## 3 Results and Discussions
### Biofilm Inhibition and MBIC Assay Results  
The biofilm's inhibition activity has been determined under an inverted microscope (40\(\times\)), and the difference in the untreated and treated biofilms with antibiotics and NDG could be evaluated clearly by observing the clearance of biofilms and the separation of planktonic cells in comparison with the untreated biofilm (Figures 7 and 8). Previous studies have explored the role of NDG in biofilm inhibition in pathogenic bacteria and provided evidence of metallic nanocomposites acting effectively against biofilm formation [32; 33; 34; 35].  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Microorganisms**} & \multicolumn{2}{c}{**Zone of Inhibitions (mm), Mean \(\pm\) SD, \(n\) = 3**} \\ \cline{2-3}  & **Ampicillin** & **NDG** \\ \hline _Klebsiella pneumoniae_ & 19.21 \(\pm\) 1.39 & 14.2 \(\pm\) 1.86 \\ \hline _Pseudomonas aeruginosa_ & 19.5 \(\pm\) 2.75 & 17.8 \(\pm\) 1.98 \\ \hline MRSA & 20.3 \(\pm\) 1.12 & 24.7 \(\pm\) 1.89 * \\ \hline _Streptococcus mutans_ & 18.21 \(\pm\) 1.33 & 22.31 \(\pm\) 1.67 * \\ \hline _Streptococcus pneumoniae_ & 19.16 \(\pm\) 1.72 & 25.45 \(\pm\) 2.89 * \\ \hline _Bacillus subtilis_ & 19.22 \(\pm\) 1.87 & 25.12 \(\pm\) 2.35 * \\ \hline \hline \end{tabular}  
* \(p<0.05\) (Significant).  
\end{table}
Table 1: Zone of inhibitions obtained during agar diffusion test by NDG as compared to ampicillin. Results were presented as mean \(\pm\) SD, \(n\) = 3.  
Figure 7: Results observed under an inverted microscope (40\(\times\)) (**A**). MRSA biofilm untreated, (**B**). MRSA inhibition treated with NDG (100 \(\upmu\)g/mL), (**C**). MRSA inhibition treated with NDG (200 \(\upmu\)g/mL), (**D**). MRSA inhibition treated with ampicillin (200 \(\upmu\)g/mL), (**E**). MRSA inhibition treated with ampicillin (1000 \(\upmu\)g/mL).  
The biofilm inhibition activity of isolates _Pseudomonas aeruginosa_ and MRSA has been performed using NDG and has been compared with standard antibiotics ampicillin. The biofilms of _Pseudomonas aeruginosa_ and MRSA were found to show a good percentage of inhibition against NDG in contrast to the standard antibiotic ampicillin. The biofilm inhibition activity of NDG against _Pseudomonas aeruginosa_ showed the least MIC of 25 \(\upmu\)g/mL and showed the highest percentage of inhibition 96.26 with an IC\({}_{50}\) value of 12.98 \(\upmu\)g/mL in comparison to standard antibiotic tested. The results with MRSA biofilm inhibition activities of NDG showing IC\({}_{50}\) value 23.11 \(\upmu\)g/mL with an inhibition percentage of 98.12 in comparison to standard antibiotic-tested results are tabulated in Table 2. Because biofilms are difficult to eliminate, they may increase the risk of infection, lengthen hospitalizations, and boost healthcare expenses. Resistance to antibiotic therapy and gene transfers between bacteria are two mechanisms linked to biofilm protection and survival, which allow the pathogen to dodge host immune responses and create chronic infections [36]. To combat bacterial resistance, scientists are exploring new antibiotic alternatives that not only prevent resistance but also reduce the use of traditional antibiotics, allowing for effective biofilm eradication [37]. Among these, the application of NDG has attracted much attention due to its antibacterial and antib biofilm properties, as revealed in later investigations, and the NDG could be used as an alternative to antibiotics against biofilm-forming bacterial strains [38; 39].  
Figure 8: Results observed under inverted microscope (40\(\times\)) (**A**). _Pseudomonas aeruginosa_ Biofilm untreated, (**B**). _Pseudomonas aeruginosa_ inhibition treated with NDG (100 \(\upmu\)g/mL), (**C**). _Pseudomonas aeruginosa_ inhibition treated with NDG (200 \(\upmu\)g/mL), (**D**). _Pseudomonas aeruginosa_ inhibition treated with ampicillin (1000 \(\upmu\)g/mL), (**E**). _Pseudomonas aeruginosa_ inhibition treated with ampicillin (2000 \(\upmu\)g/mL).