## 2 Results
### Effect of KHS-Cnd on Biofilm Formation
Comparing the images in false colors of the control sample (Figure 3A,B) with the images of the treated sample (Figure 3C,D), the disaggregating effect of KHS-Cnd was evident: the trabeculae in the control sample presented an arrangement with tighter filaments, and the meshes were tight, while the trabeculae in the treated sample had a loose filament arrangement and the meshes were larger. Images of untreated _P. aeruginosa_ 27P samples in Figure 4A showed the presence of an abundant extracellular matrix (ECM), organized in both compact (asterisk) and spongy (star) areas. The observation at higher magnification (B) of the spongy area showed in more detail that ECM was formed by interconnected laminae, rather than trabeculae (as in PA14) with curly edges (I, laminae). The 3D arrangement of interconnected laminae formed a labyrinthic system of channels (larger than that of PA14), producing an ECM spongy appearance. Figure 2: VP-SEM images of untreated _P. aeruginosa_ PA14 (**A**,**B**). (**A**) Low magnification (1.00 K) showed smooth and compact ECM areas (asterisk), spongy, and rough areas (star). (**B**) At higher magnification (3.00K), ECM showed large meshes in some areas (arrow) and smaller ones in others (dotted oval). VP-SEM of _P. aeruginosa_ PA14 treated with KHS-Cnd (**C**,**D**). (**C**) At low magnification (1.00 K), ECM appeared almost totally with a spongy appearance (star), and rare compact areas (asterisk) were present. (**D**) Higher magnification (3.00 K) showed that a 3D network of ECM trabeculae was formed by the aggregates of fine granular filaments having a “pearl necklace” aspect (arrow). Treatment with KHS-Cnd showed a less robust effect on the _P. aeruginosa_ 27P ultrastructure. The image at low magnification (C) of the treated biofilm showed a spongy appearance on all of the sample surface, namely, less compact when compared to the control sample (Figure 4A). At higher magnification of the treated biofilm (D), the edges of the laminae formed an ECM 3D network, which appeared curly as in the control sample (comparing Figure 4B of the control sample with Figure 4D of the treated sample), and the porosity did not significantly increase. Figure 3: Three-dimensional reconstruction and analysis of _P. aeruginosa_ PA14 **(A,B)** and _P. aeruginosa_ PA14 treated with KHS-Cnd (**C,D**). The magnification is 3.00 K. (**A**) The spongy surface of untreated _P. aeruginosa_ PA14 biofilm is imagined in 3D. (**B**) The circle represents the extracted area shown in false colors and related to untreated PA14. (**C**) The spongy surface of KHS-Cnd treated _P. aeruginosa_ PA14 biofilm is imagined in 3D. (**D**) The circle represents the extracted area shown in false colors and related to KHS-Cnd treated _P. aeruginosa_ PA14. High elevated areas corresponding to ECM trabeculae are colored in green. The labyrinthic channel system is represented in blue. To further quantitatively analyze the effect of KHS-Cnd on the ECM ultrastructure of _P. aeruginosa_ 27P, we used the Hitachi Map 3D Software (v.8.2., Digital surf, Besancon, France). Results are shown in Figure 5A-D for a representative experiment. The three-dimensional reconstruction of the samples and the circular area extracted were magnified and are represented in false colors in Figure 5A-D. High elevated areas corresponding to the ECM trabeculae lining the meshes are represented in green. The deep channels of the labyrinthic system are represented in blue. Despite no dramatic morphological modifications being appreciable, when comparing the images in false color, the presence of a less compact matrix (Figure 5C) and more abundant labyrinthic channels (Figure 5D) were evident in the treated sample compared to the control (Figure 5A,B). Figure 4: VP-SEM images of untreated _P. aeruginosa_ 27P (**A,B**). (**A**) At low magnification (1.00 K), ECM was organized in both compact (asterisk) and spongy areas (star). (**B**) At higher magnification (3.00 K), ECM was formed by interconnected laminae (l), rather than trabeculae. VP-SEM images of _P.