Selection and Characterization of Cyanophage Resistance in Marine Synechococcus Strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Parasite diversity drives rapid host dynamics and evolution of resistance in a bacteria‐phage system

Host–parasite evolutionary interactions are typically considered in a pairwise species framework. However, natural infections frequently involve multiple parasites. Altering parasite diversity alters ecological and evolutionary dynamics as parasites compete and hosts resist multiple infection. We investigated the effects of parasite diversity on host–parasite population dynamics and evolution using the pathogen Pseudomonas aeruginosa and five lytic bacteriophage parasites. To manipulate parasite diversity, bacterial populations were exposed for 24 hours to either phage monocultures or diverse communities containing up to five phages. Phage communities suppressed host populations more rapidly but also showed reduced phage density, likely due to interphage competition. The evolution of resistance allowed rapid bacterial recovery that was greater in magnitude with increases in phage diversity. We observed no difference in the extent of resistance with increased parasite diversity, but there was a profound impact on the specificity of resistance; specialized resistance evolved to monocultures through mutations in a diverse set of genes. In summary, we demonstrate that parasite diversity has rapid effects on host–parasite population dynamics and evolution by selecting for different resistance mutations and affecting the magnitude of bacterial suppression and recovery. Finally, we discuss the implications of phage diversity for their use as biological control agents.     

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.     

Selection and characterization of cyanophage resistance in marine Synechococcus strains

Marine viruses are an important component of the microbial food web, influencing microbial diversity and contributing to bacterial mortality rates. Resistance to cooccurring cyanophages has been reported for natural communities of Synechococcus spp.; however, little is known about the nature of this resistance. This study examined the patterns of infectivity among cyanophage isolates and unicellular marine cyanobacteria (Synechococcus spp.). We selected for phage-resistant Synechococcus mutants, examined the mechanisms of phage resistance, and determined the extent of cross-resistance to other phages. Four strains of Synechococcus spp. (WH7803, WH8018, WH8012, and WH8101) and 32 previously isolated cyanomyophages were used to select for phage resistance. Phage-resistant Synechococcus mutants were recovered from 50 of the 101 susceptible phage-host pairs, and 23 of these strains were further characterized. Adsorption kinetic assays indicate that resistance is likely due to changes in host receptor sites that limit viral attachment. Our results also suggest that receptor mutations conferring this resistance are diverse. Nevertheless, selection for resistance to one phage frequently resulted in cross-resistance to other phages. On average, phage-resistant Synechococcus strains became resistant to eight other cyanophages; however, there was no significant correlation between the genetic similarity of the phages (based on g20 sequences) and cross-resistance. Likewise, host Synechococcus DNA-dependent RNA polymerase (rpoC1) genotypes could not be used to predict sensitivities to phages. The potential for the rapid evolution of multiple phage resistance may influence the population dynamics and diversity of both Synechococcus and cyanophages in marine waters.     

Systematic exploration of Escherichia coli phage–host interactions with the BASEL phage collection

Bacteriophages, the viruses infecting bacteria, hold great potential for the treatment of multidrug-resistant bacterial infections and other applications due to their unparalleled diversity and recent breakthroughs in their genetic engineering. However, fundamental knowledge of the molecular mechanisms underlying phage–host interactions is mostly confined to a few traditional model systems and did not keep pace with the recent massive expansion of the field. The true potential of molecular biology encoded by these viruses has therefore remained largely untapped, and phages for therapy or other applications are often still selected empirically. We therefore sought to promote a systematic exploration of phage–host interactions by composing a well-assorted library of 68 newly isolated phages infecting the model organism Escherichia coli that we share with the community as the BASEL (BActeriophage SElection for your Laboratory) collection. This collection is largely representative of natural E. coli phage diversity and was intensively characterized phenotypically and genomically alongside 10 well-studied traditional model phages. We experimentally determined essential host receptors of all phages, quantified their sensitivity to 11 defense systems across different layers of bacterial immunity, and matched these results to the phages’ host range across a panel of pathogenic enterobacterial strains. Clear patterns in the distribution of phage phenotypes and genomic features highlighted systematic differences in the potency of different immunity systems and suggested the molecular basis of receptor specificity in several phage groups. Our results also indicate strong trade-offs between fitness traits like broad host recognition and resistance to bacterial immunity that might drive the divergent adaptation of different phage groups to specific ecological niches. We envision that the BASEL collection will inspire future work exploring the biology of bacteriophages and their hosts by facilitating the discovery of underlying molecular mechanisms as the basis for an effective translation into biotechnology or therapeutic applications.

This study presents the BASEL collection of phages that infect the model bacterium Escherichia coli; this resource for the community is representative of natural E. coli phage diversity and has been extensively characterized phenotypically and genomically.     

Local biotic environment shapes the spatial scale of bacteriophage adaptation to bacteria

The ecological, epidemiological, and evolutionary consequences of host-parasite interactions are critically shaped by the spatial scale at which parasites adapt to hosts. The scale of interaction between hyperparasites and their parasites is likely to be influenced by the host of the parasite and potentially likely to differ among within-host environments. Here we examine the scale at which bacteriophages adapt to their host bacteria by studying natural isolates from the surface or interior of horse chestnut leaves. We find that phages are more infective to bacteria from the same tree relative to those from other trees but do not differ in infectivity to bacteria from different leaves within the same tree. The results suggest that phages target common bacterial species, including an important plant pathogen, within plant host tissues; this result has important implications for therapeutic phage epidemiology. Furthermore, we show that phages from the leaf interior are more infective to their local hosts than phages from the leaf surface are to theirs, suggesting either increased resistance of bacteria on the leaf surface or increased phage adaptation within the leaf. These results highlight that biotic environment can play a key role in shaping the spatial scale of parasite adaptation and influencing the outcome of coevolutionary interactions.     

Outside-host phage therapy as a biological control against environmental infectious diseases

Background

Environmentally growing pathogens present an increasing threat for human health, wildlife and food production. Treating the hosts with antibiotics or parasitic bacteriophages fail to eliminate diseases that grow also in the outside-host environment. However, bacteriophages could be utilized to suppress the pathogen population sizes in the outside-host environment in order to prevent disease outbreaks. Here, we introduce a novel epidemiological model to assess how the phage infections of the bacterial pathogens affect epidemiological dynamics of the environmentally growing pathogens. We assess whether the phage therapy in the outside-host environment could be utilized as a biological control method against these diseases. We also consider how phage-resistant competitors affect the outcome, a common problem in phage therapy. The models give predictions for the scenarios where the outside-host phage therapy will work and where it will fail to control the disease. Parameterization of the model is based on the fish columnaris disease that causes significant economic losses to aquaculture worldwide. However, the model is also suitable for other environmentally growing bacterial diseases.

Results

Transmission rates of the phage determine the success of infectious disease control, with high-transmission phage enabling the recovery of the host population that would in the absence of the phage go asymptotically extinct due to the disease. In the presence of outside-host bacterial competition between the pathogen and phage-resistant strain, the trade-off between the pathogen infectivity and the phage resistance determines phage therapy outcome from stable coexistence to local host extinction.

Conclusions

We propose that the success of phage therapy strongly depends on the underlying biology, such as the strength of trade-off between the pathogen infectivity and the phage-resistance, as well as on the rate that the phages infect the bacteria. Our results indicate that phage therapy can fail if there are phage-resistant bacteria and the trade-off between pathogen infectivity and phage resistance does not completely inhibit the pathogen infectivity. Also, the rate that the phages infect the bacteria should be sufficiently high for phage-therapy to succeed.

     

Ecology of prokaryotic viruses

The finding that total viral abundance is higher than total prokaryotic abundance and that a significant fraction of the prokaryotic community is infected with phages in aquatic systems has stimulated research on the ecology of prokaryotic viruses and their role in ecosystems. This review treats the ecology of prokaryotic viruses ('phages') in marine, freshwater and soil systems from a 'virus point of view'. The abundance of viruses varies strongly in different environments and is related to bacterial abundance or activity suggesting that the majority of the viruses found in the environment are typically phages. Data on phage diversity are sparse but indicate that phages are extremely diverse in natural systems. Lytic phages are predators of prokaryotes, whereas lysogenic and chronic infections represent a parasitic interaction. Some forms of lysogeny might be described best as mutualism. The little existing ecological data on phage populations indicate a large variety of environmental niches and survival strategies. The host cell is the main resource for phages and the resource quality, i.e., the metabolic state of the host cell, is a critical factor in all steps of the phage life cycle. Virus-induced mortality of prokaryotes varies strongly on a temporal and spatial scale and shows that phages can be important predators of bacterioplankton. This mortality and the release of cell lysis products into the environment can strongly influence microbial food web processes and biogeochemical cycles. Phages can also affect host diversity, e.g., by 'killing the winner' and keeping in check competitively dominant species or populations. Moreover, they mediate gene transfer between prokaryotes, but this remains largely unknown in the environment. Genomics or proteomics are providing us now with powerful tools in phage ecology, but final testing will have to be performed in the environment.     

Isolation and characterization of bacteriophages infecting the fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum is a serious pathogen in trout aquaculture, responsible for the diseases rainbow trout fry syndrome (RTFS) and cold water disease (CWD). Bacteriophage control of F. psychrophilum may constitute a realistic approach in the treatment of these diseases; however, a detailed understanding of the phage-host interactions is needed to evaluate the potential of F. psychrophilum bacteriophages for that purpose. Twenty-two F. psychrophilum phages from Danish rainbow trout farms were isolated and characterized. The phage genome sizes differed considerably and fell into three major size classes (8.5 to 12 kb, 48 kb, and 90 kb). The phage host ranges comprised from 5 to 23 of the 28 tested F. psychrophilum strains, and 18 of the phage isolates showed unique host ranges. Each bacterial strain had a unique pattern of susceptibility to the 22 phages, and individual strains also showed large variations (up to 10(7)-fold differences) in susceptibility to specific phages. Phage burst size (7 to 162 phages infected cell(-1)) and latency period (4 to 6 h) also showed pronounced differences both between phages and, for a specific phage, between host strains. In general, the characterization documented the presence of diverse F. psychrophilum phage communities in Danish trout farms, with highly variable patterns of infectivity. The discovery and characterization of broad-host-range phages with strong lytic potential against numerous pathogenic F. psychrophilum host strains thus provided the foundation for future exploration of the potential of phages in the treatment of RTFS and CWD.     

Narrow-Host-Range Bacteriophages That Infect Rhizobium etli Associate with Distinct Genomic Types

In this work, we isolated and characterized 14 bacteriophages that infect Rhizobium etli. They were obtained from rhizosphere soil of bean plants from agricultural lands in Mexico using an enrichment method. The host range of these phages was narrow but variable within a collection of 48 R. etli strains. We obtained the complete genome sequence of nine phages. Four phages were resistant to several restriction enzymes and in vivo cloning, probably due to nucleotide modifications. The genome size of the sequenced phages varied from 43 kb to 115 kb, with a median size of ∼45 to 50 kb. A large proportion of open reading frames of these phage genomes (65 to 70%) consisted of hypothetical and orphan genes. The remainder encoded proteins needed for phage morphogenesis and DNA synthesis and processing, among other functions, and a minor percentage represented genes of bacterial origin. We classified these phages into four genomic types on the basis of their genomic similarity, gene content, and host range. Since there are no reports of similar sequences, we propose that these bacteriophages correspond to novel species.     

Genetic basis of infectivity evolution in a bacteriophage

Antagonistic coevolution between hosts and parasites is probably ubiquitous. However, very little is known of the genetic changes associated with parasite infectivity evolution during adaptation to a coevolving host. We followed the phenotypic and genetic changes in a lytic virus population (bacteriophage; phage Φ2) that coevolved with its bacterial host, Pseudomonas fluorescens SBW25. First, we show the rapid evolution of numerous unique phage infectivity phenotypes, and that both phage host range and bacterial resistance to individual phage increased over coevolutionary time. Second, each of the distinct phage phenotypes in our study had a unique genotype, and molecular evolution did not act uniformly across the phage genome during coevolution. In particular, we detected numerous substitutions on the tail fibre gene, which is involved in the first step of the host-parasite interaction: host adsorption. None of the observed mutations could be directly linked with infection against a particular host, suggesting that the phenotypic effects of infectivity mutations are probably epistatic. However, phage genotypes with the broadest host ranges had the largest number of nonsynonymous amino acid changes on genes implicated in infectivity evolution. An understanding of the molecular genetics of phage infectivity has helped to explain the complex phenotypic coevolutionary dynamics in this system.     

Diversity and Geographical Distribution of Flavobacterium psychrophilum Isolates and Their Phages: Patterns of Susceptibility to Phage Infection and Phage Host Range

Flavobacterium psychrophilum is an important fish pathogen worldwide that causes cold water disease (CWD) or rainbow trout fry syndrome (RTFS). Phage therapy has been suggested as an alternative method for the control of this pathogen in aquaculture. However, effective use of bacteriophages in disease control requires detailed knowledge about the diversity and dynamics of host susceptibility to phage infection. For this reason, we examined the genetic diversity of 49 F. psychrophilum strains isolated in three different areas (Chile, Denmark, and USA) through direct genome restriction enzyme analysis (DGREA) and their susceptibility to 33 bacteriophages isolated in Chile and Denmark, thus covering large geographical (>12,000 km) and temporal (>60 years) scales of isolation. An additional 40 phage-resistant isolates obtained from culture experiments after exposure to specific phages were examined for changes in phage susceptibility against the 33 phages. The F. psychrophilum and phage populations isolated from Chile and Denmark clustered into geographically distinct groups with respect to DGREA profile and host range, respectively. However, cross infection between Chilean phage isolates and Danish host isolates and vice versa was observed. Development of resistance to certain bacteriophages led to susceptibility to other phages suggesting that “enhanced infection” is potentially an important cost of resistance in F. psychrophilum, possibly contributing to the observed co-existence of phage-sensitive F. psychrophilum strains and lytic phages across local and global scales. Overall, our results showed that despite the identification of local communities of phages and hosts, some key properties determining phage infection patterns seem to be globally distributed.     

Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy

Phage therapy, treating bacterial infections with bacteriophages, could be a future alternative to antibiotic treatment of bacterial infections. There are, however, several problems to be solved, mainly associated to the biology of phages, the interaction between phages and their bacterial hosts, but also to the vast variation of pathogenic bacteria which implies that large numbers of different phages are going to be needed. All of these phages must under present regulation of medical products undergo extensive clinical testing before they can be applied. It will consequently be of great economic importance that effective and versatile phages are selected and collected into phage libraries, i.e., the selection must be carried out in a way that it results in highly virulent phages with broad host ranges. We have isolated phages using the Escherichia coli reference (ECOR) collection and compared two methods, spot testing and efficiency of plating (EOP), which are frequently used to identify phages suitable for phage therapy. The analyses of the differences between the two methods show that spot tests often overestimate both the overall virulence and the host range and that the results are not correlated to the results of EOP assays. The conclusion is that single dilution spot tests cannot be used for identification and selection of phages to a phage library and should be replaced by EOP assays. The difference between the two methods can be caused by many factors. We have analysed if the differences and lack of correlation could be caused by lysis from without, bacteriocins in the phage lysate, or by the presence of prophages harbouring genes coding for phage resistance systems in the genomes of the bacteria in the ECOR collection.     

The evolution of bacterial resistance against bacteriophages in the horse chestnut phyllosphere is general across both space and time

Insight to the spatial and temporal scales of coevolution is key to predicting the outcome of host–parasite interactions and spread of disease. For bacteria infecting long-lived hosts, selection to overcome host defences is just one factor shaping the course of evolution; populations will also be competing with other microbial species and will themselves be facing infection by bacteriophage viruses. Here, we examine the temporal and spatial patterns of bacterial adaptation against natural phage populations from within leaves of horse chestnut trees. Using a time-shift experiment with both sympatric and allopatric phages from either contemporary or earlier points in the season, we demonstrate that bacterial resistance is higher against phages from the past, regardless of spatial sympatry or how much earlier in the season phages were collected. Similarly, we show that future bacterial hosts are more resistant to both sympatric and allopatric phages than contemporary bacterial hosts. Together, our results suggest the evolution of relatively general bacterial resistance against phages in nature and are contrasting to previously observed patterns of phage adaptation to bacteria from the same tree hosts over the same time frame, indicating a potential asymmetry in coevolutionary dynamics.     

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium–phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey–predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.     

Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.     

A Study of 33 Bacteriophages of Rhizobium meliloti

A total of 33 Rhizobium meliloti bacteriophages were studied. Of those, 21 were isolated in northern France from field soil in which Medicago sativa L. was grown. The other 12 phages were obtained by UV light and mitomycin C induction from 46 R. meliloti strains. Rhizobiophages were characterized by their morphology, host range, serological properties, restriction endonuclease patterns, DNA-DNA homologies, and DNA molecular weights. Five morphotypes were observed showing tailed phages with icosahedral heads. The categories of morphotypes included the Myoviridae (11 phages), Siphoviridae (3 morphotypes and 20 phages), and Podoviridae (2 phages). Type NM1 phage (Siphoviridae) is highly unusual because of the presence of transverse bars on the phage tail. Soil phages had broad host ranges, whereas phages isolated from bacterial cultures showed more or less narrow host ranges. Restriction endonuclease patterns and DNA-DNA hybridization experiments showed that the five phage type genomes were unrelated. Molecular weights of phage type DNAs were estimated, and they corresponded to values expected for capsid sizes, except for phage NM8. Type M11S (Siphoviridae) did not correspond to any other described Rhizobium phages and represents a new species.     

Individual bacteria in structured environments rely on phenotypic resistance to phage

Bacteriophages represent an avenue to overcome the current antibiotic resistance crisis, but evolution of genetic resistance to phages remains a concern. In vitro, bacteria evolve genetic resistance, preventing phage adsorption or degrading phage DNA. In natural environments, evolved resistance is lower possibly because the spatial heterogeneity within biofilms, microcolonies, or wall populations favours phenotypic survival to lytic phages. However, it is also possible that the persistence of genetically sensitive bacteria is due to less efficient phage amplification in natural environments, the existence of refuges where bacteria can hide, and a reduced spread of resistant genotypes. Here, we monitor the interactions between individual planktonic bacteria in isolation in ephemeral refuges and bacteriophage by tracking the survival of individual cells. We find that in these transient spatial refuges, phenotypic resistance due to reduced expression of the phage receptor is a key determinant of bacterial survival. This survival strategy is in contrast with the emergence of genetic resistance in the absence of ephemeral refuges in well-mixed environments. Predictions generated via a mathematical modelling framework to track bacterial response to phages reveal that the presence of spatial refuges leads to fundamentally different population dynamics that should be considered in order to predict and manipulate the evolutionary and ecological dynamics of bacteria–phage interactions in naturally structured environments.

Bacteriophages represent a promising avenue to overcome the current antibiotic resistance crisis, but evolution of phage resistance remains a concern. This study shows that in the presence of spatial refuges, genetic resistance to phage is less of a problem than commonly assumed, but the persistence of genetically susceptible bacteria suggests that eradicating bacterial pathogens from structured environments may require combined phage-antibiotic therapies.