## 4 Discussion  
Bacterial colonization on surgical implants may be treated with bactericides; however, the presence of biofilms presents a barrier to the treatment, mainly because they impede drug penetration and act as reservoirs for bacterial colony formation with protection from the host's innate immune system. Hence, the removal of the biofilm alone may eradicate the bacterial colonies, since they will become more susceptible to the immune system, while subsequent treatment with bactericides may result in complete eradication [42]. Therefore, in the present study, we investigated the dissolution action of BromAc (bromelain and \(N\)-acetylcysteine) on biofilm formed by _Pseudomonas aeruginosa_ using three different strains in order to study the efficacy, with variability shown within the strains. Bromelain as a single agent was very effective at certain concentrations, showing some variability within the strains. Strain ATCC 31461 was the most sensitive, with a BD MAX of 75%, whilst PA01 3981 was less sensitive, with a BD MAX of 45%. Treatment with NAC \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline  & & **ATCC 27853** & & **PA01 3981** & & **ATCC 31461** \\ \hline
**NAC** & **BR** & & & & & \\ **(mg/mL)** & **(ug/mL)** & **BD MAX (\%)** & **CI** & **BD MAX (\%)** & **CI** \\ \hline
15 & 25 & 87 & 0.025 & - & - & 81 & 0.403 \\
25 & 12.5 & - & - & 95.5 & 0.001 & 80 & 0.357 \\
25 & 25 & - & - & 97 & 0.001 & 85 & 0.395 \\
50 & 6.25 & 85 & 0.008 & 88 & 0.009 & - & - \\
50 & 12.5 & 80.5 & 0.03 & 95.3 & 0.003 & 79 & 0.581 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Comparison of maximum biofilm dissolution (BD MAX 80% and above) using a combination of agents in the three strains of _Pseudomonas aeruginosa_. The sensitivity to \(N\)-acetylcysteine with 50 mg/mL + bromelain 12.5 mg/mL indicates that strain PA01 3981 was the most sensitive, whilst the other two strains were almost equally sensitive. CI = combination Index. \begin{table}
\begin{tabular}{c c c c c} \hline _Pseudomonas aeruginosa_ & **BR 100 mg/mL** & **OA** & **NAC 100 mg/mL** & **OA** \\ \hline ATCC 27853 & 66 & 2 & \(-\)54 & 3 \\ PA01 3981 & 45 & 3 & 3 & 2 \\ ATCC 31461 & 75 & 1 & 57 & 1 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Comparison of maximum biofilm dissolution (BD MAX) activity levels of bromelain (BR) and \(N\)-acetylcysteine (NAC). OA = order of activity. Note that negative values indicate the synthesis or growth of biofilm. showed that strain ATC31461 was the most sensitive (BD MAX = 57%), whilst ATCC 27853 was not only the least sensitive but showed biofilm growth (enhancement) in the presence of NAC (BD MAX = \(-54\%\)). These variations may be due to differences in the compositions of the biofilms, as well as other variables within the strain. Bromelain may act as a proteolytic agent, hydrolyzing the peptide and glycosidic bonds found within the glycoproteins and proteins in the biofilm and simultaneously hydrolyzing complex carbohydrates [33]. These multiple enzymic actions may be due to bromelain's complex composition, having numerous enzymes [32]. Hence, it reduced the expression of the biofilm as a single agent. However, the reducing action of NAC (antioxidant) largely depends on the disulfide bridges found within the biofilm and favored in strain ATCC 31461, whilst in strain ATCC 27854, it acted in an opposite manner, providing more than 50% extra growth. This may indicate that this particular strain may be able to use the amino thiol as a source of sulfur and nitrogen for protein synthesis, as well as to enhance the formation of glycosidic linkages with subsequent bacterial multiplication and ensuing biofilm formation; however, this needs further investigation. The abundance of biofilm is directly related to the density of the invading bacteria. The destruction of the bacteria may relate directly to the abundance of biofilm.