## 4 Discussion
Differential increased susceptibility to different classes of antibiotics has also been described in other _lifq_ deletion strains such as _Pseudomonas aeruginosa_[40], _E. coli_[41], _Salmonella_ Typhimuriumium [42] and _Proteus mirabilis_[43], indicating that the regulatory functions of Hfq enable bacteria to adapt to multiple antibiotic challenges. Similarly, some studies have demonstrated that some Hfq functions involved in antibiotic susceptibility are indirectly regulated via multiple sRNAs [44; 45], thus explaining the pleiotropic phenotypes observed in _hfq_ knockouts of different species. For its part, the knowledge on the impact of Hfq on antibiotic persistence is still limited. Previous studies have reported discrepant results regarding the persister cells formation in _Hfq_ knockouts backgrounds. While in _A. veroniii_ the deletion of _lifq_ reduced the persister cells formation at different extents, under treatment with ciprofloxacin, cefotaxime, chloramphenicol and tetracycline [5], in _E. coli_, the deletion of _lifq_ increased persister cells production by 11-fold under treatment with ampicillin [8]. Another study with the uropathogenic _E. coli_ strain UIT89 revealed that the sRNA RyhB mediates the persister cells production to levofloxacin, cefotaxime and gentamicin in an Hfq-independent manner; however, the single deletion of _lifq_ also has a detrimental effect on persister cells formation [7]. Together, our results and the data obtained from other studies reporting differential phenotypes for _hfq_ mutants highlight the complexity of the persistence phenomenon governed by many different and heterogeneous molecular mechanisms and factors. Among these factors, the bacterial genetic background that evolved from their different lifestyles also played a pivotal role. Since greater ATP levels are predictive of bactericidal antibiotic efficacy, and taking into account that the downregulation of transcription of genes involved in energy production and the ATP depletion are conditions that explain bacterial tolerance to antibiotics [38; 39; 46], we hypothesized that the _Y. ruckeri Alftq_ strain would be affected in persister cells formation. Our persister assays were conducted with five classes of antibiotics with bactericidal properties, whose choice was also based on their aquaculture usage. As we expected, the _lifq_ deletion mutant showed defects in persister cells formation to different extents for all the antibiotics tested. This result is consistent with previous reports where \(\Delta r\)_yhB_ (sRNA) and \(\Delta\)_phoU_ (regulator for phosphate metabolism) mutants from _E. coli_ with hyperactive metabolic states have significant defects in persistence [7; 24]. A previous study showed that metabolic stimulation can be used to eliminate persisters [47], suggesting that Hfq might be a potential target for anti-persisters strategies. As the central stringent-response regulator, ReLA represents the main (p)ppGpp synthetase, whose deficiency affects the production of persister cells [48]. We observed a significant reduction in persister cells production when the \(\Delta\)_relA_ mutant was treated with the five antibiotics (Figure 4). The reduction in persister levels of the \(\Delta\)_relA_ strain was comparable to that of the \(\Delta\)_relA_ strain for each antibiotic treatment, being more drastic than the effect observed in the \(\Delta\)_lfq_ strain, indicating that _relA_ is epistatic over _hfq_. This epistatic effect was further confirmed when the ATP levels were measured from stationary cultures without antibiotic treatments, as well as the replication analysis (Figure 5). As mentioned above, ReLA is the major synthetase of (p)ppGpp, the second messenger that plays a crucial role in persister formation. By controlling diverse physiological processes, (p)ppGpp leads to dormancy or slow growth in bacteria [29; 49]. In addition, it is known that, in _E. coli_, RelA fulfills functions independent of its (p)ppGpp synthetase activity.