## 3 Discussion  
The vascular graft bacterial infections are associated with high morbidity that may lead to prolonged hospitalization, organ failure, amputation, and death and exert significant financial burden on the healthcare system [29, 30]. The ability of _S. aureus_ to attach to the foreign body surface and develop a biofilm is an important determinant of resistance to antibiotic prophylaxis. Antimicrobial compounds did not generally demonstrate significant activity in established biofilms due to either a lack of penetration, drug inactivation, or due to the state of bacterial cell division within the biofilm [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31]. In order to prove efficacy in preventing bacterial adhesion and biofilm formation _in vivo_ and _in vitro_ a RIP derivative, FS8, was used. For comparison purposes, the antibiotic tiegcycline was chosen for its current utilization in clinical practice against staphylococci [17, 18, 19, 32]. At first we established an _in vitro_ system to elucidate the bactericidal activity of tiegcycline with or without FS8 in a planktonic model and in an adherent-cell biofilm model. Afterwards, we used a preclinical surgical implant infection in rats that involved the inoculation of MRSA strain in the presence of a collagen-sealed polyester vascular grafts implanted into the subcutaneous pocket surgically placed in the back of these animals. Our _in vitro_ studies showed that biofilm was strongly affected by the presence of FS8, and in its presence, tiegcycline showed MICs and MBCs much lower than those obtained in the absence of FS8. These results were confirmed by the _in vivo_ experiments, where the use of FS8-graft-bonded with or without combination with the conventional antibiotic caused significantly lower bacterial loads. In fact, similarly to tiegcycline, FS8 caused a significant reduction in bacterial load on the vascular graft tissue when compared with control untreated animals and when tiegcycline and FS8 were combined \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Group \({}^{a}\)**} & **Graft-bonded drug \({}^{b}\)** & **Intraperitoneal preoperative drug \({}^{c}\)** & **Quantitative graft culture (CFU/mL) \({}^{d}\)** & **\(p\)-value (comparison with untreated control group)** & **\(p\)-value (comparison with double-treated group)** \\ \hline Uncontaminated control & & - & \(<\)10 & & \\ Untreated control & & - & \(7.5\times 10^{6}\)\(\pm 2.1\times 10^{6}\) & & \\ Group 1 & - & Tigecycline & \(4.4\times 10^{4}\)\(\pm 1.2\times 10^{4}\) & \(<\)0.001 & \(<\)0.001 \\ Group 2 & FS8 & - & \(6.8\times 10^{4}\)\(\pm 2.0\times 10^{4}\) & \(<\)0.001 & \(<\)0.001 \\ Group 3 & FS8 & Tigecycline & \(3.7\times 10^{1}\)\(\pm 0.7\times 10^{1}\) & \(<\)0.001 & \(<\)0.001 \\ \hline \hline \multicolumn{6}{l}{\({}^{a}\)_Staphylococcus aureus_, strain Smith diffuse (SD); \({}^{b}\) The Dacron graft segments were impregnated with 10 mg/L of FS8; \({}^{c}\) Each rat received intraperitoneally 2 mg/Kg of tiegcycline; \({}^{d}\) The limit of detection for the method was \(\leq\)10 CFU/mL.} \\ \end{tabular}
\end{table} 
Table 2: Activity of tiegcycline and FS8 against _Staphylococcus aureus_, strain Smith diffuse (SD) in a subcutaneous rat pouch model of infection. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**RP-HPLC retention time (min)**} & \multirow{2}{*}{**Purity (\%)**} & \multicolumn{2}{c}{**Mass**} & **Binding to the Calculated** & **Caluated** & **Found** & **grafts (\(\mathbf{\mu}\)g/cm\({}^{2}\))** \\ \cline{3-5}  & & & & \\ \hline
14.43 & 97.85 & 836.393 & 836.388 & 0.67 \(\pm\)0.04 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Analytical data of FS8. resulted in significant bacterial growth inhibition even if high concentrations of organisms are locally inoculated in the area of prosthesis implantation. In summary, not only did FS8 by itself reduce bacterial load, it also enhanced the effect of tigecycline.