Introduction: One of the mechanisms that bacteria use to stay safe from antimicrobial agents is to forma biofilm. In a biofilm, bacteria are less likely to be swept off to unfavorable conditions and are less susceptible to antibiotics. Many outbreaks of pathogens have been attributed to biofilms [1]. Lactoferrin is an 80 kDa iron-binding glycoprotein that involves a diverse range of biological activities including strong antimicrobial and antiviral properties [2]. Methods: The bovine lactoferrin (BL) (purity 95%) was prepared from Xi\\\'an Sgonek Biological Technology Company, China. The bacterial strains included Pseudomonas aeruginosa PTCC 1707, Escherichia coli ATCC 25922 and Staphylococcus aureus PTCC1337 were procured from microbial collection, Ferdowsi University of Mashhad, Mashhad, Iran. MIC: was carried out using the micro broth dilution method as recommended CLSI. Dilutions of BL from 0.125 to 8 mg/mL were prepared in MHB. Growth medium without inoculum and growth medium with Gentamicin (10 µg/mL) were used for negative and positive controls. Biofilm formation:10 µL microbial suspensions with 190 µL of MHB were added to the 96 microwells and incubated at 37˚C for 24 h. Then the microwells washed with sterilized distilled water three times. 200 μl of 0.025% Safranin solution was added to the wells and washed again with water after 2 minutes. Finally, 200 μl of ethanol-acetone solution (50% V/V) was added to each well, and after 15 minutes, OD was read at 630 nm. The growth medium without inoculum was used for negative control (ODC). Bacterial strains were classified as: ODT ≤ODC= non forming biofilm, ODC < ODT ≤(2× ODC)=weakly adherent; (2× ODC) < ODT ≤(4× ODC)=moderately adherent, and (4× ODC)< ODT =strongly adherent [3]. Inhibition of biofilm formation:10 µL microbial suspensions (OD630 = 0.08-0.13) with 190 µL of dilutions of BL were added to the 96 microwell plates and incubated at 37˚C for 24 h. Then the microwells were empty and washed with water and filled with 200 μl of 0.025% Safranin solution than washed with water after 2 minutes. Finally, 200 μl of ethanol-acetone solution (50% V/V) was add and after 15 minutes, OD was read at 630 nm. Growth medium without inoculum, microbial suspension, different concentration of lactoferrin were used as media, negative and lactoferrin controls [3]. % inhibition = [(OD negative control-OD media control)-(OD test-OD lactoferrin control)] /(OD negative control-OD media control) ×100 Results were analyzed by Minitab and differences among the means were determined by one-way ANOVA for significance at p <0.05. Results: The results showed that MIC for S. aureus obtained 6.5 mg/mL, and for P. aeruginosa and E. coli obtained bigger than 8 mg/mL. The biofilm was created by E. coli and S. aureus strongly and was moderately by P. aeruginosa. The results showed that with increasing the concentrations from 7 to 8 mg/mL of BL, prevention of biofilm formation was observed. The lowest inhibitory effects of BL on biofilm formation were observed against E. coli (84.13 %). Conclusion: BL can reduce bacterial growth, as pointed out here and by others, and inhibit bacterial adhesion and biofilm formation; thus, it should be considered as a helpful antimicrobial therapeutic agent. Moreover, our findings suggest the possibility of using lactoferrin as an anti-biofilm agent in the food and pharmaceutical industries.

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