## 3 Results and Discussion
### Biofilm Inhibition and Eradication Ability  
In our study, the strain biofilm production ability was classified as reported by [PERSON] et al., 2007 [44] in no biofilm, weak, moderate, and strong producers. The results obtained showed that clinical and, as expected, reference strains [83] were strong biofilm producers. To verify the efficacy in preventing bacterial biofilm, sub-inhibiting concentrations (0.01 or 0.1 ug/mL) of nanocarriers, the free-form antibiotic, or LO were put in contact during biofilm formation for 24 h.  
As shown in Table 4, when the preparations were incubated during the bacterial biofilm formation step, all failed to inhibit its production by _Staphylococcus_ spp. isolates. Interestingly, only LVX repressed, in a significant manner, the amount of biofilm produced by the _S. epidermidis_ strain. Several studies have reported the antib biofilm activity of LVX, against Gram-positive and Gram-negative bacteria, but often in association with other compounds [84]. Recently, to combat surface-adherent bacteria and potential biofilm \begin{table}
\begin{tabular}{l c c c c c c} \hline  & \multicolumn{2}{c}{**MIC (\(\mu\)g/mL)**} & \multicolumn{3}{c}{**MBC (\(\mu\)g/mL)**} \\  & **NEs** & **NEsL** & **LVX** & **NEs** & **NEsL** & **LVX** \\ \hline _S. aureus_ ATCC 6538P & 2000 & 0.08 & 0.08 & 2000 & 0.08 & 0.08 \\ _S. aureus_ DA & 2000 & 0.63 & 0.63 & 2000 & 0.63 & 0.63 \\ _S. aureus_ HC & 2000 & 0.31 & 0.08 & 2000 & 0.31 & 0.08 \\ _S. epidermidis_ ATCC 35984 & 1000 & 0.31 & 0.31 & 1000 & 0.63 & 0.31 \\ \hline \end{tabular}
\end{table} 
Table 3: MIC and MBC values of NEs, NEsL and LVX. formation by _S. epidermidis_, a method has been developed to impregnate LVX antibiotic into the outer polymer of the catheter [85]. The effect on pre-formed biofilms was evaluated after 7 h of incubation with nanocarriers, the free-form antibiotic, or LO at sub-MIC concentrations (0.01 or 0.1 \(\upmu\)g/mL). In our research, the low sub-inhibiting concentration of LVX (0.01 and 0.1 \(\upmu\)g/mL) failed to act against _Staphylococcus_ spp. reference strains in sessile form even if the antibiotic was loaded in nanocarriers. Interestingly, at the same concentration of 0.1 \(\upmu\)g/mL an ameliorate, but non-significant, activity of both LVX and LO was observed against both clinical strains. It has been reported that fluoroquinolones are able to eradicate biofilms in vitro [86]. Recently, [PERSON] et al., 2024, demonstrated that free LVX remove _S. aureus_ ATCC 6538P and _S. epidermidis_ ATCC 35984 pre-formed biofilm in a dose-dependent manner [87] at about 8 and 256 \(\upmu\)g/mL, respectively. Therefore, our results could be attributed to lower concentrations used in our experiments, unable to produce an inhibitory action. The most prominent eradicating effect was obtained against the biofilms formed by clinical _S. aureus_ DA and _S. aureus_ HC with percentages of eradication of 71.0 \(\pm\) 10.0 and 41.0 \(\pm\) 0.3, respectively, by NEs loaded with LVX. Noteworthily, NEs showed eradicating ability against both _S. aureus_ HC and, even if in a not significant manner, against _S. epidermidis_ strains which could suggest a potential action by the LO. As suggested by other authors, LO might be able to contrast the formation of pathogenic biofilm [19] but in addition, in the NE formulation, preserve and, above all, strengthen biological activities. As suggested by [PERSON] et al., 2018 [88], oil-loaded nanoemulsions could exhibit excellent biofilm penetration ability owing to the enhanced interactions between the nanocarrier surface and biofilm matrices compared to hydrophobic oils alone. Recently, [PERSON] et al., 2023, demonstrated the activity of pseudopyronine analog-loaded nanoemulsions against preformed sessile forms of _S. aureus_, suggesting the facilitated solubilization and effective delivery of the polymer deep into the biofilm matrix [89]. [PERSON] et al., 2000 reported that resistance to LVX cannot be attributed to its low diffusion, but rather to its failure to kill bacteria in the biofilm [90].