## 2 Results and Discussion
### Antibacterial and Antibofilm Activity  
The potential antibacterial activity of honey were associated with its physicochemical properties just as much as various chemical components are derived from the plant secretions, pollens, propolis and from bee that is collecting it [47]. MH exhibited a relatively increased antimicrobial activity against Gram-positive bacteria compared to Gram-negative bacteria (Table 3). Currently, tested sample of MH showed a higher antimicrobial effect towards Gram-positive than negative bacterial strains. This is in agreement with previous reports where generally Gram-positive bacteria exhibited higher level of sensitivity towards MH than Gram-negative bacterial isolates [12, 48].  
The intrinsic property of honey to work against pathogenic bacteria is the essence of multiple factors. These factors either work individually or collectively to achieve the required function/s. The major factors that are playing an important role would include, phenolic compounds, H\({}_{2}\)O\({}_{2}\), honey pH and the osmotic pressure produced by honey itself [49]. It was claimed that the prominent antibacterial efficacy of MH is mainly linked to the quantity of MGO present in the honey [40]. A study explored the antimicrobial effect of MH has shown its activity against various bacterial species, including _Streptococcus pyogenes_, _Streptococcus mutans_, _Staphylococcus aureus_, _Enterobacter cloacae_, _Proteus mirabilis_ and _Pseudomonas aeruginosa_[46].  
Biofilm formation can project great hindrance against antibiotics, as they furiously remain stable and would not be eradicated in severe infections [50]. The majority of antibacterial and anti-biofilm agents are known to be present in most medicinal plants and their compounds [51]. Even though all extracts have strong anti-bacterial potential, the lowest anti-biofilm potential of extracts must be discovered in the current investigation.  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{2}{c}{**Tested Bacteria**} & \multicolumn{2}{c}{**Tested Honey Sample (10\%)**} \\  & \multicolumn{2}{c}{**Zone of Inhibition (mm)**} \\ \hline Gram-positive & _B. subtilis_ & 9.54 \(\pm\) 0.3 \\  & _S. aureus_ & 6.55 \(\pm\) 0.24 \\ \hline Gram-negative & _E. coli_ & 4.6 \(\pm\) 0.25 \\  & _P. aeruginosa_ & 3.7 \(\pm\) 0.15 \\ \hline \hline \end{tabular} All results are presented as the mean \(\pm\) standard deviation (\(n=3\)).  
\end{table}
Table 3: Zone of inhibition of various bacterial isolates treated with manuka honey.  
The MH sample's biofilm percentage inhibition was assessed (Table 4), and \(\frac{1}{2}\) MIC values were also calculated (Table 4). A similar pattern of these findings was obtained in the case of anti-bacterial activity was noted, and the MH sample exerted more anti-biofilm potential against _S. aureus_ and _B. subtilis_, with percentage of inhibition as 67.13 and 56.52 %, respectively. Recent research has shown that metabolites, such as alkaloids, tannins, polyphenols, flavonoids, steroids and terpenoids are effective anti-bacterial and anti-biofilm agents. Even the function of catechin and polyphenol fractions as powerful biofilm inhibitors has been investigated [52]. The presence of these compounds in MH could be the reason for the observed anti-biofilm activity in the current study.