Elina Laanto

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 strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio) as a model system, the ancestral rhizoid morphotypes were associated with a 25–100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types), did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.

High Temperature and Bacteriophages Can Indirectly Select for Bacterial Pathogenicity in Environmental Reservoirs

The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.

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.

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 phage had G + C content of 30.2%, and the closest similarities regarding the structural proteins were found in Cellulophaga phage phiSM. Under controlled experimental conditions, two host fish species, rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio), were used to study the success of phage therapy to prevent F. columnare infections. The survival of both fish species was significantly higher in the presence of the phage. Hundred percent of the zebrafish and 50% of the rainbow trout survived in the phage treatment (survival without phage 0 and 8.3%, respectively). Most importantly, the rainbow trout population was rescued from infection by a single addition of the phage into the water in a flow-through fish tank system. Thus, F. columnare could be used as a model system to test the benefits and risks of phage therapy on a larger scale.

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. Our data suggest that the farming environment may select for bacterial strains that have high virulence at both long and short time scales, and it seems that these strains have also evolved increased ability for interference competition. Our results are consistent with the suggestion that selection pressures at fish farms can cause rapid changes in pathogen populations, which are likely to have long-lasting evolutionary effects on pathogen virulence. A better understanding of these evolutionary effects will be vital in prevention and control of disease outbreaks to secure food production.

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 conserved spacers, phage-matching spacers appear in the variable ends of both loci over time. The spacers mostly target the terminal end of the phage genomes, which also exhibit the most variation across time, resulting in arms-race-like changes in the protospacers of the coevolving phage population.Arms races between phage and bacteria are well known from lab experiments, but insight from field systems is limited. Here, the authors show changes in the resistance and CRISPR loci of bacteria and the infectivity, host range and genome size of phage over multiple years in an aquaculture environment.

Virus found in a boreal lake links ssDNA and dsDNA viruses

Significance We describe the phage FLiP, an ssDNA virus with an icosahedral capsid and an internal lipid membrane. The FLiP genome shows limited similarity to known sequences, although an ssDNA replication mechanism was implied by genome analysis. However, because the capsid protein fold indicates relatedness with the dsDNA viruses of the PRD1–adenovirus lineage, FLiP exhibits a unique combination of structural and replication modules. It is suggested that the capsid protein structure could be used to complement the sequence data when classifying viruses and in detecting their deep evolutionary relationships, especially in the absence of sequence similarities. Furthermore, these findings demonstrate the value of characterizing unknown viruses from diverse environmental sources to understand the diversity of the microbial world. 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 limited sequence similarity to other known viruses. The genetic data imply that this virus might use replication mechanisms similar to those found in other ssDNA replicons. However, the structure of the viral major capsid protein, elucidated at near-atomic resolution using cryo-electron microscopy, is strikingly similar to that observed in dsDNA viruses of the PRD1–adenovirus lineage, characterized by a major capsid protein bearing two β-barrels. The strong similarity between FLiP and another member of the structural lineage, bacteriophage PM2, extends to the capsid organization (pseudo T = 21 dextro) despite the difference in the genetic material packaged and the lack of significant sequence similarity.

New enveloped dsRNA phage from freshwater habitat.

Cystoviridae is a family of bacteriophages with a tri-segmented dsRNA genome enclosed in a tri-layered virion structure. Here, we present a new putative member of the Cystoviridae family, bacteriophage ϕNN. ϕNN was isolated from a Finnish lake in contrast to the previously identified cystoviruses, which originate from various legume samples collected in the USA. The nucleotide sequence of the virus reveals a strong genetic similarity (~80 % for the L-segments, ~55 % for the M-segments and ~84 % for the S-segments) to Pseudomonas phage ϕ6, the type member of the virus family. However, the relationship between ϕNN and other cystoviruses is more distant. In general, proteins located in the internal parts of the virion were more conserved than those exposed on the virion surface, a phenomenon previously reported among eukaryotic dsRNA viruses. Structural models of several putative ϕNN proteins propose that cystoviral structures are highly conserved.

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 situ sectioning. Here, the first HIM‐images of bacterial colonies and phage–bacterium interactions are presented at different stages of the infection as they occur on an agar culture. The feasibility of neon and helium milling is also demonstrated to reveal the subsurface structures of bacterial colonies on agar substrate, and in some cases also structure inside individual bacteria after cross‐sectioning. The study concludes that HIM offers great opportunities to advance the studies of microbial imaging, in particular in the area of interaction of viruses with cells.

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

ABSTRACT FL-1, a myophage of Flavobacterium, was found to have a 53-kb genome with 87 putative coding sequences.