## 4 Discussion  
Bacterial infections still represent a major health problem, amplified by the phenomenon of antibiotic resistance. Micromycetes have long been a reservoir of antibacterial drugs, but higher fungi, i.e., mushroom-forming fungi or macromycetes, are now emerging as a promising source of antimicrobial compounds [9; 10]. Generally, antibacterial activity of crude extracts is assessed by microdilution assays; however, this approach provides only a global quantitative assessment of the potential of the extract, but does not bring any information about the number of molecules involved in this antimicrobial activity. Furthermore, results can be affected by antagonist or synergistic effects. For these reasons, we decided to screen extracts by TLC-direct bioactivity, which combines preliminary planar chromatographic separation of crude extracts and subsequent application of bacterial suspensions on eluted TLC plates [11]. This strategy enables us to evaluate the number of compounds involved in bioactivity, to assess if different bacterial strains are inhibited by the same or distinct compounds, and to get an idea about the polarity of active compounds according to their elution on the chromatographic plate. Though we have chosen to order antibacterial activity according to fungal taxonomy (Tables 11-11), no correlation emerges from our work that would assign any antimicrobial activity to a known fungal lineage. Some chemical structures may be restricted to a limited number of species or genera, but they could equally well have evolved in response to ecological constraints shared by organisms taxonomically unrelated. In fact, since the function of most bioactive compounds from macromycetes is still unknown for their establishment or maintenance in their respective ecological niche, molecules inhibiting bacterial growth in vitro (on TLC plates or in petri dishes) may just have no such function in situ or they may not be subjected to direct selection pressure. Consistent with this view, the distribution of lethal amatoxins among the _Amantia_, _Galerina_, and _Lepiota_ genera, shows that (very) active chemical structures have evolved repeatedly in distinct fungal lineages, without an obvious link with ecological features or evolutionary origins [12]. In addition, as experienced here with _Neobelets erythropus_ or _Scleroderma citrinum_, environmental factors such as biotopes or seasons seem to affect both the qualitative and quantitative contents of antibacterial compounds, further blurring any correlation between taxonomy and activity of these molecules. Notwithstanding these open questions about the physiological function of these active metabolites for the fungus, the data obtained here show that most--if not all--mushrooms produce few to several compounds endowed with antibacterial properties, with high or low selectivity, confirming our initial hypothesis. Our results highlighted that low-polarity metabolites seem to contribute significantly to antibacterial effects. Stearic acid in particular was detected in many samples, yielding an intense inhibition band in the upper part of several bioautograms (Rf around 0.75). This observation was first considered with caution, as it is known that some non-polar products may generate false positives, as hydrophobicity of the compound can act as a repellent for the aqueous bacterial suspension. However, we observed that even more lipophilic fatty acids did not affect the growth of bacteria in our system, corroborating a genuine activity of some low-polarity compounds. It turns out that the antibacterial action of stearic acid and other related fatty acids is well documented [13; 14]. It was demonstrated that free fatty acids induce their antibacterial action through several mechanisms, including cell disruption, interference with cellular energy production, enzyme inhibition, and others.