## 2 Results  
### Effect of KHS-Cnd on Biofilm Formation  
Phenotypic features of the clinical strains and PA14 reference strain are summarized in Table 1.  
Preliminary experiments were performed to assess the effect of KHS-Cnd on planktonic growth of six _P. aeruginosa_ strains, five clinical strains, and the hyperbiofilm producer PA14 reference strain [23]. The phenotypic characterization of clinical and PA14 strains are summarized in Table 1. The detected results showed that the peptide did not affect bacterial viability up to a concentration of 40 uM, except for the 32P strain where an antimicrobial activity was evidenced at the highest used concentration. Considering these results, biofilm experiments were performed by testing KHS-Cnd at a concentration of 40 uM for all strains, and only for 32P at a 20 uM concentration. The effect of KHS-Cnd was investigated either during biofilm development by adding it to the medium at the beginning of the cultivation (time zero, pre-adhesion period), or after biofilm formation (24 h of bacterial culture, mature biofilm). As the control, bacteria were cultured in BHI medium without the peptide. Results of KHS-Cnd on the pre-adhesion period are presented in Figure 1, panel A. Results were expressed as the percentage of biofilm formed in the presence of KHS-Cnd compared to the bacteria grown only in the BHI medium. KHS-Cnd showed an anti-biofilm activity in five out of six tested strains, with a percentage of inhibition ranging between 60% and 20%. The strongest inhibition (60%) was achieved on the isolate 32P and on the reference strain PA14. On clinical strain 40P, instead, a significant increase in the biofilm formation was highlighted. Furthermore, it is worth noting that 40P was the strain exhibiting a lower biofilm biomass at 24 h compared to the others (Table 1). \begin{table}
\begin{tabular}{c c c c c} \hline
**Bacterial** & **Colony** & **Protease Activity** & **Biofilm 24 h a** & **Biofilm 48 h b** \\
**Strain** & **Phenotype** & **(OD 400 nm)** & **(OD 590 nm)** & **(OD 590 nm)** \\ \hline PA14 & smooth & 2.037 \(\pm\) 0.020 & 3.561 \(\pm\) 0.357 & 13.470 \(\pm\)1.403 \\
23P & smooth & 1.988 \(\pm\) 0.057 & 3.175 \(\pm\) 0.851 & 0.738 \(\pm\) 0.373 \\
27P & smooth & 2.175 \(\pm\) 0.036 & 1.429 \(\pm\) 0.643 & 3.049 \(\pm\) 0.796 \\
31P & mucoid & 1.915 \(\pm\) 0.001 & 1.741 \(\pm\) 0.154 & 5.133 \(\pm\) 0.946 \\
32P & smooth & 2.439 \(\pm\) 0.078 & 1.117 \(\pm\) 0.163 & 5.597 \(\pm\) 1.390 \\
40P & irregular colony & 2.064 \(\pm\) 0.058 & 0.970 \(\pm\) 0.201 & 2.172 \(\pm\) 0.194 \\  & edges & & & \\ \end{tabular}
\end{table} 
Table 1: Phenotypic characterization of clinical and PA14 strains. Figure 1: Effect of KHS-Cnd on biofilm formation by different clinical strains and the PA14 reference strain. Panel (**A**): Effect of KHS-Cnd on biofilm formation. KHS-Cnd was added to the culture medium at time zero (0 h, pre-adhesion period) and the biofilm was analyzed after overnight incubation. In the ordinate axis, the percentage of bacterial biofilm production is reported. Data are expressed as the percentage of biofilm formed in the presence of 40 uM KHS-Cnd (except for 32P where 20 uM KHS-Cnd was used) compared with the untreated bacteria. Each data point is composed of three independent experiments, each performed at least in three replicates. Panel (**B**): Effect of KHS-Cnd on the mature biofilm. KHS-Cnd was added to the culture medium after 24 h of biofilm growth (24 h of bacterial culture) and the biofilm was analyzed after overnight incubation. In the ordinate axis, the percentage of residual biofilm is reported. Data are expressed as the percentage of residual biofilm after 24 h of treatment with 40 uM KHS-Cnd (except for 32P where 20 uM KHS-Cnd was used) compared with the control sample. Error bars indicate the standard deviations of all of the measurements. Statistical difference was determined by the Student’s _t_-test: * \(p\) < 0.05; ** \(p\) < 0.01, compared with the control. KHS-Cnd was also tested on the mature biofilm (24 h of bacterial culture).