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 coinfection. Growth of the strains was studied in single and in co-cultures in liquid medium, and interference competition (growth-inhibiting ability) on agar. Results: The individual strains differed in their virulence, growth and ability for interference competition. Number of coinfecting strains significantly influenced the virulence of infection, with three-strain coinfection differing from the two-strain and single infections. Differences in virulence seemed to associate with the identity of the coinfecting bacterial strains, and their pairwise interactions. This indicates that benefits of competitive ability (production of growth-inhibiting compounds) for virulence are highest when multiple strains co-occur, whereas the high virulence in coinfection may be independent from in vitro bacterial growth. Conclusions: Intraspecific competition can lead to plastic increase in virulence, likely caused by faster utilization of host resources stimulated by the competitive interactions between the strains. However, disease outcome depends both on the characteristics of individual strains and their interactions. Our results highlight the importance of strain interactions in disease dynamics in environments where various pathogen genotypes co-occur.peerReviewe

Recognition of six additional cystoviruses : Pseudomonas virus phi6 is no longer the sole species of the family Cystoviridae

Cystoviridae is a family of bacterial viruses (bacteriophages) with a tri-segmented dsRNA genome. It includes a single genus Cystovirus, which has presently only one recognised virus species, Pseudomonas virus phi6. However, a large number of additional dsRNA phages have been isolated from various environmental samples, indicating that such viruses are more widespread and abundant than previously recognised. Six of the additional dsRNA phage isolates (Pseudomonas phages phi8, phi12, phi13, phi2954, phiNN and phiYY) have been fully sequenced. They all infect Pseudomonas species, primarily plant pathogenic Pseudomonas syringae strains. Due to the notable genetic and structural similarities with Pseudomonas phage phi6, we propose that these viruses should be included into the Cystovirus genus (and consequently into the Cystoviridae family). Here, we present an updated taxonomy of the family Cystoviridae and give a short overview of the properties of the type member phi6 as well as the putative new members of the family.Peer reviewe

Complete genome sequence of an aquaculture associated phage FL-1 infecting Flavobacterium species

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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. Additional bioinformatic analyses reveal several species in the phylum Bacteroidetes that host a similar type of putative prophage, including bacteria that are important animal and human pathogens. Furthermore, a survey of F. columnare Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers indicate a shared evolutionary history between F. columnare strains and the fF4 phage, and another putative prophage in the F. columnare strain ATCC 49512, named p49512. First, CRISPR spacer content from the two CRISPR loci (types II-C and VI-B) of the fF4 lysogen F. columnare ATCC 23463 revealed a phage terminase protein-matching spacer in the VI-B locus. This spacer is also present in two Chinese F. columnare strains. Second, CRISPR analysis revealed four F. columnare strains that contain unique spacers targeting different regions of the putative prophage p49512 in the F. columnare strain ATCC 49512, despite the geographical distance or genomovar of the different strains. This suggests a common ancestry for the F. columnare prophages and different host strains

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.Peer reviewe

Complete genome sequence of the fish pathogen Flavobacterium columnare strain B185, originating from Finland

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Mucosal Environment Induces Phage Susceptibility in Streptococcus mutans

Pathogenic bacteria are attracted toward mucosa, as it is their way of entry into the body. However, we know surprisingly little about the phage-bacterium interactions in the mucosal environment. Here, we explored the effect of the mucosal environment on growth characteristics and phage-bacterium interactions in Streptococcus mutans, a causative agent of dental caries. We found that although mucin supplementation increased bacterial growth and survival, it decreased S. mutans biofilm formation. More importantly, the presence of mucin had a significant effect on S. mutans phage susceptibility. In two experiments done in Brain Heart Infusion Broth, phage M102 replication was detected only with 0.2% mucin supplementation. In 0.1 × Tryptic Soy Broth, 0.5% mucin supplementation led to a 4-log increase in phage titers compared with the control. These results suggest that the mucosal environment can have a major role in the growth, phage sensitivity, and phage resistance of S. mutans, and underline the importance of understanding the effect of mucosal environment on phage-bacterium interactions

Bacteriophage Adherence to Mucus Mediates Preventive Protection against Pathogenic Bacteria

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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 practices and medical intervention of diseases. Here, we explored effects of timing of multiple infections on virulence in two common aquaculture parasites, the bacterium Flavobacterium columnare and the fluke Diplostomum pseudospathaceum. We exposed fish hosts first to flukes and then to bacteria in two separate experiments, altering timing between the infections from few hours to several weeks. We found that both short‐term and long‐term differences in timing of the two infections resulted in significant, genotype‐specific decrease in bacterial virulence. Second, we developed a mathematical model, parameterized from our experimental results, to predict the implications of sequential infections for epidemiological progression of the disease, and levels of fish population suppression, in an aquaculture setting. Predictions of the model showed that sequential exposure of hosts can decrease the population‐level impact of the bacterial epidemic, primarily through the increased recovery rate of sequentially infected hosts, thereby substantially protecting the population from the detrimental impact of infection. However, these effects depended on bacterial strain–fluke genotype combinations, suggesting the genetic composition of the parasite populations can greatly influence the degree of host suppression. Overall, these results suggest that host infection history can have significant consequences for the impact of infection at host population level, potentially shaping parasite epidemiology, disease dynamics and evolution of virulence in farming environments

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 dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies obtain at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the phage-bacterium interactions on mucosal surfaces may select for the diversification of bacterial immune systems.Peer reviewe