To develop effective live attenuated bacterial vaccines, rifampicin-resistant strategy has been successfully used to
develop the two commercially available vaccines in aquaculture: Edwardsiella ictaluri (AquaVac-ESC) and Flavobacterium columnare (AquaVac-COL) [33,34]. However, it is not clear whether the rifampicin-resistant strategy or other antibiotic-resistant strategy could also be used to attenuate A. hydrophila for the purpose of novel vaccine development. Novobiocin, a natural antibiotic produced by the actinomycete Streptomyces niveus, is a member of the order Actinobacteria [35]. Novobiocin works as a natural inhibitor of bacterial DNA gyrase, resulting in bacterial cell-death [36]. DNA gyrase, an ATP-dependent enzyme that acts by creating a transient double-stranded DNA break, is essential for efficient DNA replication, transcription, and recombination by catalyzing the negative supercoiling of DNA [37]. Recently, novobiocin-resistance strategy has been successfully used to attenuate Gram-positive Streptococcus iniae and Gram-negative E. ictaluri [38,39]. Therefore, the objectives of this study were to determine: (1) whether resistance to rifampicin or to novobiocin or to a combination of the two antibiotics (hereafter called N + R) could be used to attenuate virulent
A. hydrophila; and (2) whether attenuated A. hydrophila could be used as live vaccine to protect channel catfish or Nile tilapia frominfections by its virulent parent A. hydrophila.