Auxiliary metabolic genes encoded by bacteriophages can influence host metabolic function during infection. In temperate phages, auxiliary metabolic genes (AMGs) may increase host fitness when integrated as prophages into the host genome. However, little is known about the contribution of prophage-encoded AMGs to host metabolic properties. In this study, we examined a temperate bacteriophage, and its piezotolerant Pseudomonas sp. host obtained from sediment samples collected from the Kermadec Trench at similar to 10 000 m water depth. Both the phage and host were present throughout the sediment profiles from the surface to 30 cm into the sediment, covering large gradients of environmental conditions. The host and phage each carried one chitinase gene, which differed from each other, suggesting that chitin degradation plays a role in their substrate supply. We demonstrated that prophage-encoded chitinase supported host chitin degradation and growth in the presence of chitin. Furthermore, prophage induction dynamics were strongly substrate-dependent, suggesting that the host controls the lysis-lysogeny switch in response to the presence of chitin, thus optimizing the trade-off between the loss of cells from prophage induction and prophage enhancement of host performance. Overall, the results demonstrate prophage-encoded AMGs as collaborative goods for their hosts and emphasize the potential role of phage-host interactions in benthic biogeochemical cycling, as well as for the capability of deep-sea bacteria to efficiently adapt and thrive at a wide range of environmental conditions.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

This article presents a study that aimed to identify the stages of bladder cancer based on gene expression data. The dataset used in the study was obtained from the GDC repository and included 406 cases of bladder cancer and 431 files from the TCGA-BLCA project. The study categorized the cases into three classes based on disease stages: Stage 2, Stage 3, and Stage 4. The methodology employed R programming language and the KnowSeq library for the study development. The authors identified genes that showed significant differences in expression among the classes and created a matrix of differentially expressed genes (DEG). Machine learning models, including feature selection algorithms and classification models such as KNN and SVM, were constructed to predict the bladder cancer stages. The results revealed that the mRMR feature selection algorithm performed the best, and the 8 most relevant genes were used to build the classification models.

Background Flavobacterium psychrophilum is the causative agent of the bacterial cold-water disease and rainbow trout fry syndrome. Owing to the issues associated with increasing use of antibiotics to control the diseases, phage therapy has been proposed as an alternative method to control Flavobacterium infection within the industry. Materials and Methods We explored two simple and fast in vitro strategies for the isolation of evolved F. psychrophilum phages, using three well-characterized phages FpV4, FpV9, and FPSV-S20. Results During in vitro serial transfer experiments, 12 evolved phages were selected 72-96 h after phage exposure in the first or second week. Phenotype analysis showed improvement of host range and efficiency of plating and adsorption constants. Comparative genomic analysis of the evolved phages identified 13 independent point mutations causing amino acid changes mostly in hypothetical proteins. Conclusions These results confirmed the reliability and effectivity of two strategies to isolate evolved F. psychrophilum phages, which may be used to expand phage-host range and target phage-resistant pathogens in phage therapy applications against Flavobacterium infections.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, 27 phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage-resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage-resistant phenotypes.

SummaryThe fish pathogen Flavobacterium psychrophilum infects farmed salmonids worldwide, and application of bacteriophages has been suggested for controlling disease outbreaks in aquaculture. Successful application of phages requires detailed knowledge about the variability in phage susceptibility of the host communities. In this study, we analysed the genetic diversity of F. psychrophilum hosts and phages from the Baltic Sea area to identify genetic determinants of phage‐host interaction patterns. A host range analysis of 103 phages tested against 177 F. psychrophilum strains (18 231 phage–host interactions) identified nine phage clusters, infecting from 10% to 91% of the strain collection. The core genome‐based comparison of 35 F. psychrophilum isolates revealed an extremely low overall genomic diversity (>99.5% similarity). However, a small subset of 16 ORFs, including genes involved in the type IX secretion system (T9SS), gliding motility and hypothetical cell‐surface related proteins, exhibited a highly elevated genetic diversity. These specific genetic variations were linked to variability in phage infection patterns obtained from experimental studies, indicating that these genes are key determinants of phage susceptibility. These findings provide novel insights on the molecular mechanisms determining phage susceptibility in F. psychrophilum and emphasizes the importance of phages as drivers of core genomic diversity in this pathogen.

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly provided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence.

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.

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

Zonula occludens toxin (Zot) is a conserved protein in filamentous vibriophages and has been reported as a putative toxin in Vibrio cholerae. Recently, widespread distribution of zot-encoding prophages was found among marine Vibrio species, including environmental isolates. However, little is known about the dynamics of these prophages beyond V. cholerae. In this study, we characterized and quantified the zot-encoding filamentous phage VAIϕ, spontaneously induced from the fish pathogen V. anguillarum. VAIϕ contained 6117 bp encoding 11 ORFs, including ORF8pVAI, exhibiting 27%–73% amino acid identity to Inovirus Zot-like proteins. A qPCR method revealed an average of four VAIϕ genomes per host genome during host exponential growth phase, and PCR demonstrated dissemination of induced VAIϕ to other V. anguillarum strains through re-integration in non-lysogens. VAIϕ integrated into both chromosomes of V. anguillarum by recombination, causing changes in a putative ORF in the phage genome. Phylogenetic analysis of the V. anguillarum Inoviridae elements revealed mosaic genome structures related to mainly V. cholerae. Altogether, this study contributes to the understanding of Inovirus infection dynamics and mobilization of zot-like genes beyond human pathogenic vibrios, and discusses their potential role in the evolution of the fish pathogen V. anguillarum.

Background: Bacteriophages (phages) have been proposed as an alternative to antibiotics and surface disinfectants for treatment of Flavobacterium psychrophilum biofilms and fish infections in aquaculture settings. The aim of the study was to estimate the minimal phage:host ratio (PHR) required for the control of in vitro biofilm formation and mortalities caused by F. psychrophilum in experimentally infected fish. Materials and Methods: F. psychrophilum cells in different stages of biofilm formation were exposed to the lytic phage FPSV-D22 at different PHRs. Results: Our results show that an initial PHR of 0.01 is sufficient for more than an 80% inhibition of attachment and colonization, and disruption of maturated F. psychrophilum biofilms, whereas greater ratios resulted in almost complete interruption of the different biofilm stages. Interestingly, a similar response was observed in a phage therapy trial with live rainbow trout (Oncorhynchus mykiss), where treatment of F. psychrophilum-infected fish by injection of serial bacteriophage doses resulted in significantly (***p <= 0.001) higher survival already at a PHR of 0.02. Conclusions: These results indicate that phages have the potential to be effective for control and treatment of F. psychrophilum infections in fish farms even when applied in concentrations lower than previously expected.

Small regulatory RNAs (sRNAs) are molecules that play an important role in the regulation of gene expression. sRNAs in bacteria can affect important processes, such as metabolism and virulence. Previous studies showed a significant role of sRNAs in the Vibrio species, but knowledge about Vibrio parahaemolyticus is limited. Here, we examined the conservation of sRNAs between V. parahaemolyticus and other human Vibrio species, in addition to investigating the conservation between V. parahaemolyticus strains differing in pandemic origin. Our results showed that only 7% of sRNAs were conserved between V. parahaemolyticus and other species, but 88% of sRNAs were highly conserved within species. Nonetheless, two sRNAs coding to RNA-OUT, a component of the Tn10/IS10 system, were exclusively present in pandemic strains. Subsequent analysis showed that both RNA-OUT were located in pathogenicity island-7 and would interact with transposase VPA1379, according to the model of pairing of IS10-encoded antisense RNAs. According to the location of RNA-OUT/VPA1379, we also investigated if they were expressed during infection. We observed that the transcriptional level of VPA1379 was significantly increased, while RNA-OUT was decreased at three hours post-infection. We suggest that IS10 transcription increases in pandemic strains during infection, probably to favor IS10 transposition and improve their fitness when they are facing adverse conditions.

Vibrio anguillarum is a marine pathogenic bacterium that causes vibriosis in fish and shellfish. Although prophage-like sequences have been predicted in V. anguillarum strains, many are not characterized, and it is not known if they retain the functional capacity to form infectious particles that can infect and lysogenize other bacterial hosts. In this study, the genome sequences of 28 V. anguillarum strains revealed 55 different prophage-related elements. Chemical and spontaneous induction allowed a collection of 42 phage isolates, which were classified in seven different groups according to a multiplex PCR assay. One shared prophage sequence, p41 (group III), was present in 17 V. anguillarum strains, suggesting that this specific element is very dynamically exchanged among V. anguillarum populations. Interestingly, the host range of genetically identical phages was highly dependent on the strains used for proliferation, indicating that phenotypic properties of phages were partly regulated by the host. Finally, experimental evidence displayed that the induced phage ɸVa_90-11-287_p41 was able to lysogenize V. anguillarum strain Ba35, and subsequently spontaneously become released from the lysogenized cells, demonstrating an efficient transfer of the phage among V. anguillarum strains. Altogether, the results showed large genetic and functional diversity and broad distribution of prophages in V. anguillarum, and demonstrated the potential of prophages as drivers of evolution in V. anguillarum strains.

Vibrio anguillarum is a marine bacterium that can cause vibriosis in many fish and shellfish species. Although phage therapy has been proposed as an alternative treatment, the defense mechanisms against phage infection in V. anguillarum and their impact on host function are not fully understood. Here, we examined phage defense strategies in four V. anguillarum strains during exposure to the broad-host-range bacteriophage KVP40. Whole-genome sequences of phage-resistant V. anguillarum isolates showed mutations causing premature stop codons, frameshifts and amino acid changes in the OmpK phage receptor. Moreover, certain phage-resistant variants recovered susceptibility to phage infection following re-culturing, suggesting alternative protection mechanisms, such as formation of biofilm, receptor downregulation and phage inactivation by proteases. Also, the lack of phage production by some strains despite strong phage control suggested an abortive infection mechanism was in play. In addition, examination of the virulence properties and extracellular enzyme secretion of the phage-resistant variants suggested that phage resistance was associated with reduced virulence in V. anguillarum. Altogether, the results identified a variety of phage resistance mechanisms in V. anguillarum including both mutational and non-mutational defenses and demonstrated a significant fitness loss associated with mutational changes, which may explain the selection for alternative defense mechanisms.

A rare nontailed virus designated NO16 was isolated against Vibrio anguillarum, a major aquaculture pathogen for both fish and shellfish. Here, we announce the 10,594-bp genome sequence of Vibrio phage NO16 with a 23-gene content.

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout (Oncorhynchus mykiss), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F. psychrophilum, or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD50) of 26 geographically disparate F. psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F. psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD50 (8.9 × 103 to 3.1 × 106 CFU). No association between MLST ST and virulence was found because of a high variation in LD50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD50 < 105 CFU), moderate (LD50 = 105-106 CFU), and weak (LD50 > 106 CFU) virulence were similar in phenotypic characteristics in vitro. However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum. Univariate correlation studies found only non-significant associations between LD50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.

Bacteriophages are an important factor in bacterial evolution. Some reports suggest that lytic bacteriophages can select for resistant mutant strains with reduced virulence. The present study explores the role of the CHOED bacteriophage in the diversification and virulence of its host Vibrio anguillarum. Nine phage-resistant strains were analyzed for their phenotype and different virulence factors, showing alterations in their fitness, motility, biofilm formation, lipopolysaccharide profiles and/or protease activity. Seven of the nine phage-resistant strains showed virulence reduction in a Sparus aurata larvae model. However, this is not generalized since two of the resistant strains show equal virulence compared with the parental strain. The genomic analysis of representative resistant strains displayed that the majority of the mutations are specific for each isolate, affecting genes related to lipopolysaccharide biosynthesis, quorum sensing, motility, toxin and membrane transport. The observed mutations were coherent with the phenotypic and virulence differences observed. These results suggest that the CHOED phage acts as a selective pressure on V. anguillarum, allowing proliferation of resistant strains with different genotypes, phenotypes and degrees of virulence, contributing to bacterial diversification.

We present here the draft genome sequences of six Vibrio diazotrophicus strains, which were isolated from deep subseafloor sediments of the Baltic Sea. The genomic sequences contained several virulence and antibiotic resistance genes. These genome sequences provide insights into the genetic composition and evolution of the genus Vibrio in marine environments.