Genetic-based evaluation of management units for sustainable vendace (Coregonus albula) fisheries in a large lake system

Abstract The goal of the processing industry, trade and consumers is to get eco-labelled freshwater fish products from sustainable fisheries into the market as soon as possible. The fourth largest natural lake system in Europe, the Saimaa lake system supports a fishery for vendace (Coregonus albula). Certification of the fishery requires an understanding of population structure to help determine the number and spatial extent of management units. In this study, we analysed the genetic diversity of local vendace populations in the Saimaa lake system and aimed to identify the conservation and management units of vendace. Within the Saimaa, the genetic divergence between local populations of ve…

Additional file 6 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

The concatenated tree based on six housekeeping MLSA genes (including trpB; right) and five MLSA gene sequences (rpoD, dnaK, tuf, gyrB, atpA; left). The arrow shows disagreement concerning the position of strain G2 (B399) between the two trees. (PDF 77 kb)

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

Summary 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…

Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria

The mucosal surfaces of animals are habitat for microbes, including viruses. Bacteriophages—viruses that infect bacteria—were shown to be able to bind to mucus. This may result in a symbiotic relationship in which phages find bacterial hosts to infect, protecting the mucus-producing animal from bacterial infections in the process. Here, we studied phage binding on mucus and the effect of mucin on phage-bacterium interactions. The significance of our research is in showing that phage adhesion to mucus results in preventive protection against bacterial infections, which will serve as basis for the development of prophylactic phage therapy approaches. Besides, we also reveal that exposure to m…

ICTV Virus Taxonomy Profile : Finnlakeviridae

Finnlakeviridae is a family of icosahedral, internal membrane-containing bacterial viruses with circular, single-stranded DNA genomes. The family includes the genus, Finnlakevirus, with the species, Flavobacterium virus FLiP. Flavobacterium phage FLiP was isolated with its Gram-negative host bacterium from a boreal freshwater habitat in Central Finland in 2010. It is the first described single-stranded DNA virus with an internal membrane and shares minimal sequence similarity with other known viruses. The virion organization (pseudo T=21 dextro) and major capsid protein fold (double-β-barrel) resemble those of Pseudoalteromonas phage PM2 (family Corticoviridae), which has a double-stranded …

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…

Occurrence of antibiotics and risk of antibiotic resistance evolution in selected Kenyan wastewaters, surface waters and sediments

Active pharmaceutical ingredients, especially antibiotics, are micropollutants whose continuous flow into hydrological cycles has the potential to mediate antibiotic resistance in the environment and cause toxicity to sensitive organisms. Here, we investigated the levels of selected antibiotics in four wastewater treatment plants and the receiving water bodies. The measured environmental concentrations were compared with the proposed compound-specific predicted no-effect concentration for resistance selection values. The concentration of doxycycline, amoxicillin, sulfamethoxazole, trimethoprim, ciprofloxacin and norfloxacin within the influents, effluents, surface waters and river sediments…

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

supplementary information and data from Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentration of antibiotic affects the co-infection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum, in their fish host. We found that antibiotic in fee…

Phage Specificity of the Freshwater Fish Pathogen Flavobacterium columnare

ABSTRACT Flavobacteria and their phages were isolated from Finnish freshwaters and fish farms. Emphasis was placed on finding phages infecting the fish pathogen Flavobacterium columnare for use as phage therapy agents. The host ranges of the flavobacterial phages varied, phages infecting F. columnare being more host specific than the other phages.

Prophages and Past Prophage-Host Interactions Revealed by CRISPR Spacer Content in a Fish Pathogen

The role of prophages in the evolution, diversification, or virulence of the fish pathogen Flavobacterium columnare has not been studied thus far. Here, we describe a functional spontaneously inducing prophage fF4 from the F. columnare type strain ATCC 23463, which is not detectable with commonly used prophage search methods. We show that this prophage type has a global distribution and is present in strains isolated from Finland, Thailand, Japan, and North America. The virions of fF4 are myoviruses with contractile tails and infect only bacterial strains originating from Northern Finland. The fF4 resembles transposable phages by similar genome organization and several gene orthologs. Addit…

Long-term genomic coevolution of host-parasite interaction in the natural environment

Antagonistic coevolution of parasite infectivity and host resistance may alter the biological functionality of species, yet these dynamics in nature are still poorly understood. Here we show the molecular details of a long-term phage–bacterium arms race in the environment. Bacteria (Flavobacterium columnare) are generally resistant to phages from the past and susceptible to phages isolated in years after bacterial isolation. Bacterial resistance selects for increased phage infectivity and host range, which is also associated with expansion of phage genome size. We identified two CRISPR loci in the bacterial host: a type II-C locus and a type VI-B locus. While maintaining a core set of conse…

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Sex and sexual ornamentation associated with survival of the cyprinid fish, Rutilus rutilus, under disease stress

By conveying information of disease resistance, sexual signals may be used as cues for adaptive mate choice. Here we report observations on survival of laboratory-maintained, wild-collected, sexually mature, ready-to-spawn cyprinid fish, Rutilus rutilus (roach), under accidental epidemic attributed to Flavobacterium psychrophilum, diagnosed using species-specific PCR. The fish were maintained in a single tank. During the 27 days observation period, both the proportion of fish surviving the infection as well as the length-adjusted mean survival time of the fish that died was the highest among the high-ornamented males with large breeding tubercles, intermediary among the low-ornamented males…

High Nutrient Concentration Can Induce Virulence Factor Expression and Cause Higher Virulence in an Environmentally Transmitted Pathogen

Environmentally transmitted opportunistic pathogens shuttle between two substantially different environments: outside-host and within-host habitats. These environments differ from each other especially with respect to nutrient availability. Consequently, the pathogens are required to regulate their behavior in response to environmental cues in order to survive, but how nutrients control the virulence in opportunistic pathogens is still poorly understood. In this study, we examined how nutrient level in the outside-host environment affects the gene expression of putative virulence factors of the opportunistic fish pathogen Flavobacterium columnare. The impact of environmental nutrient concen…

Thermal Tolerance is linked with Virulence in a Fish Pathogen

ABSTRACTAlthough increase in temperatures may boost the number of pathogens, a complex process involving the interaction of a susceptible host, a virulent strain, and environmental factors would influence disease virulence in unpredictable ways. Here we explored if the virulence of an environmentally growing opportunistic fish pathogen,Flavobacterium columnare, would be malleable to evolutionary changes via correlated selection on thermal tolerance. Virulence among the strains increased over years, but tolerance to higher temperatures was associated with reduced virulence. Our results suggest that observed increase in frequency of columnaris epidemics over the last decade is most likely ass…

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

AbstractIncreasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, prot…

supplementary information and data from Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentration of antibiotic affects the co-infection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum, in their fish host. We found that antibiotic in fee…

Additional file 1: Figure S1. of Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

Bacterial densities (mean ± S.E.M.) of dose controls of strains A, B and C of Flavobacterium columnare in the growth experiment. The average optical density of the cultures is on the Y-axis, and culture time on the X-axis. (DOC 65 kb)

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Mucin induces CRISPR-Cas defense in an opportunistic pathogen

It is unknown what circumstances promote particular bacterial defenses against bacterial viruses (phages). Almeida & Hoikkala et al. show that mucin, derived from mucus, greatly accelerates CRISPR-Cas defenses against phage in an opportunistic pathogen. Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a …

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Aims:  Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Methods and Results:  Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in…

Cooperation between CRISPR-Cas types enables adaptation in an RNA-targeting system

AbstractCRISPR-Cas immune systems adapt to new threats by acquiring spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition, despite apparent variation in spacer content between strains. It has been suggested that such loci may use acquisition machinery from co-occurring CRISPR-Cas systems. Here, using a lytic dsDNA phage, we observe spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient subtype VI-B locus and a complete subtype II-C locus. We characterize acquisition events in both loci and show that the RNA-targeting VI-B locus acquires spacers in trans using acquisit…

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…

Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage

So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution…

Rich resource environment of fish farms facilitates phenotypic variation and virulence in an opportunistic fish pathogen

Phenotypic variation is suggested to facilitate the persistence of environmentally growing pathogens under environmental change. Here, we hypothesized that the intensive farming environment induces higher phenotypic variation in microbial pathogens than natural environment, because of high stochasticity for growth and stronger survival selection compared to the natural environment. We tested the hypothesis with an opportunistic fish pathogen Flavobacterium columnare isolated either from fish farms or from natural waters. We measured growth parameters of two morphotypes from all isolates in different resource concentrations and two temperatures relevant for the occurrence of disease epidemic…

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 …

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Higher resource level promotes virulence in an environmentally transmitted bacterial fish pathogen

Diseases have become a primary constraint to sustainable aquaculture, but remarkably little attention has been paid to a broad class of pathogens: the opportunists. Opportunists often persist in the environment outside the host, and their pathogenic features are influenced by changes in the environment. To test how environmental nutrient levels influence virulence, we used strains of Flavobacterium columnare, an environmentally transmitted fish pathogen, to infect rainbow trout and zebra fish in two different nutrient concentrations. To separate the effects of dose and nutrients, we used three infective doses and studied the growth of bacteria in vitro. High nutrient concentration promoted …

Interactions among bacterial strains and fluke genotypes shape virulence of co-infection.

Most studies of virulence of infection focus on pairwise host–parasite interactions. However, hosts are almost universally co-infected by several parasite strains and/or genotypes of the same or different species. While theory predicts that co-infection favours more virulent parasite genotypes through intensified competition for host resources, knowledge of the effects of genotype by genotype (G × G) interactions between unrelated parasite species on virulence of co-infection is limited. Here, we tested such a relationship by challenging rainbow trout with replicated bacterial strains and fluke genotypes both singly and in all possible pairwise combinations. We found that virulence (host mo…

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 su…

Intensive aquaculture selects for increased virulence and interference competition in bacteria

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare , change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003–2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbre…

The use of phage FCL-2 as an alternative to chemotherapy against columnaris disease in aquaculture

Flavobacterium columnare, the causative agent of columnaris disease in fish, causes millions of dollars of losses in the US channel catfish industry alone, not to mention aquaculture industry worldwide. Novel methods are needed for the control and treatment of bacterial diseases in aquaculture to replace traditionally used chemotherapies. A potential solution could be the use of phages, i.e., bacterial viruses, host-specific and self-enriching particles that can be can easily distributed via water flow. We examined the efficacy of phages to combat columnaris disease. A previously isolated phage, FCL-2, infecting F. columnare, was characterized by sequencing. The 47 142 bp genome of the phag…

Complete Genome Sequence of Fish Pathogen Flavobacterium columnare Strain B185, Originating from Finland

We report a complete genome sequence of a Finnish isolate of the fish pathogen Flavobacterium columnare. Using PacBio RS II sequencing technology, the complete circular genome of F. columnare strain B185 with 3,261,404 bp was obtained.

Quantity and Quality of Aquaculture Enrichments Influence Disease Epidemics and Provide Ecological Alternatives to Antibiotics

Environmental heterogeneity is a central component influencing the virulence and epidemiology of infectious diseases. The number and distribution of susceptible hosts determines disease transmission opportunities, shifting the epidemiological threshold between the spread and fadeout of a disease. Similarly, the presence and diversity of other hosts, pathogens and environmental microbes, may inhibit or accelerate an epidemic. This has important applied implications in farming environments, where high numbers of susceptible hosts are maintained in conditions of minimal environmental heterogeneity. We investigated how the quantity and quality of aquaculture enrichments (few vs. many stones

Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite.

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentrations of antibiotic affects the co-infection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum , in their fish host. We found that antibiotic in …

supplementary information and data from Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentration of antibiotic affects the co-infection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum, in their fish host. We found that antibiotic in fee…

Additional file 2: Table S1. of Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

Effect of the treatment (single or coinfection) on the growth parameters of Flavobacterium columnare. (DOC 29Â kb)

Additional file 3: Table S2. of Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

The treatment and control groups in Flavobacterium columnare infection experiment. (DOC 70Â kb)

Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

Background In nature, organisms are commonly coinfected by two or more parasite strains, which has been shown to influence disease virulence. Yet, the effects of coinfections of environmental opportunistic pathogens on disease outcome are still poorly known, although as host-generalists they are highly likely to participate in coinfections. We asked whether coinfection with conspecific opportunistic strains leads to changes in virulence, and if these changes are associated with bacterial growth or interference competition. We infected zebra fish (Danio rerio) with three geographically and/or temporally distant environmental opportunist Flavobacterium columnare strains in single and in coinf…

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium

AbstractStudies on species’ responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to…

Rearing background and exposure environment together explain higher survival of aquaculture fish during a bacterial outbreak

1.Parasitic diseases represent one of the greatest challenges for aquaculture worldwide and there is an increasing emphasis on ecological solutions to prevent infections. One proposed solution is enriched rearing, where traditional stimulus‐poor rearing tanks are equipped with different types of structures to increase habitat complexity. Such spatial enrichment is known to increase survival of fish during parasite epidemics, but the underlying mechanisms are still unclear. 2.We studied whether enriched rearing affected infection of an important fish pathogen Flavobacterium columnare in young Atlantic salmon (Salmo salar) and sea‐migrating brown trout (Salmo trutta). First, we used natural b…

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Complete Genome Sequence of an Aquaculture-Associated Phage, FL-1, Infecting Flavobacterium spp

Flavobacterium spp. are abundant and widespread in freshwater environments (1, 2). Despite the prevalence of the members in this genus, the phages infecting Flavobacterium spp. are less known. So far, most of the phages studied in detail infect fish pathogenic members of the genus, such as F. psychrophilum (3). The phage FL-1 and its Flavobacterium sp. host strain B183 were previously isolated from a water sample from a fish farm in Central Finland. Transmission electron microscopy revealed FL-1 to be a member of the family Myoviridae (4). In addition to the isolation host, FL-1 also infects multiple Flavobacterium sp. isolates and strains of the fish pathogen F. columnare, the causative ag…

Bacteriophage imaging : past, present and future

The visualization of viral particles only became possible after the advent of the electron microscope. The first bacteriophage images were published in 1940 and were soon followed by many other publications that helped to elucidate the structure of the particles and their interaction with the bacterial hosts. As sample preparation improved and new technologies were developed, phage imaging became important approach to morphologically classify these viruses and helped to understand its importance in the biosphere. In this review we discuss the main milestones in phage imaging, how it affected our knowledge on these viruses and recent developments in the field. peerReviewed

Additional file 1 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Site, year of isolation, source of isolation (fish or water), location of isolation, host species, sequence type (ST), and allelic profile data for the 83Â F. columnare strains from Finland analyzed by MLST. (DOCX 46 kb)

Antibacterial Efficiency of Surface-Immobilized Flavobacterium-Infecting Bacteriophage

Control of bacterial diseases by bacteriophages (phages) is gaining more interest due to increasing antibiotic resistance. This has led to technologies to attach phages on surfaces to form a biomaterial that can functionally display phages that interact with bacteria, to carry out successful infection cycles. Such a material could be applied in many environments, where the target pathogens are expected. Although this approach has been applied successfully in a few studies already, the basis of the antibacterial effect by the immobilized phages is unclear, and the interpretation of the results depends on the study. Here, we studied the phage attachment density, their detachment rate and infe…

Contamination of Surface Water and River Sediments by Antibiotic and Antiretroviral Drug Cocktails in Low and Middle-Income Countries: Occurrence, Risk and Mitigation Strategies

Presence of antimicrobial cocktails in the hydrological cycles is of interest because of their potential to mediate antimicrobial resistance within the natural environment. In this study, we determined the concentrations of selected antibiotics and antiretroviral drugs (ARVDs) in wastewater treatment plant (WWTP) effluent, effluent suspended particulate matter (SPM), surface waters and river sediments in Kenya in order to determine the extent of pollution within the sampled environment. Target analysis for the most common antibiotics and ARVDs was done. Sulfamethoxazole (SMX), ciprofloxacin (CIP), trimethoprim (TMP), norfloxacin (NOR), zidovidine (ZDV), lamivudine (3TC) and nevirapine (NVP)…

Broad thermal tolerance is negatively correlated with virulence in an opportunistic bacterial pathogen

Predicting the effects of global increase in temperatures on disease virulence is challenging, especially for environmental opportunistic bacteria, because pathogen fitness may be differentially affected by temperature within and outside host environment. So far, there is very little empirical evidence on the connections between optimal temperature range and virulence in environmentally growing pathogens. Here, we explored whether the virulence of an environmentally growing opportunistic fish pathogen, Flavobacterium columnare, is malleable to evolutionary changes via correlated selection on thermal tolerance. To this end, we experimentally quantified the thermal performance curves (TPCs) f…

A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Background Columnaris disease caused by Flavobacterium columnare is a serious problem in aquaculture, annually causing large economic losses around the world. Despite considerable research, the molecular epidemiology of F. columnare remains poorly understood. Methods We investigated the population structure and spatiotemporal changes in the genetic diversity of F. columnare population in Finland by using a multilocus sequence typing (MLST) and analysis (MLSA) based on DNA sequence variation within six housekeeping genes. A total of 83 strains of F. columnare were collected from eight different areas located across the country between 2003 and 2012. Results Partial sequencing of six housekee…

Bioimaging: Imaging Bacterial Colonies and Phage-Bacterium Interaction at Sub-Nanometer Resolution Using Helium-Ion Microscopy (Adv. Biosys. 8/2017)

Gliding Motility and Expression of Motility-Related Genes in Spreading and Non-spreading Colonies of Flavobacterium columnare

Gliding motility facilitates the movement of bacteria along surfaces in many Bacteroidetes species and results in spreading colonies. The adhesins required for the gliding are secreted through a gliding motility-associated protein secretion system, known as the type IX secretion system (T9SS). The fish pathogen Flavobacterium columnare produces spreading (rhizoid [Rz], soft [S]) and non-spreading (rough [R]) colony types, of which only the spreading Rz type is virulent. In this study, we explored the spreading behavior of these colony types by microscopic imaging and measured the expression of genes associated with gliding motility and T9SS (gldG, gldH, gldL, sprA, sprB, sprE, sprF, sprT, a…

The Influence of Infective Dose on the Virulence of a Generalist Pathogen in Rainbow Trout (Oncorhynchus mykiss) and Zebra Fish (Danio rerio)

Pathogen density and genetic diversity fluctuate in the outside-host environment during and between epidemics, affecting disease emergence and the severity and probability of infections. Although the importance of these factors for pathogen virulence and infection probability has been acknowledged, their interactive effects are not well understood. We studied how an infective dose in an environmentally transmitted opportunistic fish pathogen, Flavobacterium columnare, affects its virulence both in rainbow trout, which are frequently infected at fish farms, and in zebra fish, a host that is not naturally infected by F. columnare. We used previously isolated strains of confirmed high and low …

Adapting a Phage to Combat Phage Resistance

Phage therapy is becoming a widely recognized alternative for fighting pathogenic bacteria due to increasing antibiotic resistance problems. However, one of the common concerns related to the use of phages is the evolution of bacterial resistance against the phages, putatively disabling the treatment. Experimental adaptation of the phage (phage training) to infect a resistant host has been used to combat this problem. Yet, there is very little information on the trade-offs of phage infectivity and host range. Here we co-cultured a myophage FCV-1 with its host, the fish pathogen Flavobacterium columnare, in lake water and monitored the interaction for a one-month period. Phage resistance was…

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 Flavobacter…

The forgotten tale of Brazilian phage therapy

The use of bacteriophages to treat bacterial infections (known as phage therapy) is considered a possible solution to the antimicrobial resistance crisis. However, phage therapy is not a new concept. The discovery of phages in the early 20th century was closely tied to clinical practice, and phage therapy quickly spread around the world. The use of phage therapy in South America in the previous century is still shrouded in mystery and has been mentioned only briefly in recent scientific literature. Research on Brazilian reference collections of medical texts showed that Brazil was an important, but so far little-known, player of phage therapy, uncovering interesting priority claims and miss…

Comparing the different morphotypes of a fish pathogen - implications for key virulence factors in Flavobacterium columnare

Background: Flavobacterium columnare (Bacteroidetes) is the causative agent of columnaris disease in farmed freshwater fish around the world. The bacterium forms three colony morphotypes (Rhizoid, Rough and Soft), but the differences of the morphotypes are poorly known. We studied the virulence of the morphotypes produced by F. columnare strain B067 in rainbow trout ( Onconrhynchus mykiss ) and used high-resolution scanning electron microscopy to identify the fine structures of the cells grown in liquid and on agar. We also analysed the proteins secreted extracellularly and in membrane vesicles to identify possible virulence factors. Results: Only the Rhizoid morphotype was virulent in rain…

Virus found in a boreal lake links ssDNA and dsDNA viruses.

Viruses have impacted the biosphere in numerous ways since the dawn of life. However, the evolution, genetic, structural, and taxonomic diversity of viruses remain poorly understood, in part because sparse sampling of the virosphere has concentrated mostly on exploring the abundance and diversity of dsDNA viruses. Furthermore, viral genomes are highly diverse, and using only the current sequence-based methods for classifying viruses and studying their phylogeny is complicated. Here we describe a virus, FLiP (Flavobacterium-infecting, lipid-containing phage), with a circular ssDNA genome and an internal lipid membrane enclosed in the icosahedral capsid. The 9,174-nt-long genome showed limite…

Prevalence of genetically similarFlavobacterium columnarephages across aquaculture environments reveals a strong potential for pathogen control

SummaryIntensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria.Flavobacterium columnarecauses columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126F. columnarestrains and 63 phages againstF. columnarefrom Finland and Sweden. 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 228 bact…

ICTV Virus Taxonomy Profile: Finnlakeviridae

Finnlakeviridae is a family of icosahedral, internal membrane-containing bacterial viruses with circular, single-stranded DNA genomes. The family includes the genus, Finnlakevirus, with the species, Flavobacterium virus FLiP. Flavobacterium phage FLiP was isolated with its Gram-negative host bacterium from a boreal freshwater habitat in Central Finland in 2010. It is the first described single-stranded DNA virus with an internal membrane and shares minimal sequence similarity with other known viruses. The virion organization (pseudo T=21 dextro) and major capsid protein fold (double-β-barrel) resemble those of Pseudoalteromonas phage PM2 (family Corticoviridae), which has a double-stranded…

Cooperation between Different CRISPR-Cas Types Enables Adaptation in an RNA-Targeting System

CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome.

Mucin induces CRISPR-Cas defence in an opportunistic pathogen

AbstractParasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogenFlavobacterium columnareto its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium…

Additional file 4 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

The fluorescence peak profiles for the ARISA genotypes analysed with ABI Prism 3130xl Genetic Analyser and the GeneMapper v.5.0 software (Applied Biosystems, Carlsbad, California, USA). For the 83 strains isolated from Finland, we also determined the ARISA (automated ribosomal intergenic spacer analysis) genotypes following the procedure described by Suomalainen et al. [8]. However, the previously published method was modified so that ABI Prism 3130xl Genetic Analyser is used instead of LI-COR 4200 automatic sequencer The analysis revealed that ARISA genotypes associate uniformly with the clusters from the MLSA scheme. Briefly, the PCR reaction mixture (total volume 10 ul) contained 1X Drea…

Additional file 3 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Neighbor Joining phylogenetic trees based on the individual sequences of six MLST loci (trpB, rpoD, gyrB, dnaK, atpA,and tuf). MEGA v5.2 was used to evaluate the models for nucleotide substitution for each protein-coding locus and to construct the phylogenetic trees for Finnish F. columnare strains. The F. columnare type strain NCIMB 2248T isolated in the USA and two reference strains JIP39/87 and ATCC49512, both isolated in France, were also included in the phylogenetic analysis. The best model indicated by the lowest Bayesian Information Criterion (BIC) value was used to generate the Neighbor-Joining tree based on 1000 replicates. The T92 model was selected for dnaK, tuf, and gyrB, while …

Additional file 2 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Target genes and primers for the housekeeping genes of F. columnare. The loci used for the MLST/MLSA scheme are shown in bold font. Length refers to the length of the target sequence. * Reference for 16S rDNA primers [52]. (DOCX 20 kb)

Imaging Bacterial Colonies and Phage-Bacterium Interaction at Sub-Nanometer Resolution Using Helium-Ion Microscopy

Imaging of microbial interactions has so far been based on well‐established electron microscopy methods. This study presents a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). In biological imaging, HIM has advantages over traditional scanning electron microscopy with its sub‐nanometer resolution, increased surface sensitivity, and the possibility to image nonconductive samples. Furthermore, by controlling the He beam dose or by using heavier Ne ions, the HIM instrument provides the possibility to mill out material in the samples, allowing for subsurface imaging and in …

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 Flavobacteri…

Half a Century of Research on Membrane-Containing Bacteriophages: Bringing New Concepts to Modern Virology

Half a century of research on membrane-containing phages has had a major impact on virology, providing new insights into virus diversity, evolution and ecological importance. The recent revolutionary technical advances in imaging, sequencing and lipid analysis have significantly boosted the depth and volume of knowledge on these viruses. This has resulted in new concepts of virus assembly, understanding of virion stability and dynamics, and the description of novel processes for viral genome packaging and membrane-driven genome delivery to the host. The detailed analyses of such processes have given novel insights into DNA transport across the protein-rich lipid bilayer and the transformati…

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Additional file 2 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Target genes and primers for the housekeeping genes of F. columnare. The loci used for the MLST/MLSA scheme are shown in bold font. Length refers to the length of the target sequence. * Reference for 16S rDNA primers [52]. (DOCX 20 kb)

supplementary information and data from Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentration of antibiotic affects the co-infection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum, in their fish host. We found that antibiotic in fee…

Phage-driven loss of virulence in a fish pathogenic bacterium

Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four s…

Genome size evolution in macroparasites.

Reduction in genome size has been associated not only with a parasitic lifestyle in intracellular microparasites but also in some macroparasitic insects and nematodes. We collected the available data on genome size for flatworms, annelids, nematodes and arthropods, compared those with available data for the phylogenetically closest free-living taxa and found evidence of smaller genome sizes for parasites in six of nine comparisons. Our results suggest that despite great differences in evolutionary history and life cycles, parasitism as a lifestyle promotes convergent genome size reduction in macroparasites. We discuss factors that could be associated with small genome size in parasites whic…

Genetic-based evaluation of management units for sustainable vendace (Coregonus albula) fisheries in a large lake system

The goal of the processing industry, trade and consumers is to get eco-labelled freshwater fish products from sustainable fisheries into the market as soon as possible. The fourth largest natural lake system in Europe, the Saimaa lake system supports a fishery for vendace (Coregonus albula). Certification of the fishery requires an understanding of population structure to help determine the number and spatial extent of management units. In this study, we analysed the genetic diversity of local vendace populations in the Saimaa lake system and aimed to identify the conservation and management units of vendace. Within the Saimaa, the genetic divergence between local populations of vendace was…

Cryo-EM structure of ssDNA bacteriophage ΦCjT23 provides insight into early virus evolution.

AbstractThe origin of viruses remains an open question. While lack of detectable sequence similarity hampers the analysis of distantly related viruses, structural biology investigations of conserved capsid protein structures facilitate the study of distant evolutionary relationships. Here we characterize the lipid-containing ssDNA temperate bacteriophage ΦCjT23, which infects Flavobacterium sp. (Bacteroidetes). We report ΦCjT23-like sequences in the genome of strains belonging to several Flavobacterium species. The virion structure determined by cryogenic electron microscopy reveals similarities to members of the viral kingdom Bamfordvirae that currently consists solely of dsDNA viruses wit…

Additional file 5 of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Phylogenetic tree based on the 16Â s rDNA sequence data obtained from the representatives of Finnish F .columnare genotypes (A-H) studied in this study and other F .columnare sequences obtained from the GenBank. The tree was constructed by a UPGMA clustering method with a resampling of 1,000 bootstrap replicates and the Jukes Cantor model. Two strains representative of each ARISA genotype/MLSA cluster studied in this study were used for tree construction (identical sequences removed for clarity of representation). (PDF 191 kb)

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

of A multilocus sequence analysis scheme for characterization of Flavobacterium columnare isolates

Data from: Intensive aquaculture selects for increased virulence and interference competition in bacteria

Although increased disease severity driven by intensive farming practices is problematic in food production, the role of evolutionary change in disease is not well understood in these environments. Experiments on parasite evolution are traditionally conducted using laboratory models, often unrelated to economically important systems. We compared how the virulence, growth and competitive ability of a globally important fish pathogen, Flavobacterium columnare, change under intensive aquaculture. We characterized bacterial isolates from disease outbreaks at fish farms during 2003-2010, and compared F. columnare populations in inlet water and outlet water of a fish farm during the 2010 outbreak…

Data from: Interactions among bacterial strains and fluke genotypes shape virulence of co-infection

Most studies of virulence of infection focus on pairwise host-parasite interactions. However, hosts are almost universally co-infected by several parasite strains and/or genotypes of the same or different species. While theory predicts that co infection favours more virulent parasite genotypes through intensified competition for host resources, knowledge of effects of genotype by genotype (G×G) interactions between unrelated parasite species on virulence of co infection is limited. Here we tested such relationship by challenging rainbow trout with replicated bacterial strains and fluke genotypes both singly and in all possible pairwise combinations. We found that virulence (host mortality) …

Data from: Rearing background and exposure environment together explain higher survival of aquaculture fish during a bacterial outbreak

1. Parasitic diseases represent one of the greatest challenges for aquaculture worldwide and there is an increasing emphasis on ecological solutions to prevent infections. One proposed solution is enriched rearing, where traditional stimulus-poor rearing tanks are equipped with different types of structures to increase habitat complexity. Such spatial enrichment is known to increase survival of fish during parasite epidemics, but the underlying mechanisms are still unclear. 2. We studied whether enriched rearing affected infection of an important fish pathogen Flavobacterium columnare in young Atlantic salmon (Salmo salar) and sea-migrating brown trout (Salmo trutta). First, we used natural…

Data from: Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

Background In nature, organisms are commonly coinfected by two or more parasite strains, which has been shown to influence disease virulence. Yet, the effects of coinfections of environmental opportunistic pathogens on disease outcome are still poorly known, although as host-generalists they are highly likely to participate in coinfections. We asked whether coinfection with conspecific opportunistic strains leads to changes in virulence, and if these changes are associated with bacterial growth or interference competition. We infected zebra fish (Danio rerio) with three geographically and/or temporally distant environmental opportunist Flavobacterium columnare strains in single and in coinf…

Data from: Minor environmental concentrations of antibiotics can modify bacterial virulence in co-infection with a non-targeted parasite

Leakage of medical residues into the environment can significantly impact natural communities. For example, antibiotic contamination from agriculture and aquaculture can directly influence targeted pathogens, but also other non-targeted taxa of commensals and parasites that regularly co-occur and co-infect the same host. Consequently, antibiotics could significantly alter interspecific interactions and epidemiology of the co-infecting parasite community. We studied how minor environmental concentration of antibiotic affects the coinfection of two parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum, in their fish host. We found that antibiotic in feed…

Data from: Broad thermal tolerance is negatively correlated with virulence in an opportunistic bacterial pathogen

Predicting the effects of global increase in temperatures on disease virulence is challenging, especially for environmental opportunistic bacteria, because pathogen fitness may be differentially affected by temperature within and outside host environment. So far, there is very little empirical evidence on the connections between optimal temperature range and virulence in environmentally growing pathogens. Here we explored if the virulence of an environmentally growing opportunistic fish pathogen, Flavobacterium columnare, is malleable to evolutionary changes via correlated selection on thermal tolerance. To this end, we experimentally quantified the thermal performance curves (TPCs) for max…

CRISPR-Cas in aquaculture pathogens from Aquaculture as a source of empirical evidence for coevolution between CRISPR-Cas and phage.

Contains strain-specific information on the CRISPR-Cas content of 24 bacterial species relevant to aquaculture

Data from: Higher resource level promotes virulence in an environmentally transmitted bacterial fish pathogen

Diseases have become a primary constraint to sustainable aquaculture, but remarkably little attention has been paid to a broad class of pathogens: the opportunists. Opportunists often persist in the environment outside the host and their pathogenic features are influenced by changes in the environment. To test how environmental nutrient levels influence virulence, we used strains of Flavobacterium columnare, an environmentally transmitted fish pathogen, to infect rainbow trout and zebra fish in two different nutrient concentrations. To separate the effects of dose and nutrients, we used three infective doses and studied the growth of bacteria in vitro. High nutrient concentration promoted b…

Data from: Sequential infection can decrease virulence in a fish-bacterium-fluke interaction: implications for aquaculture disease management

Hosts are typically infected with multiple strains or genotypes of one or several parasite species. These infections can take place simultaneously, but also at different times, i.e. sequentially, when one of the parasites establishes first. Sequential parasite dynamics are common in nature, but also in intensive farming units such as aquaculture. However, knowledge of effects of previous exposures on virulence of current infections in intensive farming is very limited. This is critical as consecutive epidemics and infection history of a host could underlie failures in management practises and medical intervention of diseases. Here, we explored effects of timing of multiple infection on viru…

Original data for manuscript: Quantity and Quality of Aquaculture Enrichments Influence Disease Epidemics and Provide Ecological Alternatives to Antibiotics

The data processed and analyzed in this study are given in a single file: Karvonen et al. exposure data.xlsx. For detailed description of the material, methods and results of the study, see the article.

Research data of an article: "Application of high resolution melting assay (HMR) to study temperature dependent infraspecific competition in an pathogenic bacterium."

Studies on species’ responses to climate change have focused largely on the direct effect of abiotic factors and in particular temperature, neglecting the effects of biotic interactions in determining the outcome of climate change projections. Many microbes rely on strong interference competition; hence the fitness of many pathogenic bacteria could be a function of both their growth properties and intraspecific competition. However, due to technical challenges in distinguishing and tracking individual strains, experimental evidence on intraspecific competition has been limited so far. Here, we developed a robust application of the high-resolution melting (HRM) assay to study head-to-head co…

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

This dataset consists of three phage therapy experiments to prevent Flavobacterium columnare infections in rainbow trout. Experiments I and II were done in stagnant water, and Experiment III in a flow-through system. See the documentation file attached with the dataset for full description of the methods (also found in the original article).

Dataset for Mucin induces CRISPR-Cas defense in an opportunistic pathogen

This dataset contains raw data points of (1) phage-bacterium co-evolution experiment (flavobacterium columnare colony counts, phage counts, bacterial growth characteristics and number of CRISPR-Cas spacers) and (2) from competition experiment between Flavobacterium columnare and Aeromonas sp. For detailed method description, see the attached Documentation file.