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Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, twenty-seven phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence, and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage resistant phenotypes.

Increasing problems with antibiotic resistance have directed interest toward phage therapy in the aquaculture industry. However, phage resistance evolving in target bacteria is considered a challenge. To investigate how phage resistance influences the fish pathogen Flavobacterium columnare, two wild-type bacterial isolates, FCO-F2 and FCO-F9, were exposed to phages (FCO-F2 to FCOV-F2, FCOV-F5, and FCOV-F25, and FCO-F9 to FCL-2, FCOV-F13, and FCOV-F45), and resulting phenotypic and genetic changes in bacteria were analyzed. Bacterial viability first decreased in the exposure cultures but started to increase after 1 to 2 days, along with a change in colony morphology from original rhizoid to rough, leading to 98% prevalence of the rough morphotype. Twenty-four isolates (including four isolates from no-phage treatments) were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion, and biofilm formation, protease activity, whole-genome sequencing, and virulence in rainbow trout fry. The rough isolates arising in phage exposure were phage resistant with low virulence, whereas rhizoid isolates maintained phage susceptibility and high virulence. Gliding motility and protease activity were also related to the phage susceptibility. Observed mutations in phage-resistant isolates were mostly located in genes encoding the type IX secretion system, a component of the Bacteroidetes gliding motility machinery. However, not all phage-resistant isolates had mutations, indicating that phage resistance in F. columnare is a multifactorial process, including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections since phage resistance is associated with decreases in bacterial virulence. IMPORTANCE Phage resistance of infectious bacteria is a common phenomenon posing challenges for the development of phage therapy. Along with a growing world population and the need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by Flavobacterium columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce the development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-susceptible isolates and thus not a challenge for phage therapy against columnaris disease. This is valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Phage-based approaches have gained increasing interest as sustainable alternative strategies to antibiotic treatment or as prophylactic measures against disease outbreaks in aquaculture. The potential of three methods (oral, bath, and injection) for delivering a two-component phage mixture to rainbow trout fry for controlling Flavobacterium psychrophilum infections and reduce fish mortality was investigated using bacteriophages FpV4 and FPSV-D22. For the oral administration experiment, bacteriophages were applied on feed pellets by spraying (1.6 × 108 PFU g–1) or by irreversible immobilization (8.3 × 107 PFU g–1), using the corona discharge technology (Fixed Phage Ltd.). The fish showed normal growth for every group and no mortality was observed prior to infection as well as in control groups during the infection. Constant detection of phages in the intestine (∼103 PFU mg–1) and more sporadic occurrence in kidney, spleen, and brain was observed. When fish were exposed to F. psychrophilum, no significant effect on fish survival, nor a direct impact on the number of phages in the sampled organs, were detected. Similarly, no significant increase in fish survival was detected when phages were delivered by bath (1st and 2nd bath: ∼106 PFU ml–1; 3rd bath: ∼105 PFU ml–1). However, when phages FpV4 and FPSV-D22 (1.7 × 108 PFU fish–1) were administered by intraperitoneal injection 3 days after the bacterial challenge, the final percent survival observed in the group injected with bacteriophages FpV4 and FPSV-D22 (80.0%) was significantly higher than in the control group (56.7%). The work demonstrates the delivery of phages to fish organs by oral administration, but also suggests that higher phage dosages than the tested ones may be needed on feed pellets to offer fish an adequate protection against F. psychrophilum infections.