## 3 Results
### Hfq Is Required for Antibiotics Persistence in Y. ruckeri  
To analyze the role of Hfq in antibiotic persistence of _Y. ruckeri_, susceptibility of stationary-phase cells of the \(\Delta\)_Hfq_ and the wild-type strains was examined during the exposure to lethal concentrations of bactericidal antibiotics (time-kill curves) as an indicator for persister cells production. Survival was significantly diminished in the \(\Delta\)_Hfq_ strain upon treatment with ampicillin for 48 h (3-fold), oxolinic acid for 72 h (4-fold), ciprofloxacin for 72 h (\(\sim\)4-fold), polymyxin B for 48 h (\(\sim\)3-fold) and florfenicol for 42 h (\(\sim\)2-fold) (Figure 3a-e), indicating that the persister cells production is affected in the \(\Delta\)_Hfq_ strain compared with the wild type. Since the age of inoculum strongly influences persister cells formation [6,24,25], we analyzed whether Hfq also mediates the persister cells production (24 h) from bacteria at exponential phase of growth (OD\({}_{600}\) of 0.3). We observed that the survival of the \(\Delta\)_Hfq_ strain was also decreased, but to a lesser extent, compared to the wild type (Figure 3f). Since persisters have also been described as dormant subpopulations arising within bacterial biofilms that are characteristically highly tolerant to antibiotics [26], we also analyzed the  
Figure 2: Growth curves of the WT and \(\Delta\)_Hfq_ strains exposed to sublethal concentrations of antibiotics. WT and \(\Delta\)_Hfq_ strains were grown in TSB medium supplemented with 0.01 mg/mL ampicillin **(a)**, 0.0001 mg/mL oxolinic acid **(b)**, 0.001 mg/mL ciprofloxacin **(c)**, 0.001 mg/mL florfenicol **(d)** and 0.002 mg/mL polymyxin B **(e)**. OD\({}_{600}\) was measured every 30 min until the bacteria reached the stationary phase (16 h). Data represent the means \(\pm\) standard deviations (\(n=3\)). time-kill curves using resuspended cells that had just been sampled from 24 h-biofilms from both \(\Delta\)_lifq_ and the wild-type strain. As previously reported in other persister assays with cells suspension from biofilm cultures [27], we observed that cells harvested from biofilms produce more persister cells than those from liquid cultures. However, persister cells production in the \(\Delta\)_lifq_ strain was also decreased for all the antibiotics treatments for 24 h, in comparison with the wild-type (Figure 3g). These results indicate that Hfq was involved in antibiotic persistence in bacteria harvested from liquid and biofilm cultures. The Effect of Hfq on Persister Cells Production Is Further Aggravated in the \(\Delta\)_relA_ ((p)ppGpp synthetase) Genetic Background  
To better understand the role of Hfq in antibiotic persistence and since it is well known that (p)ppGpp is a key regulator of persistence [28], we evaluated whether the role of Hfq in persister cells production is dependent on ReLA, the (p)ppGpp synthetase described as a central regulator of persistence [29]. To address this purpose, we constructed the single and double knockout mutants for the _relA_ gene, namely, the \(\Delta\)_relA_ and \(\Delta\)_relA_\(\Delta\)_lifq_ strains. We determined the persister levels of the single and double knockout mutants \(\Delta\)_relA_, \(\Delta\)_lifq_, \(\Delta\)_relA_\(\Delta\)_lifq_ and the wild-type strain from stationary phase cultures treated with lethal concentrations of ampicillin (1 mg mL\({}^{-1}\)), ciprofloxacin (0.05 mg mL\({}^{-1}\)), florfenicol (0.01 mg mL\({}^{-1}\)), oxolinic acid (0.05 mg mL\({}^{-1}\)) and polymyxin B (1 mg mL\({}^{-1}\)) for 24 h. We  
Figure 3: Time-kill curves and persister cells production of the WT and \(\Delta\)_lifq_ strains exposed to antibiotics. Stationary cells of the WT and \(\Delta\)_lifq_ strains were exposed to 1 mg mL\({}^{-1}\) ampicillin (**a**), 0.05 mg mL\({}^{-1}\) oxolinic acid (**b**), 0.05 mg mL\({}^{-1}\) ciprofloxacin (**c**), 0.01 mg mL\({}^{-1}\) forfenicol (**d**) or 1 mg mL\({}^{-1}\) polymyxin B (**e**), and the survival of persister cells was measured as CFUs per 1 mL at different time points.