## 2 Materials and Methods
Sample were adjusted to 10\({}^{8}\) cells/mL in TSB and exposed to antibiotics (1 mg mL\({}^{-1}\) ampicillin, 0.05 mg mL\({}^{-1}\) ciprofloxacin, 0.01 mg mL\({}^{-1}\) forfenicol, 0.05 mg mL\({}^{-1}\) oxaliplatin acid or 1 mg mL\({}^{-1}\) polyvinyl B), at desired time points and washed in PBS before plating on TSA without antibiotics to determine CFU count. Growth curves: Bacteria were cultured in TSB media at 26 \({}^{\circ}\)C overnight, diluted into 100 mL of fresh TSB medium at a ratio of 1:100, and incubated with shaking at 200 rpm until stationary phase. When required, the cultures were treated with sublethal concentrations of each antibiotic (ampicillin, oxolinic acid, ciprofloxacin, florenicol and polymyxin B). The OD\({}_{600}\) was measured every 30 min until stationary phase of growth (16 h), using a Synergy H1 microplate reader (Biotek). The experiments were repeated three times. Intracellular ATP levels and NAD\({}^{+}\)/NADH ratios: Bacteria were grown in TSB until OD\({}_{600}\) 0.9 and the quantification of ATP and NAD\({}^{+}\)/NADH was performed with the ATP Fluorometric Assay Kit and NAD\({}^{+}\)/NADH quantification kit, respectively (BioVision \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strain** & **Characteristics** & **Reference** \\ \hline _Y. ruckeri_ CD2 & Wild-type strain of _Y. ruckeri_ & [18] \\ _Alfq_ & _Y. ruckeri_ CD2 lacking _lfq_ gene & [18] \\ \(\Delta\)_relA_ & _Y. ruckeri_ CD2 lacking _relA_ gene & This study \\ \(\Delta\)_relA_ \(A\) _lfq_ & _Y. ruckeri_ CD2 lacking _relA_ and _lfq_ genes & This study \\ \hline
**Plasmid** & **Genotype** & **Reference** \\ \hline pCM433 & \begin{tabular}{c} Ap\({}^{R}\), Cm\({}^{R}\), Tc\({}^{R}\), oriT, tet, cat, _sacB_ and ColEI ori; \\ broad-host-range sacb-based allelic exchange vector \\ pCM433-_lfq_ \\ pCM433-_lfq_ \\ pCM433-_relA_ \\ pBR322 & \
\begin{tabular}{c} ApR, TcR, ColEl Ori. \\ \end{tabular} &
\begin{tabular}{c} [20] \\ \end{tabular} \\ pHfq & _Infq_ region of _Y. ruckeri_ CD2 cloned into pBR322 & This study \\ pDiGc & _bla_ GFP pBAD DsRed ori f1 Amp\({}^{R}\) & [21] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Bacterial strains and plasmids used in this study. Research Products, Milpitas, CA, USA), as previously described [18]. The ATP levels and NAD\({}^{+}\)/NADH ratios were normalized to the bacterial cell concentration. Measurement of bacterial replication by fluorescence dilution: The procedure was performed as previously described [18] with some modifications. Bacterial strains carrying pDiGc plasmid [21] were aerobically grown overnight at 26 \({}^{\circ}\)C in TSB medium supplemented with 4% arabinose to allow production of red fluorescent protein. These bacteria were purified by cell sorting using FACSAria III (Becton Dickinson, New Jersey, USA). Bacteria (6 \(\times\) 10\({}^{6}\) cells) were resuspended in 2 mL of TSB and incubated at 26 \({}^{\circ}\)C for 12 h. Aliquots of 200 \(\mu\)L were taken at different times (0, 3, 6, 9 and 12 h), and bacteria were analyzed using a FACSCalibur flow cytometer (Becton Dickinson). Data were analyzed using FlowJo 8.6.3 software. Statistics: The data was statistically analyzed with Student's \(T\)-test. Values of \(p<\) 0.05 were considered statistically significant.