Bacteriophage protein Dap1 regulates evasion of antiphage immunity and Pseudomonas aeruginosa virulence impacting phage therapy in mice.

Bacteriophages have evolved diverse strategies to overcome host defence mechanisms and to redirect host metabolism to ensure successful propagation. Here we identify a phage protein named Dap1 from Pseudomonas aeruginosa phage PaoP5 that both modulates bacterial host behaviour and contributes to phage fitness. We show that expression of Dap1 in P. aeruginosa reduces bacterial motility and promotes biofilm formation through interference with DipA, a c-di-GMP phosphodiesterase, which causes an increase in c-di-GMP levels that trigger phenotypic changes. Results also show that deletion of dap1 in PaoP5 significantly reduces genome packaging. In this case, Dap1 directly binds to phage HNH endonuclease, prohibiting host Lon-mediated HNH degradation and promoting phage genome packaging. Moreover, PaoP5Δdap1 fails to rescue P. aeruginosa-infected mice, implying the significance of dap1 in phage therapy. Overall, these results highlight remarkable dual functionality in a phage protein, enabling the modulation of host behaviours and ensuring phage fitness.

Quorum sensing gene lasR promotes phage vB_Pae_PLY infection in Pseudomonas aeruginosa.

BACKGROUND: Quorum sensing (QS) is a cell density-based intercellular communication system that controls virulence gene expression and biofilm formation. In Pseudomonas aeruginosa (P. aeruginosa), the LasR system sits at the top of the QS hierarchy and coordinates the expression of a series of important traits. However, the role of lasR in phage infection remains unclear. This study aims to investigate the role of lasR QS in phage infection. METHODS: The P. aeruginosa phage was isolated from sewage, and its biological characteristics and whole genome were analyzed. The adsorption receptor was identified via a phage adsorption assay. Following lasR gene knockout, the adsorption rate and bactericidal activity of phage were analyzed. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to explore how lasR promoting phage infection. RESULTS: The lytic phage vB_Pae_PLY was isolated and lipopolysaccharide (LPS) was identified as its adsorption receptor. The adsorption rate and bactericidal activity of vB_Pae_PLY were reduced after lasR knockout. RT-qPCR results showed that the expression of galU, a key gene involved in LPS synthesis, was down-regulated, and several genes related to type IV pili (T4P) were also down-regulated in the lasR mutant PaΔlasR. CONCLUSIONS: The study showed that QS lasR may promote phage vB_Pae_PLY infection by involving in the synthesis of LPS and T4P. This study provides an example of QS in promoting phage infection and deepens the understanding of phage-bacteria interactions.

Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

A phage tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria.

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control.

Coevolutionary analysis of Pseudomonas syringae-phage interactions to help with rational design of phage treatments.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges in vitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Lysis Physiology of Pseudomonas aeruginosa Infected with ssRNA Phage PRR1.

The phage PRR1 belongs to the Leviviridae family, a group of ssRNA bacteriophages that infect Gram-negative bacteria. The variety of host cells is determined by the specificity of PRR1 to a pilus encoded by a broad host range of IncP-type plasmids that confer multiple types of antibiotic resistance to the host. Using P. aeruginosa strain PAO1 as a host, we analyzed the PRR1 infection cycle, focusing on cell lysis. PRR1 infection renders P. aeruginosa cells sensitive to lysozyme approximately 20 min before the start of a drop in suspension turbidity. At the same time, infected cells start to accumulate lipophilic anions. The on-line monitoring of the entire infection cycle showed that single-gene-mediated lysis strongly depends on the host cells' physiological state. The blockage of respiration or a reduction in the intracellular ATP concentration during the infection resulted in the inhibition of lysis. The same effect was observed when the synthesis of PRR1 lysis protein was induced in an E. coli expression system. In addition, lysis was strongly dependent on the level of aeration. Dissolved oxygen concentrations sufficient to support cell growth did not ensure efficient lysis, and a coupling between cell lysis initiation and aeration level was observed. However, the duration of the drop in suspension turbidity did not depend on the level of aeration.

Development of an enzyme-linked phage receptor-binding protein assay (ELPRA) based on a novel biorecognition molecule- receptor-binding protein Gp130 of Pseudomonas aeruginosa bacteriophage Henu5.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818-0.991), and the specificity was 0.905 (0.682-0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave.

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

PqsA mutation-mediated enhancement of phage-mediated combat against Pseudomonas aeruginosa.

Phage therapy is a potential approach in the biocontrol of foodborne pathogens. However, the emergence of phage resistance and the narrow host range of most phage isolates continue to limit the antimicrobial efficacy of phages. Here, we investigated the potential of the pqsA gene, encoding the anthranilate-CoA ligase enzyme, as an adjuvant for phage therapy. The knockout of the pqsA gene significantly enhanced the bactericidal effect of phages vB_Pae_QDWS and vB_Pae_S1 against Pseudomonas aeruginosa. Under phage infection pressure, the growth of the PaΔpqsA was significantly inhibited within 8 h compared to the wild-type PAO1. Furthermore, we found that altering phage adsorption is not how PaΔpqsA responds to phage infection. Although pqsA represents a promising target for enhancing phage killing, it may not be applicable to all phages, such as types vB_Pae_W3 and vB_Pae_TR. Our findings provide new material reserves for the future design of novel phage-based therapeutic strategies.

Targeted Genome Editing of Virulent Pseudomonas Phages Using CRISPR-Cas3.

The vast number of unknown phage-encoded ORFan genes and limited insights into the genome organization of phages illustrate the need for efficient genome engineering tools to study bacteriophage genes in their natural context. In addition, there is an application-driven desire to alter phage properties, which is hampered by time constraints for phage genome engineering in the bacterial host. We here describe an optimized CRISPR-Cas3 system in Pseudomonas for straightforward editing of the genome of virulent bacteriophages. The two-vector system combines a broad host range CRISPR-Cas3 targeting plasmid with a SEVA plasmid for homologous directed repair, which enables the creation of clean deletions, insertions, or substitutions in the phage genome within a week. After creating the two plasmids separately, a co-transformation to P. aeruginosa cells is performed. A subsequent infection with the targeted phage allows the CRISPR-Cas3 system to cut the DNA specifically and facilitate or select for homologous recombination. This system has also been successfully applied for P. aeruginosa and Pseudomonas putida genome engineering. The method is straightforward, efficient, and universal, enabling to extrapolate the system to other phage-host pairs.

Accumulation of defense systems in phage-resistant strains of Pseudomonas aeruginosa.

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains.

Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University.

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d'Hérelle's first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.

Characterization of a new Pseudomonas aeruginosa Queuovirinae bacteriophage.

The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Pharmacokinetics and safety evaluation of intravenously administered Pseudomonas phage PA_LZ7 in a mouse model.

IMPORTANCE: Phage therapy is gaining traction as an alternative to antibiotics due to the rise of multi-drug-resistant (MDR) bacteria. This study assessed the pharmacokinetics and safety of PA_LZ7, a phage targeting MDR Pseudomonas aeruginosa, in mice. After intravenous administration, the phage showed an exponential decay in plasma and its concentration dropped significantly within 24 h for all dosage groups. Although there was a temporary increase in certain plasma cytokines and spleen weight at higher dosages, no significant toxicity was observed. Therefore, PA_LZ7 shows potential as an effective and safe candidate for future phage therapy against MDR P. aeruginosa infections.

Biophysical basis of filamentous phage tactoid-mediated antibiotic tolerance in P. aeruginosa.

Inoviruses are filamentous phages infecting numerous prokaryotic phyla. Inoviruses can self-assemble into mesoscale structures with liquid-crystalline order, termed tactoids, which protect bacterial cells in Pseudomonas aeruginosa biofilms from antibiotics. Here, we investigate the structural, biophysical, and protective properties of tactoids formed by the P. aeruginosa phage Pf4 and Escherichia coli phage fd. A cryo-EM structure of the capsid from fd revealed distinct biochemical properties compared to Pf4. Fd and Pf4 formed tactoids with different morphologies that arise from differing phage geometries and packing densities, which in turn gave rise to different tactoid emergent properties. Finally, we showed that tactoids formed by either phage protect rod-shaped bacteria from antibiotic treatment, and that direct association with a tactoid is required for protection, demonstrating the formation of a diffusion barrier by the tactoid. This study provides insights into how filamentous molecules protect bacteria from extraneous substances in biofilms and in host-associated infections.

Jumbo phages are active against extensively drug-resistant eyedrop-associated Pseudomonas aeruginosa infections.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

Diversity and Current Classification of dsRNA Bacteriophages.

Half a century has passed since the discovery of Pseudomonas phage phi6, the first enveloped dsRNA bacteriophage to be isolated. It remained the sole known dsRNA phage for a quarter of a century and the only recognised member of the Cystoviridae family until the year 2018. After the initial discovery of phi6, additional dsRNA phages have been isolated from globally distant locations and identified in metatranscriptomic datasets, suggesting that this virus type is more ubiquitous in nature than previously acknowledged. Most identified dsRNA phages infect Pseudomonas strains and utilise either pilus or lipopolysaccharide components of the host as the primary receptor. In addition to the receptor-mediated strictly lytic lifestyle, an alternative persistent infection strategy has been described for some dsRNA phages. To date, complete genome sequences of fourteen dsRNA phage isolates are available. Despite the high sequence diversity, similar sets of genes can typically be found in the genomes of dsRNA phages, suggesting shared evolutionary trajectories. This review provides a brief overview of the recognised members of the Cystoviridae virus family and related dsRNA phage isolates, outlines the current classification of dsRNA phages, and discusses their relationships with eukaryotic RNA viruses.