Original Article

Biofilm formation and eradication of Staphylococcus aureus: a study of culture conditions and endolysin ZAM-CS effect

Abstract

Background and Objectives: Staphylococcus aureus significantly contributes to healthcare-associated infections, with biofilm formation causing chronic, antibiotic-resistant cases. Because biofilms show high resistance to conventional antibiotics, endolysins have emerged as a promising alternative for treating antibiotic-resistant, biofilm-associated infections. This study evaluated the effects of four culture media and different incubation times on biofilm formation in methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains and assessed the anti-biofilm efficacy of a novel chimeric endolysin called ZAM-CS (catalytic domain of SAL-1 endolysin and binding domain of lysostaphin).
Materials and Methods: Biofilms were grown for 24, 48, and 72 hours in Mueller-Hinton broth (MHB), Luria broth (LB), terrific broth (TB), and tryptic soy broth (TSB). The crystal violet assay was used to assess the biomass of the biofilm. The optimal biofilm conditions were then used to test ZAM-CS’s activity at different concentrations.
Results: MSSA formed the strongest biofilms in TB. MRSA formed stable, high-biomass biofilms in TSB, TB, and LB, while MHB was the least supportive medium for both strains. ZAM-CS significantly reduced biofilm biomass in both MSSA and MRSA (up to 77%).
Conclusion: ZAM-CS’s rapid and potent anti-biofilm activity at low concentrations highlights its potential as a promising treatment against antibiotic-resistant S. aureus biofilm infections.

1. Thompson T. The staggering death toll of drug-resistant bacteria. Nature 2022; 10.1038/d41586-022-00228-x.
2. Chinemerem Nwobodo D, Ugwu MC, Oliseloke Anie C, Al-Ouqaili MTS, Chinedu Ikem J, Victor Chigozie U, et al. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J Clin Lab Anal 2022; 36(9): e24655.
3. Cheung GYC, Bae JS, Otto M. Pathogenicity and virulence of Staphylococcus aureus. Virulence 2021; 12: 547-569.
4. Stefani S, Goglio A. Methicillin-resistant Staphylococcus aureus: related infections and antibiotic resistance. Int J Infect Dis 2010; 14 Suppl 4: S19-S22.
5. Kiedrowski MR, Horswill AR. New approaches for treating staphylococcal biofilm infections. Ann N Y Acad Sci 2011; 1241: 104-121.
6. Sauer K, Stoodley P, Goeres DM, Hall-Stoodley L, Burmolle M, Stewart PS, et al. The biofilm life cycle: expanding the conceptual model of biofilm formation. Nat Rev Microbiol 2022; 20: 608-620.
7. Roilides E, Simitsopoulou M, Katragkou A, Walsh TJ. How biofilms evade host defenses. Microbiol Spectr 2015; 3: 10.1128/microbiolspec.MB-0012-2014.
8. Del Pozo JL. Biofilm-related disease. Expert Rev Anti Infect Ther 2018; 16: 51-65.
9. Shree P, Singh CK, Sodhi KK, Surya JN, Singh DK. Biofilms: Understanding the structure and contribution towards bacterial resistance in antibiotics. Med Microecol 2023; 16: 100084.
10. Michaelis C, Grohmann E. Horizontal gene transfer of antibiotic resistance genes in biofilms. Antibiotics (Basel) 2023; 12: 328.
11. Guo Y, Song G, Sun M, Wang J, Wang Y. Prevalence and therapies of antibiotic-resistance in Staphylococcus aureus. Front Cell Infect Microbiol 2020; 10: 107.
12. Murray E, Draper LA, Ross RP, Hill C. The Advantages and challenges of using endolysins in a clinical setting. Viruses 2021; 13: 680.
13. Lusiak-Szelachowska M, Weber-Dabrowska B, Gorski A. Bacteriophages and lysins in biofilm control. Virol Sin 2020; 35: 125-133.
14. Lu Y, Wang Y, Wang J, Zhao Y, Zhong Q, Li G, et al. Phage endolysin LysP108 showed promising antibacterial potential against methicillin-resistant Staphylococcus aureus. Front Cell Infect Microbiol 2021; 11: 668430.
15. Ning H, Lin H, Wang J, He X, Lv X, Ju L. Characterizations of the endolysin Lys84 and its domains from phage qdsa002 with high activities against Staphylococcus aureus and its biofilms. Enzyme Microb Technol 2021; 148: 109809.
16. Momen S, Soleimani N, Azizmohseni F, Ahmadbeigi Y, Borhani S, Amini-Bayat Z. Characterization and bioinformatic analysis of a new chimeric endolysin against MRSA with great stability. AMB Express 2024; 14: 143.
17. Liu H, Hu Z, Li M, Yang Y, Lu S, Rao X. Therapeutic potential of bacteriophage endolysins for infections caused by Gram-positive bacteria. J Biomed Sci 2023; 30: 29.
18. Chen P, Abercrombie JJ, Jeffrey NR, Leung KP. An improved medium for growing Staphylococcus aureus biofilm. J Microbiol Methods 2012; 90: 115-118.
19. Lopes LQS, Guerim P, Santos RCV, Marquezan FK, Marquezan PK. The influence of different culture media on Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus biofilm formation. Biosci J 2023; 39: e39096.
20. Wijesinghe G, Dilhari A, Gayani B, Kottegoda N, Samaranayake L, Weerasekera M. Influence of laboratory culture media on in vitro growth, adhesion, and biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus. Med Princ Pract 2019; 28: 28-35.
21. Stepanovic S, Vukovic D, Hola V, Di Bonaventura G, Djukic S, Cirkovic I, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 2007; 115: 891-899.
22. Tang J, Chen J, Liu J, Zhang R, Yang R, Chen L. Effects of different cultivation conditions on Staphylococcus aureus biofilm formation and diversity of adhesin genes. J Food Saf 2012; 32: 210-218.
23. Lade H, Park JH, Chung SH, Kim IH, Kim JM, Joo HS, et al. Biofilm formation by Staphylococcus aureus clinical isolates is differentially affected by glucose and sodium chloride supplemented culture media. J Clin Med 2019; 8: 1853.
24. Zhang Y, Cheng M, Zhang H, Dai J, Guo Z, Li X, et al. Antibacterial effects of phage Lysin LysGH15 on Planktonic cells and biofilms of diverse Staphylococci. Appl Environ Microbiol 2018; 84(15): e00886-18.
25. Abdurahman MA, Durukan I, Dincer T, Pektas S, Karatas E, Kilic AO. Staphylococcus aureus Bacteriophage 52 endolysin exhibits anti-biofilm and broad antibacterial activity against Gram-positive bacteria. Protein J 2023; 42: 596-606.
26. Arroyo-Moreno S, Begley M, Dembicka K, Coffey A. Engineering of the CHAPk staphylococcal phage endolysin to enhance antibacterial activity against stationary-phase cells. Antibiotics (Basel) 2021; 10: 722.
27. Behera M, Singh G, Vats A, Parmanand, Roshan M, Gautam D, et al. Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus. Int J Biol Macromol 2024; 254: 127969.
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IssueVol 17 No 4 (2025) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v17i4.19247
Keywords
Biofilms Staphylococcus aureus Culture media Anti-bacterial agents Lysostaphin
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How to Cite
1.
Ahmadbeigi Y, Soleimani N, Azizmohseni F, Amini-Bayat Z. Biofilm formation and eradication of Staphylococcus aureus: a study of culture conditions and endolysin ZAM-CS effect. Iran J Microbiol. 2025;17(4):586-592.