## 4 Discussion  
Vaccination against the major serotypes of _Y. ruckeri_ is used in most fish farms worldwide, although this practice is not effective in all cases [10], and the emergence of outbreaks has substantially increased since 2003 [11]. This situation has awakened interest to investigate the molecular mechanisms of virulence [10; 18], and the antimicrobial resistance in _Y. ruckeri_ isolates from aquaculture [13; 14; 30; 31]. However, studies focused on persister cells production, i.e., the formation of cells highly tolerant to killing by antibiotics, have not been reported to date. In the present study, we investigated the contribution of the sRNA chaperone Hfq from _Y. ruckeri_ in persistence to multiple antibiotics, using an isolate from a salmon farm in Chile. Among the most frequently used antimicrobials to treat ERM and other bacterial diseases affecting aquaculture, we can mention oxytterracycline and florfenicol [13; 14; 15; 16]. Other antibiotics used, but to a lesser extent, are oxolinite acid and amoxicillin [15; 16]. ERM outbreaks have been commonly related to the bacterial resistance to these antibiotics [13; 17; 32]. However, persister cells could also be responsible for outbreaks and the recalcitrance of infections, since they remain viable and repopulate the fish tanks forming biofilms when the level of antibiotics drops [26]. Persisters are thought to be less sensitive to antimicrobials because they are not undergoing cellular processes that antibiotics can affect, i.e., these cells are in an arrest-growth state that enables antibiotic tolerance. Hfq is a global regulator that impacts growth and metabolism in many bacteria, including some human pathogens of the _Yersinia_ genus [22; 23; 33]. However, contrary to what is commonly observed in those bacteria, the _hftq_ deletion in _Y. ruckeri_ resulted in faster growth than the wild-type (Figure 1a), as well as in a more active metabolic state (Figure 1b-d). These results suggest global modulatory roles for Hfq in the restraint of growth and/or energy metabolism in this  
Figure 5: Bacterial replication and metabolic status of the \(\Delta\)_relA_ and \(\Delta\)_relA_\(\Delta\)_lfq_ strains. (**a**) WT, \(\Delta\)_lfq_, \(\Delta\)_relA_ and \(\Delta\)_relA_\(\Delta\)_lfq_ strains were grown in TSB to OD\({}_{600}\) of 0.9 and the intracellular ATP levels were determined. Asterisks represent statistical differences with respect to the WT strain (**_p_ < 0.001; ***_p_ = 0.0001). Data represent the means \(\pm\) standard deviations (\(n\) = 3). (**b**) Flow cytometric detection of DsRed and EGFP fluorescence at different time points from the \(\Delta\)_relA_ and \(\Delta\)_relA_\(\Delta\)_lfq_ strains (carrying pDiGc plasmid) cultured in TSB medium for 12 h (\(n\) = 30,000 events analyzed at each time point). bacterium. To our knowledge, similar phenotypes have been previously described in this and other species lacking Hfq-dependent sRNAs [18; 34; 35], but not in _Hfq_ knockout mutants from other species, where the phenotypes are even dissimilar. For example, in _Aeromonas veronii_, the knockout of the _Hfq_ gene caused a slower growth rate at the exponential phase and produced less cell mass at the stationary phase [5]. By contrast, the knockouts of _Hfq_ in other species such as _Y. pestis_[33], _E. coli_[36] and _S. Typhimuriumium_[37] were also affected on growth but in _Y. pseudotuberculosisis_ displayed no effect [33]. Although Hfq is highly conserved among bacteria, homologs from different species may exhibit variations in their functions intimately linked to the lifestyle or niches that bacteria inhabit. As we expected, given that reduced ATP levels have been associated with diminished antibiotic efficacy and vice versa [38; 39], the _Y. ruckeri Alftq_ strain was more susceptible to most of the antibiotics tested under the sub-lethal concentrations evaluated (Figure 2, Table 2).