Co-evolution Of Phages Research Articles

Lytic Activity Spectra of Bacteriophages

One of the central topics in bacteriophage research is the host specificity. It depends on the success of completing viral life cycle stages, including adsorption, penetration of the genetic material of the virus into the cell and its replication, assembly of phage particles and cell lysis. Laboratory assessments of the spectrum of lytic activity of phages are inextricably linked to significant methodological biases, and the often used spot test method can be associated with a large percentage of false-positive results. Along with the variety of types of phage specificity, there is temporal variability. The co-evolution of phages and bacteria leads to the acquisition of resistance to viruses by bacteria and the accumulation of mutations in the genomes of bacteriophages aimed at overcoming this resistance. At the same time, the adaptation of bacteriophages to bacteria that are evolutionarily distant from the isolation hosts is barely possible. This barrier is based on the peculiarities of metabolism, cell wall structures and mechanisms for the implementation of matrix processes. The spatial factor of phage specificity is manifested in the greater breadth of the spectra of lytic activity of bacteriophages on local samples of bacteria compared to the spectra assessed on samples of isolates from habitats geographically distant from the place of virus isolation.

Bacteriophages in the Dairy Industry: A Problem Solved?

Bacteriophages (or phages) represent one of the most persistent threats to food fermentations, particularly large-scale commercial dairy fermentations. Phages infecting lactic acid bacteria (LAB) that are used as starter cultures in dairy fermentations are well studied, and in recent years there have been significant advances in defining the driving forces of LAB-phage coevolution. The means by which different starter bacterial species defend themselves against phage predation and the chromosomal or plasmid location of the genes encoding these defense mechanisms have dictated the technological approaches for the development of robust starter cultures. In this review, we highlight recent advances in defining phage-host interactions and how phage resistance occurs in different bacterial species. Furthermore, we discuss how these insights continue to transform the dairy fermentation industry and how they also are anticipated to guide food fermentations involving plant-based alternatives in the future.

Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance.

Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria–phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.

Parallel phage resistance - virulence trade - offs during clinical phage therapy and in vitro

With rising antibiotic resistance, modern medicine needs new approaches for tackling bacterial infections. Phage therapy uses the viruses of pathogenic bacteria to clear the infection. Unlike antibiotics, phage can evolve if bacteria become resistant to maintain or even increase their infectivity (coevolution). While laboratory studies can give insight into complex bacteria-phage interactions, whether they act as a true representation of phage therapy in patients is unknown. Here, we compared phage therapy in a patient to that of in vitro experiments. The patient had been admitted with aPseudomonas aeruginosainfection and was successfully treated with a phage cocktail. Bacteria were isolated before and during phage therapy, allowing us to follow bacteria-phage coevolution in the patient while doing experiments on the same clones in vitro. In vivo and in vitro, bacteria rapidly evolved resistance with little or no evidence of bacteria - phage coevolution. Although resistance mechanisms differed, parallel resistance - virulence trade-offs were found in vivo and in vitro. Therefore, phage resistance could increase treatment success and our results indicate to what extent bacteria - phage evolutionary dynamics can be predicted from in vitro experiments.

Systematic evaluation of horizontal gene transfer between eukaryotes and viruses.

Gene exchange between viruses and their hosts acts as a key facilitator of horizontal gene transfer and is hypothesized to be a major driver of evolutionary change. Our understanding of this process comes primarily from bacteria and phage co-evolution, but the mode and functional importance of gene transfers between eukaryotes and their viruses remain anecdotal. Here we systematically characterized viral-eukaryotic gene exchange across eukaryotic and viral diversity, identifying thousands of transfers and revealing their frequency, taxonomic distribution and projected functions. Eukaryote-derived viral genes, abundant in the Nucleocytoviricota, highlighted common strategies for viral host-manipulation, including metabolic reprogramming, proteolytic degradation and extracellular modification. Furthermore, viral-derived eukaryotic genes implicate genetic exchange in the early evolution and diversification of eukaryotes, particularly through viral-derived glycosyltransferases, which have impacted structures as diverse as algal cell walls, trypanosome mitochondria and animal tissues. These findings illuminate the nature of viral-eukaryotic gene exchange and its impact on the evolution of viruses and their eukaryotic hosts.

Survival Comes at a Cost: A Coevolution of Phage and Its Host Leads to Phage Resistance and Antibiotic Sensitivity of Pseudomonas aeruginosa Multidrug Resistant Strains.

The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (∼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection.

Phages and their lysins: Toolkits in the battle against foodborne pathogens in the postantibiotic era.

Worldwide, foods waste caused by putrefactive organisms and diseases caused by foodborne pathogens persist as public health problems even with a plethora of modern antimicrobials. Our over reliance on antimicrobials use in agriculture, medicine, and other fields will lead to a postantibiotic era where bacterial genotypic resistance, phenotypic adaptation, and other bacterial evolutionary strategies cause antimicrobial resistance (AMR). This AMR is evidenced by the emergence of multiple drug-resistant (MDR) bacteria and pan-resistant (PDR) bacteria, which produces cross-contamination in multiple fields and poses a more serious threat to food safety. A "red queen premise" surmises that the coevolution of phages and bacteria results in an evolutionary arms race that compels phages to adapt and survive bacterial antiphage strategies. Phages and their lysins are therefore useful toolkits in the design of novel antimicrobials in food protection and foodborne pathogens control, and the modality of using phages as a targeted vector against foodborne pathogens is gaining momentum based on many encouraging research outcomes. In this review, we discuss the rationale of using phages and their lysins as weapons against spoilage organisms and foodborne pathogens, and outline the targeted conquest or dodge mechanism of phages and the development of novel phage prospects. We also highlight the implementation of phages and their lysins to control foodborne pathogens in a farm-table-hospital domain in the postantibiotic era.

Spatial patterns in phage-Rhizobium coevolutionary interactions across regions of common bean domestication

Bacteriophages play significant roles in the composition, diversity, and evolution of bacterial communities. Despite their importance, it remains unclear how phage diversity and phage-host interactions are spatially structured. Local adaptation may play a key role. Nitrogen-fixing symbiotic bacteria, known as rhizobia, have been shown to locally adapt to domesticated common bean at its Mesoamerican and Andean sites of origin. This may affect phage-rhizobium interactions. However, knowledge about the diversity and coevolution of phages with their respective Rhizobium populations is lacking. Here, through the study of four phage-Rhizobium communities in Mexico and Argentina, we show that both phage and host diversity is spatially structured. Cross-infection experiments demonstrated that phage infection rates were higher overall in sympatric rhizobia than in allopatric rhizobia except for one Argentinean community, indicating phage local adaptation and host maladaptation. Phage-host interactions were shaped by the genetic identity and geographic origin of both the phage and the host. The phages ranged from specialists to generalists, revealing a nested network of interactions. Our results suggest a key role of local adaptation to resident host bacterial communities in shaping the phage genetic and phenotypic composition, following a similar spatial pattern of diversity and coevolution to that in the host.

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

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.IMPORTANCEListeria 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.

A generalised model for generalised transduction: the importance of co-evolution and stochasticity in phage mediated antimicrobial resistance transfer.

Antimicrobial resistance is a major global challenge. Of particular concern are mobilizable elements that can transfer resistance genes between bacteria, leading to pathogens with new combinations of resistance. To date, mathematical models have largely focussed on transfer of resistance by plasmids, with fewer studies on transfer by bacteriophages. We aim to understand how best to model transfer of resistance by transduction by lytic phages. We show that models of lytic bacteriophage infection with empirically derived realistic phage parameters lead to low numbers of bacteria, which, in low population or localised environments, lead to extinction of bacteria and phage. Models that include antagonistic co-evolution of phage and bacteria produce more realistic results. Furthermore, because of these low numbers, stochastic dynamics are shown to be important, especially to spread of resistance. When resistance is introduced, resistance can sometimes be fixed, and at other times die out, with the probability of each outcome sensitive to bacterial and phage parameters. Specifically, that outcome most strongly depends on the baseline death rate of bacteria, with phage-mediated spread favoured in benign environments with low mortality over more hostile environments. We conclude that larger-scale models should consider spatial compartmentalisation and heterogeneous microenviroments, while encompassing stochasticity and co-evolution.

Formation of phage lysis patterns and implications on co-propagation of phages and motile host bacteria.

Coexistence of bacteriophages, or phages, and their host bacteria plays an important role in maintaining the microbial communities. In natural environments with limited nutrients, motile bacteria can actively migrate towards locations of richer resources. Although phages are not motile themselves, they can infect motile bacterial hosts and spread in space via the hosts. Therefore, in a migrating microbial community coexistence of bacteria and phages implies their co-propagation in space. Here, we combine an experimental approach and mathematical modeling to explore how phages and their motile host bacteria coexist and co-propagate. When lytic phages encountered motile host bacteria in our experimental set up, a sector-shaped lysis zone formed. Our mathematical model indicates that local nutrient depletion and the resulting inhibition of proliferation and motility of bacteria and phages are the key to formation of the observed lysis pattern. The model further reveals the straight radial boundaries in the lysis pattern as a telltale sign for coexistence and co-propagation of bacteria and phages. Emergence of such a pattern, albeit insensitive to extrinsic factors, requires a balance between intrinsic biological properties of phages and bacteria, which likely results from coevolution of phages and bacteria.

Prophage Hunter: an integrative hunting tool for active prophages.

Identifying active prophages is critical for studying coevolution of phage and bacteria, investigating phage physiology and biochemistry, and engineering designer phages for diverse applications. We present Prophage Hunter, a tool aimed at hunting for active prophages from whole genome assembly of bacteria. Combining sequence similarity-based matching and genetic features-based machine learning classification, we developed a novel scoring system that exhibits higher accuracy than current tools in predicting active prophages on the validation datasets. The option of skipping similarity matching is also available so that there's higher chance for novel phages to be discovered. Prophage Hunter provides a one-stop web service to extract prophage genomes from bacterial genomes, evaluate the activity of the prophages, identify phylogenetically related phages, and annotate the function of phage proteins. Prophage Hunter is freely available at https://pro-hunter.bgi.com/.

Spontaneously induced prophages are abundant in a naturally evolved bacterial starter culture and deliver competitive advantage to the host

BackgroundIn complex microbial ecosystems such as the marine environment, the gastrointestinal tract, but also in mixed culture fermentations, bacteriophages are frequently found to be a part of the microbial community. Moreover, prophages or prophage-like elements are frequently identified in sequenced bacterial genomes. The mixed undefined starter cultures represent an ecosystem which is shaped by long term evolution under relatively defined environmental conditions and provides an interesting model to study co-evolution of phages and their hosts as well as the impact of diversity on microbial community stability.ResultsIn the present study we investigated the presence, identity and behaviour of prophages in lactococci being part of a complex cheese starter culture. Genome analysis of representative strains of the 7 genetic lineages of Lactococcus lactis constituting the culture indicated the presence of prophages in all strains. Exposure of potential lysogens to mitomycin C confirmed the release of ~ 1010·ml− 1 phage particles from all tested strains. Furthermore, phages were also released in substantial amounts due to spontaneous induction: more than 108·ml− 1 phage particles were present in cultures under non-inducing conditions. This observation suggests continuous release of phage particles by the lactococci. The released bacteriophages exhibited an unusual morphology. For most strains tested, tailless icosahedral phage heads were found. The competitive advantage of lysogens compared to their cured derivatives and their high abundance in the culture suggests that the released tailless bacteriophages play an important role in the ecosystem.ConclusionsThe results of this study indicate that chromosomal genetic elements are active participants in the stable complex microbial community of the starter culture. We show that prophages are abundant in such a community, are produced continuously in large amounts and, despite the huge metabolic burden imposed on the cells by phage particle production, provide a selective advantage to the host.

Prophage-mediated defence against viral attack and viral counter-defence.

Temperate phages are common, and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses that infect mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages revealed at least five distinct prophage-expressed viral defence systems that interfere with the infection of lytic and temperate phages that are either closely related (homotypic defence) or unrelated (heterotypic defence) to the prophage. Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defence systems include a single-subunit restriction system, a heterotypic exclusion system and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, which acts as a highly effective counter-defence system. Prophage-mediated viral defence offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defence promotes phage co-evolution.

The driving force of prophages and CRISPR-Cas system in the evolution of Cronobacter sakazakii

Cronobacter sakazakii is an important foodborne pathogens causing rare but life-threatening diseases in neonates and infants. CRISPR-Cas system is a new prokaryotic defense system that provides adaptive immunity against phages, latter play an vital role on the evolution and pathogenicity of host bacteria. In this study, we found that genome sizes of C. sakazakii strains had a significant positive correlation with total genome sizes of prophages. Prophages contributed to 16.57% of the genetic diversity (pan genome) of C. sakazakii, some of which maybe the potential virulence factors. Subtype I-E CRISPR-Cas system and five types of CRISPR arrays were found in the conserved site of C. sakazakii strains. CRISPR1 and CRISPR2 loci with high variable spacers were active and showed potential protection against phage attacks. The number of spacers from two active CRISPR loci in clinical strains was significant less than that of foodborne strains, it maybe a reason why clinical strains were found to have more prophages than foodborne strains. The frequently gain/loss of prophages and spacers in CRISPR loci is likely to drive the quick evolution of C. sakazakii. Our study provides a new insight into the co-evolution of phages and C. sakazakii.

Plasmid carriage can limit bacteria-phage coevolution.

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria–phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria–phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.

Research highlights for issue 6: the CRISPR/Cas revolution.

Research highlights for issue 6: the CRISPR/Cas revolution.

Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities.

Bacteria–phage coevolution, the reciprocal evolution between bacterial hosts and the phages that infect them, is an important driver of ecological and evolutionary processes in microbial communities. There is growing evidence from both laboratory and natural populations that coevolution can maintain phenotypic and genetic diversity, increase the rate of bacterial and phage evolution and divergence, affect community structure, and shape the evolution of ecologically relevant bacterial traits. Although the study of bacteria–phage coevolution is still in its infancy, with open questions regarding the specificity of the interaction, the gene networks of coevolving partners, and the relative importance of the coevolving interaction in complex communities and environments, there have recently been major advancements in the field. In this review, we sum up our current understanding of bacteria–phage coevolution both in the laboratory and in nature, discuss recent findings on both the coevolutionary process itself and the impact of coevolution on bacterial phenotype, diversity and interactions with other species (particularly their eukaryotic hosts), and outline future directions for the field.

Membrane-like protein involved in phage adsorption associated with phage-sensitivity in the bloom-forming cyanobacterium Microcystis aeruginosa

The cyanobacterium, Microcystis aeruginosa, contains a large number of defense genes (Makarova et al., 2011); thus, it is a good model to study the co-evolution of phage and bacteria. Here, we isolated and characterized two phage-resistant M. aeruginosa mutants that came from a phage intermediate-sensitive culture. To determine the mutation conferring resistance, a protein expression pattern analysis was performed comparing phage-sensitive and -resistant sub-strains using SDS-PAGE. There were no apparent differences in expression patterns in the soluble fraction; however, a ∼90kDa protein in the hydrophobic fraction from the phage-sensitive sub-strain was observed. Using a successive thermal asymmetric interlaced-PCR, the entire sequence encoding the protein, assigned ISP90, as well as its neighboring regions (ca. 7.8kb) was determined. ISP90 contained no conserved domains and was predicted to be a membrane-associated protein. No mutations were detected in the nucleotide sequences coding ISP90 and diversification of ISP90 regions within this species were observed. Diversification of ISP90 regions within this species suggests a possible genomic island that may be subjected to selective pressures from phages. The ISP90 sequence involving phage resistance/sensitivity contributes to the understanding of co-evolution between M. aeruginosa and phages.

Coevolutionary diversification creates nested-modular structure in phage–bacteria interaction networks

Phage and their bacterial hosts are the most diverse and abundant biological entities in the oceans, where their interactions have a major impact on marine ecology and ecosystem function. The structure of interaction networks for natural phage-bacteria communities offers insight into their coevolutionary origin. At small phylogenetic scales, observed communities typically show a nested structure, in which both hosts and phages can be ranked by their range of resistance and infectivity, respectively. A qualitatively different multi-scale structure is seen at larger phylogenetic scales; a natural assemblage sampled from the Atlantic Ocean displays large-scale modularity and local nestedness within each module. Here, we show that such 'nested-modular' interaction networks can be produced by a simple model of host-phage coevolution in which infection depends on genetic matching. Negative frequency-dependent selection causes diversification of hosts (to escape phages) and phages (to track their evolving hosts). This creates a diverse community of bacteria and phage, maintained by kill-the-winner ecological dynamics. When the resulting communities are visualized as bipartite networks of who infects whom, they show the nested-modular structure characteristic of the Atlantic sample. The statistical significance and strength of this observation varies depending on whether the interaction networks take into account the density of the interacting strains, with implications for interpretation of interaction networks constructed by different methods. Our results suggest that the apparently complex community structures associated with marine bacteria and phage may arise from relatively simple coevolutionary origins.