burn center in North of Iran. BMC Microbiology, 25(1), 302. https://doi.org/10.1186/s12866-025-04018-z
Arciola, C. R., Baldassarri, L., & Montanaro, L. (2001). Presence of IcaA and icaD genes and slime production in a collection of staphylococcal strains from catheter-associated infections. Journal of Clinical Microbiology, 39(6), 2151–2156. https://doi.org/10.1128/jcm.39.6.2151-2156.2001
Awayid, H. S., & Qassim Mohammad, S. (2022). Prevalence and Antibiotic Resistance Pattern of Methicillin-Resistant Staphylococcus aureus Isolated from Iraqi Hospitals. Archives of Razi Institute, 77(3). https://doi.org/10.22092/ARI.2022.357391.2031
Bamneshin, K., Poudineh, M., Alibabaei, R. H., Amiri, M. R. J., Fateminasab, Z. S., Ghorbani, Z., Maleki, R., & Khaledi, A. (2024). Prevalence of IcaADBC genes, and correlation with biofilms and antibiotic resistance in S. aureus: a systematic review and meta-analysis. Germs, 14(4), 387. https://doi.org/10.18683/germs.2024.1448
Cheung, G. Y., & Otto, M. (2023). Virulence mechanisms of staphylococcal animal pathogens. International Journal of Molecular Sciences, 24(19), 14587. https://doi.org/10.3390/ijms241914587
Colaco, H. G., Barros, A., Neves-Costa, A., Seixas, E., Pedroso, D., Velho, T., Willmann, K. L., Faisca, P., Grabmann, G., & Yi, H.-S. (2021). Tetracycline antibiotics induce host-dependent disease tolerance to infection. Immunity, 54(1), 53–67. https://doi.org/10.1016/j.immuni.2020.09.011
Gale, M. S., & Gale, B. D. S. M. (2022). Diagnosis: fundamental principles and methods. Cureus, 14(9). https://doi.org/10.7759/cureus.28730
Girijan, S. K., & Pillai, D. (2021). Identification and characterization of vancomycin-resistant Staphylococcus aureus in hospital wastewaters: evidence of horizontal spread of antimicrobial resistance. Journal of Water and Health, 19(5), 785-795. https://doi.org/10.2166/wh.2021.117
Humphries, R., Bobenchik, A. M., Hindler, J. A., & Schuetz, A. N. (2021). Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing, M100. Journal of Clinical Microbiology, 59(12), 10-1128. https://doi.org/10.1128/jcm.00213-21
Hussein, R. A., Al-Kubaisy, S. H., & Al-Ouqaili, M. T. (2024). The influence of efflux pump, outer membrane permeability and β-lactamase production on the resistance profile of multi, extensively and pandrug resistant Klebsiella pneumoniae. Journal of Infection and Public Health, 17(11), 102544. https://doi.org/10.1016/j.jiph.2024.102544
Kadkhoda, H., Ghalavand, Z., Nikmanesh, B., Kodori, M., Houri, H., Maleki, D. T., Bavandpour, A. K., & Eslami, G. (2020). Characterization of biofilm formation and virulence factors of Staphylococcus aureus isolates from paediatric patients in Tehran, Iran. Iranian Journal of Basic Medical Sciences, 23(5), 691. https://doi.org/10.22038/ijbms.2020.36299.8644
Khan, F., Shukla, I., Rizvi, M., Mansoor, T., & Sharma, S. C. (2011). Detection of biofilm formation in Staphylococcus aureus. Does it have a role in treatment of MRSA infections? Trends in Medical Research, 6(2), 116–123. https://doi.org/10.3923/tmr.2011.116.123
Kim, D. H., Joo, M. S., Jang, S. R., Kim, H. J., Min, J. G., & Nam, B. H. (2025). Impact of Antibiotic Exposure on Growth and Biofilms Formation in Aeromonas salmonicida Subspecies Isolated from Atlantic Salmon (Salmo salar). Microorganisms, 13(12), 2863. https://doi.org/10.3390/microorganisms13122863
Levesque, R. (2005). SPSS programming and data management: a guide for SPSS and SAS users. Spss. https://dl.acm.org/doi/abs/10.5555/1199139
Michaelis, C., & Grohmann, E. (2023). Horizontal gene transfer of antibiotic resistance genes in biofilms. Antibiotics, 12(2), 328. https://doi.org/10.3390/antibiotics12020328
Mohammadi, M., Bahrami, N., Khajavian, M., & Faghri, J. (2020). The Occurrence of Type I, II, and III Integrons in Multi-drug Resistance and Methicillin-Resistant Staphylococcus aureus Isolates in Iran: M. Mohammadi et al. Current Microbiology, 77(8), 1653-1659. https://doi.org/10.1007/s00284-020-01956-x
Mohammed, A. L., & Al-Iedani, A. A. (2020). Molecular detection of ica gene and some surface proteins in biofilm producer of methicillin-resistant and methicillin-sensitive Staphylococcus aureus. Biochemical & Cellular Archives, 20. https://faculty.uobasrah.edu.iq/uploads/publications/1619436104.pdf
Moraes, G. F. Q., Cordeiro, L. V., & de Andrade Júnior, F. P. (2021). Main laboratory methods used for the isolation and identification of Staphylococcus spp. Revista Colombiana de Ciencias Químico-Farmacéuticas, 50(1), 5-28. https://doi.org/10.15446/rcciquifa.v50n1.95444
Nasser, A., Dallal, M. M., Jahanbakhshi, S., Azimi, T., & Nikouei, L. (2022). Staphylococcus aureus: biofilm formation and strategies against it. Current Pharmaceutical Biotechnology, 23(5), 664-678. https://doi.org/10.2174/1389201022666210708171123
Oliveira, A., & Cunha, M. D. L. R. (2010). Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci. BMC Research Notes, 3(1), 260. https://doi.org/10.1186/1756-0500-3-260
Omidi, A. H., Sabati, H., Amini, S., Zonobian, M. A., & Mohammadi, M. R. (2021). Staphylococcus aureus in the environment of healthcare centers. Cellular, Molecular and Biomedical Reports, 1(4), 147-157. https://doi.org/10.55705/cmbr.2021.403541.1156
Paul, A. J., Chandra, M., Kaur, G., Narang, D., & Gupta, D. K. (2026). Isolation and Characterization of Staphylococcus aureus and Its Biofilm Implicated in Bovine Mastitis. Indian Journal of Microbiology, 1–13. https://doi.org/10.1007/s12088-026-01542-y
Peng, Q., Tang, X., Dong, W., Sun, N., & Yuan, W. (2022). A review of biofilm formation of Staphylococcus aureus and its regulation mechanism. Antibiotics, 12(1), 12. https://doi.org/10.3390/antibiotics12010012
Pugazhendhi, A. S., Wei, F., Hughes, M., & Coathup, M. (2022). Bacterial adhesion, virulence, and biofilm formation. In Musculoskeletal Infection (pp. 19-64). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-030-83251-3_2
Sandman, Z., & Iqbal, O. A. (2024). Azithromycin. StatPearls [Internet].
Sauer, K., Stoodley, P., Goeres, D. M., Hall-Stoodley, L., Burmølle, M., Stewart, P. S., & Bjarnsholt, T. (2022). The biofilm life cycle: expanding the conceptual model of biofilm formation. Nature Reviews Microbiology, 20(10), 608-620. https://doi.org/10.1038/s41579-022-00767-0
Sharan, M., Dhaka, P., Bedi, J. S., Mehta, N., & Singh, R. (2024). Assessment of biofilm-forming capacity and multidrug resistance in Staphylococcus aureus isolates from animal-source foods: implications for lactic acid bacteria intervention. Annals of Microbiology, 74(1), 22. https://doi.org/10.1186/s13213-024-01768-5
Taj, Y., Essa, F., Aziz, F., & Kazmi, S. U. (2012). Study on biofilm-forming properties of clinical isolates of Staphylococcus aureus. The Journal of Infection in Developing Countries, 6(05), 403–409. https://doi.org/10.3855/jidc.1743
Tang, K., & Zhao, H. (2023). Quinolone antibiotics: resistance and therapy. Infection and Drug Resistance, 811–820. https://doi.org/10.2147/IDR.S401663
Thy, M., Timsit, J. F., & de Montmollin, E. (2023). Aminoglycosides for the treatment of severe infection due to resistant gram-negative pathogens. Antibiotics, 12(5), 860. https://doi.org/10.3390/antibiotics12050860
Wang, S., Zhao, Y., Breslawec, A. P., Liang, T., Deng, Z., Kuperman, L. L., & Yu, Q. (2023). Strategy to combat biofilms: a focus on biofilm dispersal enzymes. NPJ Biofilms and Microbiomes, 9(1), 63. https://doi.org/10.1038/s41522-023-00427-y
Weinstein, M. P., & Lewis, J. S. (2020). The clinical and laboratory standards institute subcommittee on antimicrobial susceptibility testing: background, organization, functions, and processes. Journal of Clinical Microbiology, 58(3), 10-1128. https://journals.asm.org/doi/full/10.1128/jcm.01864-19
Zhang, L., Tian, X., Sun, L., Mi, K., Wang, R., Gong, F., & Huang, L. (2024). Bacterial efflux pump inhibitors reduce antibiotic resistance. Pharmaceutics, 16(2), 170. https://doi.org/10.3390/pharmaceutics16020170
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Malaysian Journal of Medical Research (MJMR)
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
