## 4 Discussion
Biofilm production, shown in Figure 5, was significantly higher in mutant strains--Rub A1 (\(p<0.0001\)), Rub A2 (\(p<0.0001\)), and GLRX (\(p<0.0001\))--when compared to the wild strain. Although the biofilm increase for strain GLRX was expected according to the bibliography [34], the observed increase for mutants Rub A1 and Rub A2 were not foreseen, as the suppression in correspondent gene expression is associated with lower biofilm production [2; 9; 26]. The PQS is constituted by different genes: _pqs_ABCDE, _phm_AB and _pqs_H [35]. The expression of _pqs_A affects the PQS and, consequently, the biofilm production [36]. Our study conducted a comparison between the expression of the gene _pqs_A in three mutant PAO1 versus the wild-type PAO1. [PERSON], et al. identified the pqsD enzyme as a key enzyme whose inhibition could repress the PQS and the biofilm production in \(P\). _aeruginosa_[37]. Therefore, the possibility of the interaction between the other genes of PQS and biofilm production may be a hypothesis for the results observed. In the protease production, shown in Figure 6, only the mutant strain GLRX showed a significant increase in proteases when compared to the wild strain for the higher CIP concentration (\(p<0.05\)). These results were contradictory to those in a previous study, where the expression of the QS genes was decreased [28]. Once again, a possible explanation could be related with the iron metabolism. GLRX has been described before to have a synergy with iron metabolism [19], with this inorganic compound having a repressive effect on the production of LasA [38]. More studies are needed to explore this hypothesis. Rhamolipid production decreased when compared to the same strains without the antibiotic inoculation, as expected by the decrease observed in the production of QS genes previously [29]. There was no significant difference observed between the two concentrations of CIP used in this study in the rhamnoid production in all the strains tested. Rhamolipids are essential for the swarming motility of PAO1 and are involved in biofilm formation and bacteria protection from phagocytosis by macrophages. Additionally, these glycolipids can also act as hemolysins, making this virulence trait determinant in the pathogenesis of the bacteria. Therefore, more studies could be helpful to perceive the mechanism underlying the inhibition of these genes as a protective mechanism associated with host phagocytosis in virulent _P. aeruginosa_ strains [39]. In Figure 8, a heatmap of the results regarding the production of pyocyanins by the PAO1 strains shows that all mutant strains had a lower production of pyocyanins when compared to the wild strain, in the absence of the antibiotic. With the inoculation of antibiotics, there was a lower production of pyocyanins in all strains tested due to the bactericidal effect of the CIP [40]. Pyocyanins are inducers of host oxidative stress, increasing the intracellular levels of reactive oxygen species. It has also been suggested by some authors that pyocyanins induce biofilm formation by promoting the interactions between PAO1 cells [41]. Regarding the results for the phage assay, in Figure 9, the mutant strains show significantly lower absorbance values than the wild strain of PAO1 (\(p<0.0001\)), indicating that these three genes may be playing an important role or/and may affect the proliferation of this bacterium. Secondly, the phage cocktail used was significantly effective in all the PAO1 strains tested in this study, with lower growth density when compared to the control (\(p<0.0001\)). Finally, the distribution of the results with the phage inoculation was lower than the mean of the control of these strains; otherwise, the distribution of the results obtained between the phages and the wild strain was higher and lower than the mean of the control of the growth of this bacteria. Therefore, the genes Rub A1, Rub A2, and GLRX may be involved in the ability of this strain to resist phage infection.