## 4 Discussion  
The study aimed to investigate the impact of three mutations (Rub A1, Rub A2, and GLRx) on bacterial quorum sensing modulation, including gene expression, virulence trait production, viability compared to PAO1, susceptibility to ciprofloxacin, and susceptibility to a phage cocktail for bacterial infections. For that, we started by assessing the expression of the genes _lasl_, _las_R, _pqs_A, _nvf_R, and _rps_L in the three mutant strains and comparing to the wild strain. Figure 8: Comparison of the pyocyanin produced in _Pseudomonas aeruginosa_ wild type (PAO1) to the three mutant strains of PAO1 (rubredoxins: strains Rub A1 and Rub A2; a glutaredoxin: strain GLRX) with and without the use of the antibiotic ciprofloxacin (CIP) at the concentrations of 0.125 and 0.25 \(\upmu\)g/mL. Figure 9: The effect of the phage cocktail mix was tested for _Pseudomonas aeruginosa_ wild type (PAO1) and three mutant strains of PAO1 (rubredoxins: strains Rub A1 and Rub A2; a glutaredoxin: strain GLRX) and measured by spectrophotometry at 600 nm. The distribution of the values in absorbance, at 600 nm, was compared between the strains with and without the inoculation of the phage cocktail. Considering the role of the mutated genes, the silencing of each of them in each of the mutants studied should implicate a decrease in some of the production of virulence traits, such as protease LasA, which are regulated by the gene _las_R [28], and rhamnoidy and pyocyanin production, that is regulated by the gene _rhl_R [29]. The results in Figure 3 agree with the literature, showing an overall decrease in gene expression, in particular for genes _LasR_ and _rhl_R, both associated with antioxidant protection, for all mutants in comparison with the wild type. Worthy of mentioning is that although the 2-\(\Delta\)\(\Delta\)CT method is considered the gold standard of gene quantification [30], the interactions between the genes were not investigated in this study. For this, high-density DNA microarrays could be a useful methodology to understand the dynamic of the gene interactions and responses to the different environment conditions applied [31]. A representation in a heatmap for the viability of each tested strain in each concentration of CIP used can be observed in Figure 4. In this assay, the mutant strains Rub A2 and GLRX were significantly the most susceptible (\(p<0.0001\)) and the most resistant (\(p<0.05\)), respectively, to the CIP's lower concentration when compared to the wild strain. The mutant Rub A1 did not show a significant difference in the viability cell numbers in the lower concentration of CIP when compared to the wild strain, only in the higher CIP concentration (\(p<0.001\)). Rub A2 also showed vulnerability to the higher CIP concentration (\(p<0.0001\)). GLRX did not show significant results when compared to the wild strain at higher concentration of CIP. The inhibition of catalase and rubredoxin reductase may be related to the increase in bacterial membrane lipid peroxidation, and this fact may have contributed to an increase in the susceptibility of the mutant Rub A2 to the CIP concentrations tested [32]. The non-significant result observed in the other mutant strain, Rub A1, may be related to the fact that, although Rub A1 and Rub A2 share 80% of the amino acid sequence, the differences in the substitutions can change the molecular surface and consequently modify the electron acceptors of each Rdxs [17]. GLRX function in \(P\). _aeruginosa_ is still unclear, although [PERSON] et al. found recently that this gene is involved in the maturation of [Fe-S] clusters [19]. Since iron can act as a cofactor for many enzymes, our suggestion for the result obtained is that the iron metabolism might be affected [33] and consequently the lack of a cofactor may have changed the structure of the main target of CIP, bacterial DNA topoisomerase and gyrase [3], leading to the resistance observed in the GLRX mutant. Further studies are necessary to assess this suggestion.