## 4 Discussion  
Nowadays, climate change has been recognized as a major global challenge. Some strategies, such as the advancement of bioeconomy and sustainable forest management have proven to be crucial in tackling this global problem. The primary economic sector, driven by biological and \"Protected Geographical Indication\" products, has been acquiring a growing economic/social/environmental importance as shown by the latest trends in national agricultural metrics [2]. A plethora of natural sources, such as plants and microorganisms, stand as alternative sources of bioproducts with medicinal potential. Indeed, from these organisms, innovative compounds with biological activity can be isolated. For centuries, natural compounds have been harnessed in Chinese medicine as modulators of various signaling pathways like cell proliferation, angiogenesis, survival, and apoptosis, exhibiting different biological activities like antioxidant and anti-inflammatory properties. Chestnut (_Castanea sativa_) cultivation has been deep sitting in Italy, particularly within its hilly and mountainous regions. As of 2017, Italy emerged as the leading producer of chestnuts in the European Union, contributing 52,356 tons, representing 38% of the total chestnut production in the EU (FAOSTAT, Food and Agriculture Organization of the United States). Nevertheless, this trend has been threatened in recent decades due to diseases such as chestnut blight and chestnut gall wasp, alongside the progressive depopulation of rocky areas. In response to these issues, there has been a drive towards novel approaches to utilize various parts of the chestnut plant, such as the burrs, accounting for 10-15% of the total weight of chestnuts and currently employed in fuel and fermentable sugar production [3]. Burrs also exhibit multiple biological activities (anti-inflammatory, antioxidant, and antimicrobial) [11], hence the interest in investigating their potential as green sources of bioactive compounds. Recently, the rising levels of resistant bacteria result in a threat for public health. This condition raised growing interest in antimicrobial resistance (AMR) and several approaches to investigate the antimicrobial activity of bio-compounds extracted from natural wastes have successfully been applied. Olive mill waste and artichoke tracts were both found to be successfully active against foodborne pathogens [41; 42]. The current literature body already suggests antimicrobial activity from chestnut burrs, against _S. aureus_, _E. faecalis_, _Listeria monocytogenes_[43], and _E. coli_[44], confirmed by in vitro antimicrobial susceptibility assays. Here, however, an integrated in vitro and in silico workflow was adopted. After evaluating the antimicrobial activity of our chestnut burr extract [22; 45] on different bacterial strains, a potential bacterial target of extract compounds (gallic acid, quinic acid, protocatechuic acid, brevifolia acid, and ellagic acid) was sought. Firstly, an in vitro MIC assay was performed on both Gram-positive (_E. faecalis_ and _S. aureus_) and Gram-negative (_E. coli_ and _P. aeruginosa_) bacterial strains, yielding a measurable antimicrobial activity on _E. faecalis_ (MIC = 64 ug/mL) only. Hence, the antimicrobial activity of the extract could be attributable to the interaction of the bioactive compound(s) with a specific target solely present in _E. faecalis_ and not in all the other strains. Multiple sequence alignment of _E. faecalis_ proteins identified \"endocarditis and biofilm-associated pilus tip protein EppA\" after removing homologous and obsolete sequences. The 3D structure of EppA was created with AlphaFold. Subsequent computational screening revealed ellagic acid and brevifolia acid as potential antimicrobial compounds, with ellagic acid being the most abundant in the extract, but molecular dynamics simulations showed a stable binding only between ellagic acid binding and EppA, while brevifolia rapidly dissociated. Network interaction analyses confirmed strong binding between ellagic acid and EppA.