*3.5. Biofilm Inhibition*

The effects of cladode extracts on biofilm formation by *Staphylococcus aureus* ATCC 35556, a strong biofilm producer, was also investigated.

As shown in Figure 3, both cladode extracts (mature and immature) at concentration of 500 μg/mL did not inhibited the biofilm formation. At 1000 μg/mL, only the immature cladode extract inhibited significantly the biofilm formation with an inhibition rate of 80%. At 1500 μg/mL concentration, both extracts significantly inhibited the biofilm formation, with an inhibition rate of 71% for the mature cladode and 85% for the immature cladode.

**Figure 3.** Effects of different concentrations of *Opuntia ficus-indica* cladode extracts on biofilm formation by *Staphylococcus aureus* ATCC 35556. *Staphylococcus epidermidis* ATCC 12228 was used as negative control. Assays were carried out in triplicate and the results were expressed as mean values ± SD. Different letters indicate significant differences (*p* ≤ 0.05). One-way ANOVA with Bonferroni's multiple comparison test.

The inhibition of biofilm development at concentration of *Opuntia* cladode extracts higher than MIC demonstrated that the bacterial cells in a biofilm are more resistant to antimicrobial agents compared to the planktonic cells, which is a well-known feature. Our results concerning the inhibition of *Staphylococcus aureus* MRSA biofilm formation agree with previous works [10,37]. In our study, the inhibition of biofilm could be explained by the presence of flavonoids in cladodes extracts. In fact, flavonoids such as quercetin, apigenin, luteolin and rutin were found to be effective in the inhibition of *Staphylococcus aureus* biofilm [38,39].
