## 4 Discussion
The preliminary screenings are very useful for a rapid selection of bacteria with bioactivity against pathogens, but it is necessary to perform more than one test to obtain consistent and clear results. The pre-screening procedures allowed it to be demonstrated that among all microorganisms considered, it is possible to find bacterial strains with optimal potentiality for the inhibition of pathogens mainly occurring in aquaculture. In the preliminary phases of this study, the antibacterial activity was mainly exhibited against _A. hydrophyla_ and _P. damselae_ subsp. _pisciida_. The bacterial species affiliated to _Aeromonas_ and _Vibrio_ genera are well known causes of different pathogenic states of fishes and shellfishes [26; 43]. _P. damselae_ subspecies are emerging pathogens in aquaculture systems and cause fish photobacteriosis [44], as for _P. damselae_ subsp. _pisciida_ reported as responsible for fish photobacteriosis [5]. Interestingly, the Mediterranean isolate _Bacillus_ sp. PS62 produced an inhibition halo (\(37\pm 0.0\); data not shown) against _P. damselae_ subsp. _pisciida_, equal in diameter to that produced from the positive control, i.e., tetracycline. Similarly, the polar strains _Pseudomonas_ sp. C1S1 and _Microbacterium_ sp. C1S1A produced a zone of inhibition (\(10\pm 0.0\) mm) really near to that obtained from tetracycline (\(13\pm 0.0\) mm) against _A. hydrophilis_ (data not shown). When compared with strains of Mediterranean origin, very few Arctic isolates showed antibacterial activity, but in contrast they exhibited a broader activity against more than one pathogen. It is noteworthy, however, that the absence of response in the preliminary assays, when this occurred, could be dependent on different factors, such as the incubation temperature and the composition of culture media. Indeed, the temperature of 15 \({}^{\circ}\)C was chosen as optimal growth temperature for polar strains, but the production of bioactive molecules could be stimulated by stressful environmental factors, as such as suboptimal temperatures. The inhibitory activity may derive from the synthesis and diffusion of antibiotic molecules or from the fast use of nutrient components in the culture medium by the tested strain. For this reason, here the antibacterial activity of cell-free supernatants (presumably containing the bioactive molecules) of the most promising strains was investigated. The production of secondary metabolites with antimicrobial activity could be strongly affected by cultural conditions and media composition, which could improve the crude extract yield [45]. Due to the weak activity observed for CFS from TSB bacterial cultures, it was attempted to test the addition of glucose, sucrose and glycerol as additional carbon sources to enhance the production of compounds with antibacterial activity. The strategy resulted in being promising, as the addition of sugars led to a global increase in inhibitory activity, i.e., from inhibition halos with a diameter of less than 8 mm (TSB without sugar addition) to inhibition halos with an average diameter equal or greater than 10 mm. Anyway, only glucose and sucrose resulted in being a suitable carbon source for the enhancement of antibacterial activity, whereas the addition of glycerol did not provide remarkable results. Strains belonging to the genera _Bacillus_[46], _Pseudoalteromonas_[47], _Pseudomonas_[48], _Vibrio_[49], _Aeromonas_[50], _Alteromonas_[51], _Marinomonas_[16], and _Photobacterium_[52] are known to produce compounds with antimicrobial activity. The phylogenetic affiliation of the Mediterranean strain PS62 confirms the interest of members belonging to the _Bacillus_ genus as among most promising candidates in this field. Moreover, the origin of the strain _Bacillus_ sp. PS62 highlighted again the great potential of benthic organism-associated bacteria in the production of bioactive molecules [19; 23]. Several studies reporting the activity of _Bacillus_ isolates are available in literature.