Abstract
Yersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (Δhfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the Δhfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the Δhfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ΔrelA and ΔrelAΔhfq strains displayed similar defects in persister cells formation, but higher than Δhfq strain. Similarly, stationary cultures of the ΔrelA and ΔrelAΔhfq strains exhibited comparable levels of ATP but higher than that of the Δhfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon.
Highlights
IntroductionPersisters are a subpopulation of slowgrowing or growth-arrested bacterial cells with a decreased susceptibility to bactericidal antibiotics within a susceptible clonal population [3]
Since Hfq is a global regulator that impacts growth and metabolism in bacteria [22,23], we first analyzed these processes in Y. ruckeri cultures
The bacterial replication in the culture medium was accurately determined by a fluorescence dilution (FD) assay based on a replication reporter system, as previously described [18]
Summary
Persisters are a subpopulation of slowgrowing or growth-arrested bacterial cells with a decreased susceptibility to bactericidal antibiotics within a susceptible clonal population [3]. Hfq in persister cells production is dependent on. RelA, the (p)ppGpp synthetase dein persister cells production is dependent on RelA, the (p)ppGpp synthetase described scribed as a regulator central regulator of persistence [29]. To address this purpose, we constructed as a central of persistence [29]. We determined the persister the single and double knockout mustrains. We determined the persister levelslevels of theofsingle and double knockout mutants tants ΔrelA, Δhfq,∆hfq.
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