Prevalence of genetically similar Flavobacterium columnare phages across aquaculture environments reveals a strong potential for pathogen control.

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden in 2017. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 227 bacterial isolates (from 2013 to 2017) demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole-genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential for use in phage therapy.

Comparison of Delivery Methods in Phage Therapy against Flavobacterium columnare Infections in Rainbow Trout.

Viruses of bacteria, bacteriophages, specifically infect their bacterial hosts with minimal effects on the surrounding microbiota. They have the potential to be used in the prevention and treatment of bacterial infections, including in the field of food production. In aquaculture settings, disease-causing bacteria are often transmitted through the water body, providing several applications for phage-based targeting of pathogens, in the rearing environment, and in the fish. We tested delivery of phages by different methods (via baths, in phage-coated material, and via oral delivery in feed) to prevent and treat Flavobacterium columnare infections in rainbow trout fry using three phages (FCOV-S1, FCOV-F2, and FCL-2) and their hosts (FCO-S1, FCO-F2, and B185, respectively). Bath treatments given before bacterial infection and at the onset of the disease symptoms were the most efficient way to prevent F. columnare infections in rainbow trout, possibly due to the external nature of the disease. In a flow-through system, the presence of phage-coated plastic sheets delayed the onset of the disease. The oral administration of phages first increased disease progression, although total mortality was lower at the end of the experiment. When analysed for shelf-life, phage titers remained highest when maintained in bacterial culture media and in sterile lake water. Our results show that successful phage therapy treatment in the aquaculture setting requires optimisation of phage delivery methods in vivo.

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and the Type IX Secretion System.

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.

Interactions between Rainbow Trout Eyed Eggs and Flavobacterium spp. Using a Bath Challenge Model: Preliminary Evaluation of Bacteriophages as Pathogen Control Agents.

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.

Association of colony morphotypes with virulence, growth and resistance against protozoan predation in the fish pathogen Flavobacterium columnare.

Many opportunistic pathogens can alternate between inside- and outside-host environments during their life cycle. The opportunistic fish pathogen Flavobacterium columnare is an inhabitant of the natural microbial community and causes significant yearly losses in aquaculture worldwide. The bacterium grows in varying colony morphotypes that are associated with either virulence (rhizoid type) or resistance to starvation and phages (rough type). Rough type strains can arise spontaneously or can be induced by phage infection. To identify the determinants of morphotype fitness, we measured virulence, growth parameters, biofilm-forming ability and resistance to amoeba and ciliate predation of both morphotypes in thirteen F. columnare strains. The (phage-sensitive) rhizoid type had a fitness advantage over the rough type in virulence, growth rate and maximum population size. Phage-induced rough type was found to be significantly weakest in resisting both ciliate and amoeba predation, and produced more biofilm in the presence of amoebae, whereas the spontaneous rough types did not differ from rhizoid in biofilm production. In co-culture experiment, the ciliate population sizes were higher when co-cultured with rough type than with rhizoid type. Our results thus suggest that the resistance to phages and starvation of the F. columnare rough type may have strong a trade-off, as the performance of the ancestral rhizoid type is better under environmental conditions.

Starvation can diversify the population structure and virulence strategies of an environmentally transmitting fish pathogen.

BACKGROUND: Generalist bacterial pathogens, with the ability for environmental survival and growth, often face variable conditions during their outside-host period. Abiotic factors (such as nutrient deprivation) act as selection pressures for bacterial characteristics, but their effect on virulence is not entirely understood. "Sit and wait" hypothesis expects that long outside-host survival selects for increased virulence, but maintaining virulence in the absence of hosts is generally expected to be costly if active investments are needed. We analysed how long term starvation influences bacterial population structure and virulence of an environmentally transmitting fish pathogen Flavobacterium columnare. RESULTS: F. columnare populations in distilled water and in lake water were monitored for 5 months. During the experiment, the population structure of F. columnare diversified by rough and soft colony morphotypes appearing among the ancestral rhizoid ones. After 5 months starvation in lake water, the virulence of the starved and ancestral bacterial isolates was tested. The starved rhizoid isolates had significantly higher virulence than the ancestral rhizoid, whereas the virulence of the rough isolates was low. CONCLUSIONS: We suggest that F. columnare population diversification is an adaptation to tolerate unpredictable environment, but may also have other biological significance. Maintaining and increasing virulence ensures efficient invasion into the host especially under circumstances when the host density is low or the outside-host period is long. Changing from rhizoid into a rough morphotype has trade-offs in making bacteria less virulent and unable to exploit the host, but may ensure bacterial survival under unpredictable conditions. Our study gives an example how abiotic selection can diversify virulence of environmentally transmitting bacterial pathogen.

Environment may be the source of Flavobacterium columnare outbreaks at fish farms.

Flavobacterium columnare, causing columnaris disease, was isolated for the first time from free water and biofilms in the environment outside fish farms. Fourteen isolates were found from Central Finland from a river by a water intake of a salmonid farm and 400 m upstream of the farm. One isolate was from a lake not under the influence of any fish farming. The bacterium could not be isolated from five other lakes in Central Finland or from three lakes in Eastern Finland, none of them in use for fish farming. Among the environmental isolates there was both genetic variability and difference in virulence, but the isolates were less virulent than the isolates originating from a disease outbreak at a fish farm. The isolates were able to survive for months outside the fish host and also to change their colony morphology, a phenomenon probably used as a survival strategy for F. columnare. This indicates that waters upstream of fish farms are a potential source of columnaris outbreaks at the farms during the summer. The results support the hypothesis that fish farms and farming practices may select for the virulent strains of F. columnare occurring in environmental waters to cause the infections at the farms.

The efficacy of two immunostimulants against Flavobacterium columnare infection in juvenile rainbow trout (Oncorhynchus mykiss).

Bacterium Flavobacterium columnare is the causative agent of columnaris disease in many wild and farmed fish species. Immunostimulants are used with success in aquaculture against many pathogens, but the ability to improve innate resistance to columnaris disease has not been studied. Fingerling rainbow trout were treated with two immunostimulants, yeast beta-glucan and beta-hydroxy-beta-methylbutyrate (HMB). Selected innate immune function parameters, the production of reactive oxygen species (ROS) by whole blood and by isolated head kidney leukocytes, plasma lysozyme activity and complement bacteriolytic activity, were determined to assess the immune status of fish. The fish were then bath challenged with virulent F. columnare bacteria, and the mortality of fish was recorded. Given orally both stimulants raised the levels of immune function parameters, but did not improve survival in challenge at any concentration of the stimulants used. Intra peritoneal injection of beta-glucan increased parameter values several fold, but no beneficial effect of injected glucan on survival was noted. As a control, antibiotic medication administered prior to and during the challenge infection prevented the mortality. Innate immune mechanisms, even when induced to high levels with immunostimulants, as evidenced here, were not able to increase resistance against F. columnare. This may be connected to the external character of the infection. The results from the treatments with beta-glucan and HMB suggest that there is little prospect of preventing columnaris disease by means of immunostimulants in early life stage of rainbow trout. However, the efficacy of other immune stimulants remains open.

Saprophytism of a fish pathogen as a transmission strategy.

Fish farming creates conditions where disease transmission is enhanced and antibiotic treatments are commonly used to cure bacterial diseases to prevent severe losses due to infections. Ability to persist in such an environment has been suggested to lead to the evolution of high virulence. Columnaris disease caused by Flavobacterium columnare is a growing problem in freshwater fish farming. Transmission of the disease is poorly known, and survival of F. columnare in the rearing environment has not been studied. This paper addresses both transmission of columnaris disease and survival strategy of F. columnare. Saprophytic activity of F. columnare was studied by infecting rainbow trout fingerlings before and immediately after death and by following bacterial shedding from the fish carcasses. From fish killed immediately after infection, bacteria were shed at high rates for 5 days, and from fish exposed to F. columnare post mortem for 8 days. In another experiment, rainbow trout fingerlings were experimentally infected with F. columnare and monitored for transmission of the bacteria post infection until and after the death of the fish. The transmission of columnaris disease to living rainbow trout was the most efficient from dead fish, from which bacteria were shed into water at higher rates than from living fish. We also found that F. columnare can survive at least for 5 months in both sterilized distilled and lake water. These results show that death of the host causes no cost for F. columnare; it thrives in alive and dead fish, and in water. Saprophytism may have been a transition stage to pathogenicity of this originally harmless water bacterium, and maintained as an effective transmission and survival strategy of F. columnare. Our findings also suggest that F. columnare may be able to persist in the rearing environment during antibiotic treatments of the living fish.

Flavobacterium columnare colony types: connection to adhesion and virulence?

Four different colony morphologies were produced by Flavobacterium columnare strains on Shieh agar plate cultures: rhizoid and flat (type 1), non-rhizoid and hard (type 2), round and soft (type 3), and irregularly shaped and soft (type 4). Colonies produced on AO agar differed from these to some extent. The colony types formed on Shieh agar were studied according to molecular characteristics [Amplified Fragment Length Polymorphism (AFLP), Automated Ribosomal Intergenic Spacer Analysis (ARISA), and whole cell protein SDS-PAGE profiles], virulence on rainbow trout fingerlings, and adhesion on polystyrene and fish gills. There were no molecular differences between colony types within one strain. Type 2 was the most adherent on polystyrene, but type 1 was the most virulent. Adhesion of F. columnare strains used in this study was not connected to virulence. From fish infected with colony type 1, three colony types (types 1, 2 and 4) were isolated. Contrary to previous studies, our results suggest that strong adhesion capacity may not be the main virulence factor of F. columnare. Colony morphology change might be caused by phase variation, and different colony types isolated from infected fish may indicate different roles of the colony morphologies in the infection process of columnaris disease.