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1. Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes

   https://doi.org/10.1128/AEM.02138-20  

   Peters, Tracey Lee; Song, Yaxiong; Bryan, Daniel W.; Hudson, Lauren K.; Denes, Thomas G. (October 2020, Applied and Environmental Microbiology)

   Dudley, Edward G. (Ed.)

   ABSTRACT Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes . Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages. IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes . This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes . This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy. 

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2. Gene Co-occurrence Networks Reflect Bacteriophage Ecology and Evolution

   https://doi.org/10.1128/mBio.01870-17  

   Shapiro, Jason W.; Putonti, Catherine; Keim, Paul (May 2018, mBio)

   ABSTRACT Bacteriophages are the most abundant and diverse biological entities on the planet, and new phage genomes are being discovered at a rapid pace. As more phage genomes are published, new methods are needed for placing these genomes in an ecological and evolutionary context. Phages are difficult to study by phylogenetic methods, because they exchange genes regularly, and no single gene is conserved across all phages. Here, we demonstrate how gene-level networks can provide a high-resolution view of phage genetic diversity and offer a novel perspective on virus ecology. We focus our analyses on virus host range and show how network topology corresponds to host relatedness, how to find groups of genes with the strongest host-specific signatures, and how this perspective can complement phage host prediction tools. We discuss extensions of gene network analysis to predicting the emergence of phages on new hosts, as well as applications to features of phage biology beyond host range. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria, and they are critical drivers of bacterial evolution and community structure. It is generally difficult to study phages by using tree-based methods, because gene exchange is common, and no single gene is shared among all phages. Instead, networks offer a means to compare phages while placing them in a broader ecological and evolutionary context. In this work, we build a network that summarizes gene sharing across phages and test how a key constraint on phage ecology, host range, corresponds to the structure of the network. We find that the network reflects the relatedness among phage hosts, and phages with genes that are closer in the network are likelier to infect similar hosts. This approach can also be used to identify genes that affect host range, and we discuss possible extensions to analyze other aspects of viral ecology. 

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3. Isolation and characterization of four novel Vibrio parahaemolyticus bacteriophages from oysters

   https://doi.org/10.1371/journal.pone.0339894  

   Aldaroub, Joud; Walsky, Chrissy M; Ewell, Rylee E; Aylward, Frank O; Stevens, Ann M; Burke, Alison Kernell (December 2025, PLOS One)

   Fernández_Robledo, José A (Ed.)

   Vibrio parahaemolyticus(VP) is a bacterial pathogen found in brackish and marine water that infects many marine organisms, such as oysters and shrimp. Consumption of raw or undercooked seafood contaminated withV. parahaemolyticusis a primary cause of seafood-borne gastroenteritis in humans. Due to increasing ocean temperatures,V. parahaemolyticuscontamination of oyster beds in the United States has spread up the east and west coasts to the northern-most states. Promising new research is exploring the isolation of bacteriophages againstV. parahaemolyticuswith a long-term goal to possibly decontaminate oyster beds, thereby expanding the harvest season and allowing for safer consumption of seafood. In this study, store-bought oysters harvested from the Chesapeake Bay in Virginia were used to isolate four bacteriophages with activity against a specificV. parahaemolyticusstrain. A standard double agar overlay plaque assay was used to identify phage activity. After phage isolation, the genomes were sequenced, and transmission electron microscopy (TEM) was performed to visualize the virions. The genomes and TEM images revealed four distinct phages. Three of the phages are distinct isolates that exhibit podovirus-like morphology with short tails and genome sizes of approximately 43 kbp. One phage has siphovirus-like morphology and is a mid-sized tailed phage with a genome size of 80 kbp. Although spot tests performed with the oyster homogenates on up to 10 differentV. parahaemolyticusstrains recovered activity across a wide range of hosts, plaque assays with the isolated phages showed limited host range. Future work will be necessary to determine the viability of using the bacteriophages for elimination ofV. parahaemolyticusin harvested oysters, treatment of aquaculture seed and spat, and/or the environment. 

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4. Phage DisCo: targeted discovery of bacteriophages by co-culture

   https://doi.org/10.1128/msystems.01644-24  

   Rand, Eleanor A; Quinones-Olvera, Natalia; Jean, Kesther_D C; Hernandez-Perez, Carmen; Owen, Siân V; Baym, Michael (June 2025, mSystems)

   Reyes_Munoz, Alejandro (Ed.)

   Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology a priori is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages, and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method,phage discovery by co-culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the Phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.IMPORTANCEIn this work, we describe a targeted phage discovery method that allows immediate isolation of phages with specific traits. Currently, to find a phage with specific properties, huge libraries of phages must be collected and screened retroactively. This assay, Phage Discovery by Co-culture (Phage DisCo), works by co-culture of host strains that are identical except for one perturbation that may interfere with phage infection and a unique fluorescent marker. These strains are co-cultured with an environmental sample of interest in traditional plaque assay format, making phage characteristics easily identifiable by fluorescent signal after imaging of the screening plate. We validate that Phage DisCo can identify phages with specific properties, even when these phages are rare in samples. This approach allows rapid exploration of the diversity within phage samples with vastly streamlined processes, and we anticipate it will be widely adopted within the phage discovery field. 

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5. Diverse Marine T4-like Cyanophage Communities Are Primarily Comprised of Low-Abundance Species Including Species with Distinct Seasonal, Persistent, Occasional, or Sporadic Dynamics

   https://doi.org/10.3390/v15020581  

   Dart, Emily; Fuhrman, Jed A.; Ahlgren, Nathan A. (February 2023, Viruses)

   Cyanophages exert important top-down controls on their cyanobacteria hosts; however, concurrent analysis of both phage and host populations is needed to better assess phage–host interaction models. We analyzed picocyanobacteria Prochlorococcus and Synechococcus and T4-like cyanophage communities in Pacific Ocean surface waters using five years of monthly viral and cellular fraction metagenomes. Cyanophage communities contained thousands of mostly low-abundance (<2% relative abundance) species with varying temporal dynamics, categorized as seasonally recurring or non-seasonal and occurring persistently, occasionally, or sporadically (detected in ≥85%, 15-85%, or <15% of samples, respectively). Viromes contained mostly seasonal and persistent phages (~40% each), while cellular fraction metagenomes had mostly sporadic species (~50%), reflecting that these sample sets capture different steps of the infection cycle—virions from prior infections or within currently infected cells, respectively. Two groups of seasonal phages correlated to Synechococcus or Prochlorococcus were abundant in spring/summer or fall/winter, respectively. Cyanophages likely have a strong influence on the host community structure, as their communities explained up to 32% of host community variation. These results support how both seasonally recurrent and apparent stochastic processes, likely determined by host availability and different host-range strategies among phages, are critical to phage–host interactions and dynamics, consistent with both the Kill-the-Winner and the Bank models. 

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