Anti-biofilm potential of Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatant extracts against Staphylococcus aureus


1 Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran

2 Molecular Microbiology Research Center, Faculty of Medicine, Shahed University, Tehran, Iran

3 Department of Microbiology, Faculty of Medicine; Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran


Background: Biofilm production is an important virulence factor in Staphylococcus aureus. Most of the infections associated with biofilms of this bacterium are very difficult to treat using antibiotics. The present research studied the effects of the two probiotic Lactobacillus species L. casei and L. rhamnosus on S. aureus biofilm.
Materials and Methods: Cell-free supernatant (CFS) extracts of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 culture were prepared. The effects of sub-minimum inhibitory concentrations of the CFS extracts on cell surface hydrophobicity (CSH), initial attachment, biofilm formation, and their ability in eradicating S. aureus ATCC 33591 biofilms were assessed. In addition, the effects of CFS extracts on expression of the genes involved in formation of S. aureus biofilms (cidA, hld, sarA, icaA, and icaR) were also evaluated through real-time polymerase chain reaction.
Results: CFSs of both Lactobacillus spp. significantly reduced CSH, initial attachment, and biofilm formation and eradicated the biofilms. The above findings were supported by scanning electron microscopy results. These two Lactobacillus CFSs significantly changed the expression of all studied biofilm-related genes. Expression levels of cidA, hld, and icaR genes significantly increased by 4.4, 2.3, and 4.76 fold, respectively, but sarA and icaA genes were significantly downregulated by 3.12 and 2.3 fold.
Conclusion: The results indicated that CFS extracts of L. casei and L. rhamnosus had desirable antagonistic and anti-biofilm effects against S. aureus. Consequently, carrying out further research enables us to prepare pharmaceuticals from these CFSs in order to prevent and treat infections caused by S. aureus biofilms.


Arciola CR, Campoccia D, Montanaro L. Implant infections: Adhesion, biofilm formation and immune evasion. Nat Rev Microbiol 2018;16:397-409.  Back to cited text no. 1
Martins N, Rodrigues CF. Biomaterial-related infections. J Clin Med 2020;9:722.  Back to cited text no. 2
Singh AK, Prakash P, Achra A, Singh GP, Das A, Singh RK. Standardization and classification of in vitro biofilm formation by clinical isolates of Staphylococcus aureus. J Glob Infect Dis 2017;9:93-101.  Back to cited text no. 3
Maikranz E, Spengler C, Thewes N, Thewes A, Nolle F, Jung P, et al. Different binding mechanisms of Staphylococcus aureus to hydrophobic and hydrophilic surfaces. Nanoscale 2020;12:19267-75.  Back to cited text no. 4
Jenul C, Horswill AR. Regulation of Staphylococcus aureus Virulence. Microbiol Spectr 2019;7:1-34.  Back to cited text no. 5
Del Pozo JL. Biofilm-related disease. Expert Rev Anti Infect Ther 2018;16:51-65.  Back to cited text no. 6
Otto M. Staphylococcal Biofilms. In: Romeo T, editor. Bacterial Biofilms Berlin, Heidelberg: Springer; 2008. p. 207-28.  Back to cited text no. 7
Suresh MK, Biswas R, Biswas L. An update on recent developments in the prevention and treatment of Staphylococcus aureus biofilms. Int J Med Microbiol 2019;309:1-12.  Back to cited text no. 8
Bhola J, Bhadekar R. Invitro synergistic activity of lactic acid bacteria against multi-drug resistant staphylococci. BMC Complement Altern Med 2019;19:70.  Back to cited text no. 9
Guimarães A, Santiago A, Teixeira JA, Venâncio A, Abrunhosa L. Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum. Int J Food Microbiol 2018;264:31-8.  Back to cited text no. 10
Shokri D, Khorasgani MR, Mohkam M, Fatemi SM, Ghasemi Y, Taheri-Kafrani A. The inhibition effect of lactobacilli against growth and biofilm formation of Pseudomonas aeruginosa. Probiotics Antimicrob Proteins 2018;10:34-42.  Back to cited text no. 11
CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-11th Edition. CLSI DOCUMENTE M07-A11. Wayne, PA: Clinical and Laboratory Standards Institute; 2018.  Back to cited text no. 12
Mirani ZA, Fatima A, Urooj S, Aziz M, Khan MN, Abbas T. Relationship of cell surface hydrophobicity with biofilm formation and growth rate: A study on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Iran J Basic Med Sci 2018;21:760-9.  Back to cited text no. 13
Yan X, Gu S, Cui X, Shi Y, Wen S, Chen H, et al. Antimicrobial, anti-adhesive and anti-biofilm potential of biosurfactants isolated from Pediococcus acidilactici and Lactobacillus plantarum against Staphylococcus aureus CMCC26003. Microb Pathog 2019;127:12-20.  Back to cited text no. 14
Saidi N, Owlia P, Marashi SM, Saderi H. Inhibitory effect of probiotic yeast Saccharomyces cerevisiae on biofilm formation and expression of α-hemolysin and enterotoxin A genes of Staphylococcus aureus. Iran J Microbiol 2019;11:246-54.  Back to cited text no. 15
Merghni A, Dallel I, Noumi E, Kadmi Y, Hentati H, Tobji S, et al. Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureus strains. Microb Pathog 2017;104:84-9.  Back to cited text no. 16
Melo TA, Dos Santos TF, de Almeida ME, Junior LA, Andrade EF, Rezende RP, et al. Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa. BMC Microbiol 2016;16:250.  Back to cited text no. 17
Lv X, Miao L, Ma H, Bai F, Lin Y, Sun M, et al. Purification, characterization and action mechanism of plantaricin JY22, a novel bacteriocin against Bacillus cereus produced by Lactobacillus plantarum JY22 from golden carp intestine. Food Sci Biotechnol 2018;27:695-703.  Back to cited text no. 18
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8.  Back to cited text no. 19
Sharifi A, Mohammadzadeh A, Zahraei Salehi T, Mahmoodi P. Antibacterial, antibiofilm and antiquorum sensing effects of Thymus daenensis and Satureja hortensis essential oils against Staphylococcus aureus isolates. J Appl Microbiol 2018;124:379-88.  Back to cited text no. 20
Sharifi-Rad J, Rodrigues CF, Stojanović-Radić Z, Dimitrijević M, Aleksić A, Neffe-Skocińska K, et al. Probiotics: versatile bioactive components in promoting human health. Medicina (Kaunas) 2020;56:E433.  Back to cited text no. 21
Ginting EV, Retnaningrum E, Widiasih DA. Antibacterial activity of clove (Syzygium aromaticum) and cinnamon (Cinnamomum burmannii) essential oil against extended-spectrum β-lactamase-producing bacteria. Vet World 2021;14:2206-11.  Back to cited text no. 22
Koohestani M, Moradi M, Tajik H, Badali A. Effects of cell-free supernatant of Lactobacillus acidophilus LA5 and Lactobacillus casei 431 against planktonic form and biofilm of Staphylococcus aureus. Vet Res Forum 2018;9:301-6.  Back to cited text no. 23
Maske BL, de Melo Pereira GV, da S Vale A, de Carvalho Neto DP, Karp SG, Viesser JA, et al. A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application. Enzyme Microb Technol 2021;149:109836.  Back to cited text no. 24
Hu CH, Ren LQ, Zhou Y, Ye BC. Characterization of antimicrobial activity of three Lactobacillus plantarum strains isolated from Chinese traditional dairy food. Food Sci Nutr 2019;7:1997-2005.  Back to cited text no. 25
Taylor TM, Doores SX. Organic acids. In: Antimicrobials in Food. 4th ed. Boca Raton: CRC Press; 2020. p. 133-90.  Back to cited text no. 26
Ganji-Azad E, Javadi A, Jahanbani Veshareh M, Ayatollahi S, Miller R. Bacteria cell hydrophobicity and interfacial properties relationships: A new MEOR approach. Colloid Interfac 2021;5:49.  Back to cited text no. 27
Walencka E, Rózalska S, Sadowska B, Rózalska B. The influence of Lactobacillus acidophilus-derived surfactants on staphylococcal adhesion and biofilm formation. Folia Microbiol (Praha) 2008;53:61-6.  Back to cited text no. 28
Nguyen HT, Nguyen TH, Otto M. The staphylococcal exopolysaccharide PIA  Biosynthesis and role in biofilm formation, colonization, and infection. Comput Struct Biotechnol J 2020;18:3324-34.  Back to cited text no. 29
Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms: Properties, regulation, and roles in human disease. Virulence 2011;2:445-59.  Back to cited text no. 30
Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep 2018;8:2254.  Back to cited text no. 31