## 4 Discussion  
### Optimal Conditions for Aeromonas salmonicida subsp. salmonicida to Form Biofilms  
For decades, the professional literature has been asking the question of how the _Aeromonas salmonicida_ subsp. _salmonicida_ maintains in the fast and cold trout pond waters, or in cold, clean rivers, where the complete water change is very intensive and thus unsuitable for the multiplication of pathogenic bacteria [37]. Furunculosis and _Aeromonas salmonicida_ subsp. _salmonicida_ persist in trout ponds despite intensive zoo hygiene measures, application of biocides, desiccation, vaccination, and even antibiotic therapy, but the disease occasionally still breaks out, even during the winter seasons, and causes great economic damage [38]. Thus, _Aeromonas salmonicida_ subsp. _salmonicida_ biofilms may be the explanation for the persistent presence of furcunculosis in cold and unfavorable environments. In the doctoral dissertation of [PERSON] [25], the ability of _A. salmonicida_ subsp. _salmonicida_ to produce biofilms in elevated temperatures between 20 \({}^{\circ}\) and 28 \({}^{\circ}\)C in the presence of PUFAs (polyunsaturated fatty acids) has been investigated using crystal violet method, but the author added amino acids in minimal M9 (M9 with amino acids is marked as CM9) medium and performed the investigation on only one strain. Other works on this subject were performed on _A. salmonicida_ strains of unknown subspecies, or subspecies other than _salmonicida_, originating from a wide range of different species of fish, reptiles, amphibians, domestic and wild animals, birds, even humans, being conducted most often at mesophilic temperatures, between 20 \({}^{\circ}\) and 30 \({}^{\circ}\)C, mostly in rich nutrient media like Tryptone soya broth, and at the most incubation lasted for 24 to 48 h [16; 17; 29; 39; 40; 41; 42]. In our research, scarce media as minimal M9 without addition of amino acids, with glucose as the only organic source of energy, and ABTG medium, also without amino acids, with a slightly larger selection of salts compared to M9, and also with glucose as the only source of C atoms, has been chosen. Based on our results, it can be said that the optimal temperatures for _Aeromonas salmonicida_ subsp. _salmonicida_ to form biofilm could be defined only in relation to the presence of nutrients. Thus, 8-25 \({}^{\circ}\)C were optimal temperatures for _Aeromonas salmonicida_ subsp. _salmonicida_ to form a biofilms in a nutritionally rich environment; 8 \({}^{\circ}\)C was optimal in nutritionally poor environment, and 16 \({}^{\circ}\)C was the temperature at which only certain strains could form a biofilm in nutritionally poor media. In nutritionally poor media, the largest number of strains of _A. salmonicida_ subsp. _salmonicida_ were not able to form biofilms at 25 \({}^{\circ}\)C. It should be emphasized that the in vitro methodology did not include dynamic conditions in fast-flowing waters; it was a static biofilm investigation, but it can be a base for further research in dynamic conditions that correspond to the natural habitat of _A. salmonicida_ subsp. _salmonicida_. Several studies have concluded that other bacteria that are cold-adapted in their natural environments create better biofilms in nutrient-poor media than in -rich media [43]. Figure 7: Phylogenetic tree representing the genetic distance between tested samples based on WGS analysis. For example, _Pseudoalteromonas haloplanktis_ TAC125 forms much better biofilms (higher biomass, more compact structure, higher number of viable cells in the biofilm, higher amount of eDNA) in biofilms incubated at 15 \({}^{\circ}\)C and 0 \({}^{\circ}\)C in poor GG medium (the only source of carbon are D-Gluconic acid sodium and L glutamic acid,) than in rich Brain Heart Infusion Broth [44]. At zero degrees, _Pseudoalteromonas haloplanktis_ TAC125 formed a much better biofilm at the solid-liquid interface than a pellicle in GG medium, but at 15 degrees, it formed a better pellicle in GG.