• Sally Abou-Khadra Microbiology Department, Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Post code 44628, Sharkia Province, Egypt
  • Amina El-Amin Food Hygiene Department, Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt
  • Saad Al-Otaibi Biotechnology Department, faculty of science, Taif University, KSA
  • Hanan Fahmy Biotechnology Department, Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt



biofilm, antibiofilm, Staphylococci, milk, AgNPs


Biofilm-producing ability has been identified as a serious virulence factor in staphylococci and increases their antimicrobial resistance. This study aimed to investigate the biofilm forming ability of staphylococci isolated from cow milk samples. Moreover, we assessed the antibiofilm activity of silver nanoparticles (AgNPs) against methicillin resistant (MRSA) and biofilm forming staphylococci. The results revealed that 82.14% (23/28) and 91.66% (11/12) of the coagulase positive staphylococci (CPS) and coagulase negative Staphylococci (CNS) isolates, respectively produced biofilm on Congo red agar (CRA). In the case of the microtiter plates (MTP) method, all CPS and CNS isolates produced biofilm at different levels. These results revealed a substantial agreement between CRA and MTP results according to the kappa coefficient test (kappa value = 0.773).  Staphylococcus aureus species specific nuc gene and the determinant of methicillin resistance (mecA) gene were amplified from S. aureus isolates (n=10). The intercellular adhesion gene A (icaA), and intercellular adhesion gene D (icaD) were amplified from all the CPS and CNS isolates, but none of the isolates were positive for the biofilm associated protein (bap) gene. Antibiotic susceptibility testing showed that all isolates were oxacillin resistant. AgNPs (100 μg/mL) inhibited the growth of Staphylococci isolates (inhibition zone diameters ranged from 22 to 28 mm). AgNPs decreased biofilm formation in the biofilm forming Staphylococci isolates with percent ranged from 67.05 to 98.02% using MTP assay. In conclusion, AgNPs have antistaphylococcal activity and inhibit biofilm formation.


● 1. Bardiau M, Caplin J, Detilleux J, et al. Existence of two groups of Staphylococcus aureus strains isolated from bovine mastitis based on biofilm formation, intracellular survival, capsular profile and agr-typing.Vet Microbiol 2016; 185: 1–6.

● 2. Tel O, Aslantaş Ö, Keskin O, et al. Investigation of the antibiotic resistance and biofilm formation of Staphylococcus aureus strains isolated from gangrenous mastitis of ewes. Acta Vet Hung 2012; 60: 189-–7.

● 3. Oliveira M, Bexiga R, Nunes S, et al. Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet Microbiol 2006; 118: 133–40.

● 4. Zadoks R, Van Leeuwen W, Kreft D, et al. Comparison of Staphylococcus aureus isolates from bovine and human skin, milking equipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. J Clin microbiol 2002; 40: 3894–902.

● 5. Monzón M, Oteiza C, Leiva J, et al. Synergy of different antibiotic combinations in biofilms of Staphylococcus epidermidis. J Antimicrob Chemother 2001; 48: 793–801.

● 6. Mah T-F C and O'Toole G A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 2001; 9: 34-39.

● 7. Borriello G, Werner E, Roe F, et al. Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother 2004; 48(7): 2659–64.

● 8. Lewis K “Persister cells". Annu Rev Microbiol 2010; 64: 357–72.

● 9. Liberto M C, Matera G, Quirino A, et al. Pheno-typic and genotypic evaluation of slime production by conventional and molecular microbiological techni-ques. Microbiol Res 2009; 164: 522–8.

● 10. Darwish S F and Asfour H A. Investigation of biofilm forming ability in Staphylococci causing bovine mastitis using phenotypic and genotypic assays. Sci World J 2013; (2013) 9 pages (Article ID 378492,).

● 11. Cucarella C, Solano C, Valle J, et al. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 2001; 183: 2888–96.

● 12. Freeman D, Falkiner F, and Keane C. New method for detecting slime production by coagulase negative staphylococci. J clin pathol 1989; 42: 872–4.

● 13. Mathur T, Singhal S, Khan S, et al. Detection of biofilm formation among the clinical isolates of staphylococci: an evaluation of three different screen-ing methods. Indian J Med Microbiol 2006; 24: 25–9.

● 14. Oliveira A and Maria de Lourdes R Comparison of methods for the detection of biofilm production in coagulase-negative staphylococci. BMC Res Notes 2010; 3: 260.

● 15. Melo P d C, Ferreira L M, Nader Filho A, et al. Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Braz J Microbiol 2013; 44: 119–24.

● 16. Abd El-Aziz N K, Ammar A M, El-Naenaeey E-s Y, et al. Antimicrobial and antibiofilm potentials of cinnamon oil and silver nanoparticles against Streptococcus agalactiae isolated from bovine mastitis: New avenues for countering resistance. BMC Vet Res 2021; 17: 1–14.

● 17. Dakal T C, Kumar A, Majumdar R S, et al. Mechanistic basis of antimicrobial actions of silver nanoparticles. Front Microbiol 2016; 7: 1831.

● 18. Singh N, Rajwade J, and Paknikar K. Transcrip-tome analysis of silver nanoparticles treated Staphylococcus aureus reveals potential targets for biofilm inhibition. Colloids Surf B: Biointerfaces 2019; 175: 487–97.

● 19. Sondi I and Salopek-Sondi B. Silver nanoparti-cles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J Colloid Interface Sci 2004; 275(1): 177–82.

● 20. Bergey D H and Holt J G. Bergey’s Manual of Determinative Bacteriology. Baltimore, Maryland, 1994:350.

● 21. MacFaddin J F. Biochemical tests for identifi-cation of medical bacteria. 2000.

● 22. Egyptian organization for Standardization a, Quality, Control (EOSQC), Egyptian Standards 1008/2005, 2005.

● 23. Stepanović S, Vuković D, Dakić I, et al. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000; 40: 175–9.

● 24. Bauer A. Antibiotic susceptibility testing by a standardized single disc method. Am J clin pathol 1966; 45: 149–8.

● 25. Clinical and Institute L S. Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute CLSI supplement M100, Wayne, PA, 2017.

● 26. Liu Y, Liu C, Zheng W, et al. PCR detection of Klebsiella pneumoniae in infant formula based on 16S–23S internal transcribed spacer. Int J Food Microbiol 2008;125: 230–5.

● 27. Mason W J, Blevins J S, Beenken K, et al. Multiplex PCR protocol for the diagnosis of staphylococcal infection. J Clin Microbiol 2001; 39: 3332–8.

● 28. Brakstad O G, Aasbakk K, and Maeland J A. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol 1992; 30: 1654–60.

● 29. Martín-López J V, Díez-Gil O, Morales M, et al. Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus. Int Microbiol 2004; 7: 63–6.

● 30. Ciftci A, Findik A, Onuk E E, et al. Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis. Braz J Microbiol 2009; 40: 254–61.

● 31. Anderson J C. Veterinary aspects of staphylococci. In: Easman, C.S.F., Adlam, C. (Eds.), Staphylococci and Staphylococcal Infections. Academic Press, Great Britain,1983:196–219.

● 32. Ebrahimi A, Jafferi H, Habibian S, et al. Evaluation of anti biofilm and antibiotic potentiation activities of silver nanoparticles against some nosocomial pathogens. Iran J Pharm Sci 2018; 14: 7–14.

● 33. Divya K, Vijayan S, George T K, et al. Antimicrobial properties of chitosan nanoparticles: Mode of action and factors affecting activity. Fibers Polym 2017; 18: 221–30.

● 34. Landis J R and Koch G G. The measurement of observer agreement for categorical data. biometrics 1977; 159–74.

● 35. Ilstrup D M. Statistical methods in microbiology. Clin Microbiol Rev 1990; 3: 219–26.

● 36. Hosseinzadeh S and Dastmalchi Saei H. Staphylococcal species associated with bovine mastitis in the North West of Iran: emerging of coagulase-negative staphylococci. int. J Vet Sci Med 2014; 2: 27–34.

● 37. Mahmoud A K A, Khadr A M, Elshemy T M, et al. Role of Coagulase Positive and Coagulase Negative Staphylococci in Bovine Mastitis with Special Reference to Some of Their Virulence Genes and Antimicrobial Sensitivity. Alex J Vet Sci 2015; 46(1).

● 38. Seddek S. Bovine Mastitis (Age, Causes and Control) in Assiut Governorate. Assiut Vet Med J 1996;36: 149–62.

● 39. Nickerson S, Owens W, and Boddie R. Mastitis in dairy heifers: initial studies on prevalence and control. J Dairy Sci 1995; 78(7): 1607–18.

● 40. Zeinhom M and Abed A. Prevalence, Characterization, and Control of Staphylococcus aureus Isolated from Raw Milk and Egyptian Soft Cheese. J Vet Med Res 2021; 27(2): 152–60.

● 41. Sharma N, Singh N, and Bhadwal M. Relationship of somatic cell count and mastitis: An overview. Asian-australas J Anim Sci 2011; 24: 429–38.

● 42. Kandil A, Elhadidy M, El-Gamal A, et al. Identification of S. aureus and E. coli from dairy products intended for human con-sumption. Adv Anim Vet Sci 2018; 6: 509–13.

● 43. El-Aziz A, Norhan K, Ammar A M, et al. Environmental Streptococcus uberis associated with clinical mastitis in dairy cows: virulence traits, antimicrobial and biocide resistance, and epidemiological typing. Animals 2021; 11: 1849.

● 44. Bose S, Khodke M, Basak S, et al. Detection of biofilm producing staphylococci: need of the hour. J Clin Diagn Res 2009; 3: 1915–20.

● 45. Gharieb R M A, Saad M F, Mohamed A S, et al. Characterization of two novel lytic bacteriophages for reducing biofilms of zoonotic multidrug-resistant Staphylococcus aureus and controlling their growth in milk. LWT Food Sci Technol 2020; 124: 109145.

● 46. Wald R, Hess C, Urbantke V, et al. Characte-rization of Staphylococcus species isolated from bovine quarter milk samples. Animals 2019;9: 200.

● 47. Moon J-S, Lee A-R, Kang H-M, et al. Phenotypic and genetic antibiogram of methicillin-resistant staphylococci isolated from bovine mastitis in Korea. J Dairy Sci 2007; 90: 1176–85.

● 48. Taponen S and Pyörälä S Coagulase-negative staphylococci as cause of bovine mastitis—Not so different from Staphylococcus aureus? Vet Microbiol 2009; 134: 29–36.

● 49. Dhakal I Economic impact of clinical mastitis in the buffaloes in Nepal. Buffalo J 2002; 2: 225–34.

● 50. Ahmad A A, Ammar A M, Bendary M, et al. Phenogenotyping of closely related Methicillin resistant Staphylococcus aureus isolated from milk and meat products. Zagazig Vet J2017; 45: 394–3.

● 51. Abd El-Aziz N K, Abd El-Hamid M I, Bendary M M, et al. Existence of vancomycin resistance among methicillin resistant S aurues recovered from animal and human sources in Egypt. Slov Vet Res 2018; 55: 221–30.

● 52. Tartor YH, El-Naenaeey EY. RT-PCR detec-tion of exotoxin genes expression in multidrug resistant Pseudomonas aeruginosa. Cell Mol Biol (Noisy-le-grand). 2016;62:56–62.

● 53. Emam A, El-Diasty M, and Abdelkhalek A Prevalence of Staphyloсoссus aureus and Streptococcus agalaсtiae isolated from Raw Milk in Dakahlia Governorate, Egypt. Zagazig Vet J 2021; 49: 67–77.

● 54. Vasudevan P, Nair M K M, Annamalai T, et al. Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet microbiol 2003;92: 179–85.

● 55. Simojoki H, Hyvönen P, Ferrer C P, et al. Is the biofilm formation and slime producing ability of coagulase-negative staphylococci associated with the persistence and severity of intramammary infection? Vet Microbiol 2012; 158: 344–52.

● 56 Arciola C R, Campoccia D, Baldassarri L, et al. Detection of biofilm formation in Staphylococcus epidermidis from implant infections. Comparison of a PCR‐method that recognizes the presence of ica genes with two classic phenotypic methods. J Biomed Mater Res A: An Official Journal of The Society for Biomate-rials, The Japanese Society for Biomaterials, and The Australian Society for Biomaterials and the Korean Society for Biomaterials 2006;76: 425–30.

● 57. Mohanty S, Mishra S, Jena P, et al. An investigation on the antibacterial, cytotoxic, and antibiofilm efficacy of starch-stabilized silver nanoparticles. Nanomedicine: NBM 2012;8(6): 916–24.

● 58. Kalishwaralal K, BarathManiKanth S, Pandian S R K, et al. Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis. Colloids Surf B: Biointerfaces 2010; 79: 340–4.




How to Cite

Abou-Khadra, S., El-Amin, A., Al-Otaibi, S., & Fahmy, H. (2021). THE ANTIBACTERIAL AND ANTIBIOFILM ACTIVITIES OF SILVER NANOPARTICLES ON STAPHYLOCOCCI ISOLATES FROM COW MILK. Slovenian Veterinary Research, 58(24-Suppl), 133–42.



Original Research Article