## 4 Discussion  
The ability of microorganisms to produce biofilms is considered an important virulence factor. During biofilm formation, microorganisms secrete natural polymers called highly hydrated extracellular polymeric substances (EPS), including polysaccharides, proteins and extracellular DNA (eDNA) [51] that embed bacteria and protect them from antimicrobial agents [52]. This enables the embedded bacteria to escape the defence mechanisms even of the host, and to resist much more than their planktonic counterparts to  
Figure 4: Real-Time PCR results show the effects of HBD-2 and HBD-3 on expression of biofilm-associated genes in _P. aeruginosa_ (**a**) and _S. aureus_ (**b**) re-suspended in conditioned media in Caco-2/HBD-2 and Caco-2/HBD-3 cells, shown as relative gene expression. Data are representative of three different experiments \(\pm\) SD. Significant differences are indicated by \(\ast\)_p_ \(<\) 0.05, \(\ast\ast\)_p_ \(<\) 0.01.  
unfavourable conditions, such as a limited supply of nutrients, pH or inappropriate temperature. Increasing evidence suggests that the ability to form biofilms in many organisms involves quorum sensing (QS) regulation. QS is a cell density-dependent signalling system, in which bacteria produce and release a series of low molecular weight molecules called self-inductors which, once certain extracellular concentrations are reached, bind to their receptors present on bacterial cells, and regulate the expression of some genes fundamental for the formation of the biofilm [53]. In our experimental system, we created a line of human epithelial colorectal adenocarcinoma Caco-2 cells, stably transfected with the genes encoding the HBD-2 and HBD-3. Supernatants of transfected and untransfected cells were used to evaluate whether the presence of high concentration HBD-2 and HBD-3 may affect the intestinal biofilm formation of _S. aureus_ and _P. aeruginosa_. Biofilm production in _S. aureus_ is linked to the release of an intercellular polysaccharide adhesin (PIA), involved in bacterial adhesiveness to target surfaces [54], which is the first critical event in the onset of infection [55]. PIA is encoded by the _ica_ operon and its activity is linked to that of the biofilm associated protein (bap), a surface protein encoded by the homonymous gene [54]. The pronounced ability of _P. aeruginosa_ to develop biofilm-associated disease has drawn considerable interest over the past decade to develop strategies to target their disassembly. The QS system in _P. aeruginosa_ controls over 10% of the entire genome, mainly regulating the expression of virulence factors, swarming motility, secondary metabolite production and biofilm formation. In particular, it consists of two subordinate systems, _las_ and _rhl_. Specifically, the synthase _lasl_ produces the self-inductor, N- (3-oxododecanoyll) -L-homoserine lactone (3O-C12-HSL), which can bind and activate its transcriptional regulator, _lasR_[56; 57]. In addition, _pslA_ genes play a significant role in the formation of carbohydrate-rich structure of biofilm matrix. The activation of the _pslA_ operon is closely related to that of the _ppyR_ operon (putative transmembrane protein), a highly conserved gene responsible for the expression of the virulence genes of numerous clinical isolates, which enhance biofilm formation, and is endowed with elastase activity (as a virulence factor) [58]. In fact, it has been shown that _psl_ operon was repressed following inactivation of the _ppyR_ gene, and biofilm formation decreased [58]. For this purpose, cultures of _S. aureus_ and _P. aeruginosa_, re-suspended in the supernatants of transfected and untransfected Caco-2, were tested for their ability to form biofilms according to different methods: first, the staining assay crystal violet was performed, followed by a biofilm evaluation with conventional plate counting. A microscopy analysis was also performed with the Live/Dead system using the fluorescence microscope.