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Members of the family Sphaerolipoviridae have non-enveloped tailless icosahedral virions with a protein-rich internal lipid membrane. The genome is a linear double-stranded DNA of about 30 kbp with inverted terminal repeats and terminal proteins. The capsid has a pseudo triangulation T=28 dextro symmetry and is built of two major capsid protein types. Spike complexes decorate fivefold vertices. Sphaerolipoviruses have a narrow host range and a lytic life cycle, infecting haloarchaea in the class Halobacteria (phylum Euryarchaeota). This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Sphaerolipoviridae, which is available at ictv.global/report/sphaerolipoviridae.

Microbial communities often respond to various challenges, such as the presence of antibiotics, as a whole. Dissecting these community-level effects into separate acting entities requires the identification of organisms that carry functional genes for the observed feature. However, unculturable microbes abound in various environments, hence making the identification challenging. Here we present a cultivation-free technique that can be utilized to link functional genes with carrying bacterial species at single-cell resolution. The developed protocol is relatively simple to use, utilizes commercially available droplet microfluidics devices, does not require toxic reagents (as compared to some previous methods), eliminates invalid signals emerging from extracellular DNA, and allows simultaneous analysis of community diversity via 16S rRNA gene sequencing. The method can be customized for any given genetic trait to accurately identify its hosting subpopulation from a heterogeneous and potentially uncultivable bacterial community.

Plasmids are genetic elements that disperse horizontally between different strains and species of bacteria and a major factor in the dissemination of virulence factors and antibiotic resistance. Understanding the ecology of plasmids has a notable anthropocentric value and therefore the interactions between bacterial hosts and individual plasmids have been studied in detail. However, bacterial systems often carry multiple genetically distinct plasmids, but dynamics of these multiplasmid “clashes” has remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids in five different ecological settings. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are trade-offs in plasmid fitness that stem from the ecological situation of their initial hosts. Growth rates and maximum population densities increased in all communities and treatments over the 42-day evolution experiment although plasmid contents at the end varied notably. We show that large multiresistance conferring plasmids are unfit when the community also contains smaller plasmids with fewer resistance genes. This suggests that restraining the use to few antibiotics can make bacterial communities sensitive to others. The hosts also appear to react to the presence of multiple genetically different plasmids by enhancing fimbriae production instead of alleviating costs of individual plasmids. In general, the survivors of the here-studied multi-plasmid clash are significantly affected by the presence or absence of antibiotic selection and plasmid-free hosts of varying fitness. Therefore, these trade-offs in different settings can explain for example why some resistance plasmids have an advantage during a rapid proliferation of antibiotic sensitive pathogen whereas others dominate in alternative situations.

Water-soluble gold nanoclusters with well-defined molecular structures and stability possess particular biophysical properties making them excellent candidates for biological applications as well as for fundamental spectroscopic studies. The currently existing synthetic protocols for atomically monodisperse thiolate-protected gold nanoclusters (AuMPCs) have been widely expanded with organothiolates, yet the direct synthesis reports for water-soluble AuMPCs are still deficient. Here, we demonstrate a wet-chemistry pH-controlled synthesis of two large water-soluble nanoclusters utilizing p-mercaptobenzoic acid (pMBA), affording different sizes of plasmonic AuMPCs on the preparative scale (∼7 mg). AuMPCs are essentially homogenous in size and are stable in solution and the solid state. Number of characterization methods were used to gain detailed information about the size, symmetry, molecular composition, and structure of these systems, i.e., high-resolution transmission electron microscopy, powder X-ray diffraction, NMR, UV-vis, thermogravimetry, and polyacrylamide gel electrophoresis. On the basis of the conducted experimental analyses and computationally aided predictions it can be evidenced that both clusters exhibit twinned face-centered cubic symmetry with the molecular composition of Au210-230(pMBA)70-80 and Au426-442(pMBA)112-115, referred from now on as Au250 and Au500, respectively. For future reference, toxicity of both gold clusters in various concentrations on cultures of Gram-positive and Gram-negative bacteria was investigated. peerReviewed

Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with non-epidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a non-epidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro.

Air carries a vast number of bacteria and viruses over great distances all the time. This leads to continuous introduction of foreign genetic material to local, established microbial communities. In this perspective, I ask whether this silent rain may have a slowing effect on the overall evolutionary rates in the microbial biosphere. Arguably, the greater the genetic divergence between gene ‘donors’ and ‘recipients’, the greater the chance that the gene product has a deleterious epistatic interaction with other gene products in its genetic environment. This is due to the long-term absence of check for mutual compatibility. As such, if an organism is extensively different from other bacteria, genetic innovations are less probable to fit to the genome. Here, genetic innovation would be anything that elevates the fitness of the gene vehicle (e.g. bacterium) over its contemporaries. Adopted innovations increase the fitness of the compatible genome over incompatible ones, thus possibly tempering the pace at which mutations accumulate in existing genomes over generations. I further discuss the transfer of bacteriophages through atmosphere and potential effects that this may have on local dynamics and perhaps phage survival.

Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.

Beta-lactams are commonly used antibiotics that prevent cell-wall biosynthesis. Beta-lactam sensitive bacteria can acquire conjugative resistance elements and hence become resistant even after being exposed to lethal (above minimum inhibitory) antibiotic concentrations. Here we show that neither the length of antibiotic exposure (1 to 16 h) nor the beta-lactam type (penam or cephem) have a major impact on the rescue of sensitive bacteria. We demonstrate that an evolutionary rescue can occur between different clinically relevant bacterial species (Klebsiella pneumoniae and Escherichia coli) by plasmids that are commonly associated with extended-spectrum beta-lactamase (ESBL) positive hospital isolates. As such, it is possible that this resistance dynamic may play a role in failing antibiotic therapies in those cases where resistant bacteria may readily migrate into the proximity of sensitive pathogens. Furthermore, we engineered a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-plasmid to encode a guiding CRISPR-RNA against the migrating ESBL-plasmid. By introducing this plasmid into the sensitive bacterium, the frequency of the evolutionarily rescued bacteria decreased by several orders of magnitude. As such, engineering pathogens during antibiotic treatment may provide ways to prevent ESBL-plasmid dispersal and hence resistance evolution.

Tectiviridae are composed of tailless bacteriophages with an icosahedral capsid and an inner membrane enclosing a double-stranded 15 kb linear DNA genome. Five of the seven previously studied Tectivirus isolates infect bacteria from Bacillus cereus sensu lato group (Betatectivirus), one distantly related member (PRD1) infect Enterobactericeae (Alpatectivirus) and one recently discovered virus infect Gluconobacter cerinus (Gammatectivirus). Here we expand the host spectrum of Betatectivirus elements to four additional genera (Streptococcus, Exiguobacterium, Clostridium and Brevibacillus) and to more distantly related Bacillus species (B. pumilus and B. flexus) by studying the genomes of fourteen novel tectiviral elements. Overall, the genomes show significant conservation in gene synteny and in modules responsible for genome replication and formation of the virion core (including DNA packaging). Notable variation exists in regions encoding host attachment and lysis along with the surrounding area of a site in which mutations are known to alter phage life cycle.

The possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of conjugative plasmids include their independence of any particular receptor(s) on host bacteria and their relative immunity to bacterial defense mechanisms (such as restriction-modification systems) due to the synthesis of the complementary strand with host-specific epigenetic modifications. We show that conjugative plasmid in association with a mobilizable antibiotic resistance gene targeting CRISPR-plasmid efficiently causes ESBL-positive transconjugants to lose their resistance, and multiple gene types can be targeted simultaneously by introducing several CRISPR RNA encoding segments into the transferred plasmids. In the rare cases where the midbiotic plasmids failed to resensitize bacteria to antibiotics, the CRISPR spacer(s) and their adjacent repeats or larger regions were found to be lost. Results also revealed potential caveats in the design of conjugative engineering systems as well as workarounds to minimize these risks.

Phage therapy is attracting growing interest among clinicians as antibiotic resistance continues becoming harder to control. However, clinical trials and animal model studies on bacteriophage treatment are still scarce and results on the efficacy vary. Recent research suggests that using traditional antimicrobials in concert with phage could have desirable synergistic effects that hinder the evolution of resistance. Here, we present a novel insect gut model to study phage–antibiotic interaction in a system where antibiotic resistance initially exists in very low frequency and phage specifically targets the resistance bearing cells. We demonstrate that while phage therapy could not reduce the frequency of target bacteria in the population during positive selection by antibiotics, it alleviated the antibiotic induced blooming by lowering the overall load of resistant cells. The highly structured gut environment had pharmacokinetic effects on both phage and antibiotic dynamics compared within vitro: antibiotics did not reduce the overall amount of bacteria, demonstrating a simple turnover of gut microbiota from non-resistant to resistant population with little cost. The results imply moderate potential for using phage as an aid to target antibiotic resistant gut infections, and question the usefulness ofin vitroinferences.

Phage therapy is attracting growing interest among clinicians as antibiotic resistance continues becoming harder to control. However, clinical trials and animal model studies on bacteriophage treatment are still scarce and results on the efficacy vary. Recent research suggests that using traditional antimicrobials in concert with phage could have desirable synergistic effects that hinder the evolution of resistance. Here, we present a novel insect gut model to study phage-antibiotic interaction in a system where antibiotic resistance initially exists in very low frequency and phage specifically targets the resistance bearing cells. We demonstrate that while phage therapy could not reduce the frequency of target bacteria in the population during positive selection by antibiotics, it alleviated the antibiotic induced blooming by lowering the overall load of resistant cells. The highly structured gut environment had pharmacokinetic effects on both phage and antibiotic dynamics compared to in vitro: antibiotics did not reduce the overall amount of bacteria, demonstrating a simple turnover of gut flora from non-resistant to resistant population with little cost. The results imply moderate potential for using phage as an aid to target antibiotic resistant gut infections, and question the usefulness of in vitro inferences.

Aim: To investigate the potential evolutionary obstacles in the sustainable therapeutic use of plasmid-dependent phages to control the clinically important conjugative plasmid-mediated dissemination of antibiotic resistance genes to pathogenic bacteria. Materials & methods: The lytic plasmid-dependent phage PRD1 and the multiresistance conferring plasmid RP4 in an Escherichia coli host were utilized to assess the genetic and phenotypic changes induced by combined phage and antibiotic selection. Results & conclusions: Resistance to PRD1 was always coupled with either completely lost or greatly reduced conjugation ability. Reversion to full conjugation efficiency was found to be rare, and it also restored the susceptibility to plasmid-dependent phages. Consequently, plasmid-dependent phages constitute an interesting candidate for development of sustainable anticonjugation/antiresistance therapeutic applications.

Distribution of species across the Earth shows strong latitudinal and altitudinal gradients with the number of species decreasing with declining temperatures. While these patterns have been recognized for well over a century, the mechanisms generating and maintaining them have remained elusive. Here, we propose a mechanistic explanation for temperature-dependent rates of molecular evolution that can influence speciation rates and global biodiversity gradients. Our hypothesis is based on the effects of temperature and temperature-adaptation on stability of proteins and other catalytic biomolecules. First, due to the nature of physical forces between biomolecules and water, stability of biomolecules is maximal around + 20°C and decreases as temperature either decreases or increases. Second, organisms that have adapted to cold temperatures have evolved especially flexible (but unstable) proteins to facilitate catalytic reactions in cold, where molecular movements slow down. Both these effects should result in mutations being on average more detrimental at cold temperatures (i.e. lower mutational robustness in cold). At high temperatures, destabilizing water–biomolecule interactions, and the need to maintain structures that withstand heat denaturation, should decrease mutational robustness similarly. Decreased mutational robustness at extreme temperatures will slow down molecular evolution, as a larger fraction of new mutations will be removed by selection. Lower mutational robustness may also select for reduced mutation rates, further slowing down the rate of molecular evolution. As speciation requires the evolution of epistatic incompatibilities that prevent gene flow among incipient species, slow rate of molecular evolution at extreme temperatures will directly slow down the rate at which new species arise. The proposed mechanism can thus explain why molecular evolution is faster at warm temperatures, contributing to higher speciation rate and elevated species richness in environments characterized by stable and warm temperatures.

Bacterial antibiotic resistance is often a part of mobile genetic elements that move from one bacterium to another. By interfering with the horizontal movement and the maintenance of these elements, it is possible to remove the resistance from the population. Here, we show that a so-called plasmid-dependent bacteriophage causes the initially resistant bacterial population to become susceptible to antibiotics. However, this effect is efficiently countered when the system also contains a predator that feeds on bacteria. Moreover, when the environment contains antibiotics, the survival of resistance is dependent on the resistance mechanism. When bacteria can help their contemporaries to degrade antibiotics, resistance is maintained by only a fraction of the community. On the other hand, when bacteria cannot help others, then all bacteria remain resistant. The concentration of the antibiotic played a less notable role than the antibiotic used. This report shows that the survival of antibiotic resistance in bacterial communities represents a complex process where many factors present in real-life systems define whether or not resistance is actually lost., Mobile genetic elements such as conjugative plasmids are responsible for antibiotic resistance phenotypes in many bacterial pathogens. The ability to conjugate, the presence of antibiotics, and ecological interactions all have a notable role in the persistence of plasmids in bacterial populations. Here, we set out to investigate the contribution of these factors when the conjugation network was disturbed by a plasmid-dependent bacteriophage. Phage alone effectively caused the population to lose plasmids, thus rendering them susceptible to antibiotics. Leakiness of the antibiotic resistance mechanism allowing Black Queen evolution (i.e. a “race to the bottom”) was a more significant factor than the antibiotic concentration (lethal vs sublethal) in determining plasmid prevalence. Interestingly, plasmid loss was also prevented by protozoan predation. These results show that outcomes of attempts to resensitize bacterial communities by disrupting the conjugation network are highly dependent on ecological factors and resistance mechanisms. IMPORTANCE Bacterial antibiotic resistance is often a part of mobile genetic elements that move from one bacterium to another. By interfering with the horizontal movement and the maintenance of these elements, it is possible to remove the resistance from the population. Here, we show that a so-called plasmid-dependent bacteriophage causes the initially resistant bacterial population to become susceptible to antibiotics. However, this effect is efficiently countered when the system also contains a predator that feeds on bacteria. Moreover, when the environment contains antibiotics, the survival of resistance is dependent on the resistance mechanism. When bacteria can help their contemporaries to degrade antibiotics, resistance is maintained by only a fraction of the community. On the other hand, when bacteria cannot help others, then all bacteria remain resistant. The concentration of the antibiotic played a less notable role than the antibiotic used. This report shows that the survival of antibiotic resistance in bacterial communities represents a complex process where many factors present in real-life systems define whether or not resistance is actually lost.

Recognizing when and how rapid evolution drives ecological change is fundamental for our understanding of almost all ecological and evolutionary processes such as community assembly, genetic diversification and the stability of communities and ecosystems. Generally, rapid evolutionary change is driven through selection on genetic variation and is affected by evolutionary constraints, such as tradeoffs and pleiotropic effects, all contributing to the overall rate of evolutionary change. Each of these processes can be influenced by the presence of multiple environmental stressors reducing a population's reproductive output. Potential consequences of multistressor selection for the occurrence and strength of the link from rapid evolution to ecological change are unclear. However, understanding these is necessary for predicting when rapid evolution might drive ecological change. Here we investigate how the presence of two stressors affects this link using experimental evolution with the bacterium Pseudomonas fluorescens and its predator Tetrahymena thermophila. We show that the combination of predation and sublethal antibiotic concentrations delays the evolution of anti-predator defence and antibiotic resistance compared with the presence of only one of the two stressors. Rapid defence evolution drives stabilization of the predator-prey dynamics but this link between evolution and ecology is weaker in the two-stressor environment, where defence evolution is slower, leading to less stable population dynamics. Tracking the molecular evolution of whole populations over time shows further that mutations in different genes are favoured under multistressor selection. Overall, we show that selection by multiple stressors can significantly alter eco-evolutionary dynamics and their predictability.

Standard of care for cancer is commonly a combination of surgery with radiotherapy or chemoradiotherapy. However, in some advanced cancer patients this approach might still remaininefficient and may cause many side effects, including severe complications and even death. Oncolytic viruses exhibit different anti-cancer mechanisms compared with conventional therapies, allowing the possibility for improved effect in cancer therapy. Chemotherapeutics combined with oncolytic viruses exhibit stronger cytotoxic responses and oncolysis. Here, we have investigated the systemic delivery of the oncolytic adenovirus and paclitaxel encapsulated in extracellular vesicles (EV) formulation that, in vitro, significantly increased the transduction ratio and the infectious titer when compared with the virus and paclitaxel alone. We demonstrated that the obtained EV formulation reduced the in vivo tumor growth in animal xenograft model of human lung cancer. Indeed, we found that combined treatment of oncolytic adenovirus and paclitaxel encapsulated in EV has enhanced anticancer effects both in vitro and in vivo in lung cancer models. Transcriptomic comparison carried out on the explanted xenografts from the different treatment groups revealed that only 5.3% of the differentially expressed genes were overlapping indicating that a de novo genetic program is triggered by the presence of the encapsulated paclitaxel: this novel genetic program might be responsible of the observed enhanced antitumor effect. Our work provides a promising approach combining anticancer drugs and viral therapies by intravenous EV delivery as a strategy for the lung cancer treatment.

Prokaryotes harbor a variety of genetic replicators, including plasmids, viruses, and chromosomes, each having different effects on the phenotype of the hosting cell. Here, we propose a classification for replicators of bacteria and archaea on the basis of their horizontal-transfer potential and the type of relationships (mutualistic, symbiotic, commensal, or parasitic) that they have with the host cell vehicle. Horizontal movement of replicators can be either active or passive, reflecting whether or not the replicator encodes the means to mediate its own transfer from one cell to another. Some replicators also have an infectious extracellular state, thus separating viruses from other mobile elements. From the perspective of the cell vehicle, the different types of replicators form a continuum from genuinely mutualistic to completely parasitic replicators. This classification provides a general framework for dissecting prokaryotic systems into evolutionarily meaningful components.

Conjugative ESBL plasmids differ in their potential to rescue susceptible bacteria via horizontal gene transfer in lethal antibiotic concentrations

Bacteria live in dynamic systems where selection pressures can alter rapidly, forcing adaptation to the prevailing conditions. In particular, bacteriophages and antibiotics of anthropogenic origin are major bacterial stressors in many environments. We previously observed that populations of the bacterium Pseudomonas fluorescens SBW25 exposed to the lytic bacteriophage SBW25Φ2 and a noninhibitive concentration of the antibiotic streptomycin (coselection) achieved higher levels of phage resistance compared to populations exposed to the phage alone. In addition, the phage became extinct under coselection while remaining present in the phage alone environment. Further, phenotypic tests indicated that these observations might be associated with increased mutation rate under coselection. In this study, we examined the genetic causes behind these phenotypes by whole-genome sequencing clones isolated from the end of the experiments. We were able to identify genetic factors likely responsible for streptomycin resistance, phage resistance and hypermutable (mutator) phenotypes. This constitutes genomic evidence in support of the observation that while the presence of phage did not affect antibiotic resistance, the presence of antibiotic affected phage resistance. We had previously hypothesized an association between mutators and elevated levels of phage resistance under coselection. However, our evidence regarding the mechanism was inconclusive, as although with phage mutators were only found under coselection, additional genomic evidence was lacking and phage resistance was also observed in nonmutators under coselection. More generally, our study provides novel insights into evolution between univariate and multivariate selection (here two stressors), as well as the potential role of hypermutability in natural communities.

Achieving sufficient connectivity between populations is essential for persistence, but costs of dispersal may select against individual traits or behaviours that, if present, would improve connectivity. Existing dispersal models tend to ignore the multitude of risks to individuals: while many assess the effect of mortality costs, there is also a risk of failing to find new habitat, especially when the entire inhabitable area remains both small and fragmented. There are few known rules governing whether individuals evolve to disperse more, or less, than what is ideal for population connectivity and persistence. Here we aim to fill this gap, while also noting that evolution might not only produce suboptimal dispersal behaviour: it also influences individual heterogeneity in dispersal. Intuitively, we might expect heterogeneity to improve connectivity, as some individuals will travel far. However, we show that this is only true if dispersal distances on average are quite short; heterogeneity can also lead to reduced connectivity because it can reduce the proportion of the most profitable (‘safest’) intermediate dispersal distances. In general, our results show that conditions typically associated with conservation concerns (small and fragmented habitats inhabited by a species with a low birth rate) are also ones that are most likely to lead to suboptimal dispersal traits. This prompts the question of assisted dispersal in cases of urgent conservation concern.

Sexual reproduction is problematic to explain due to its costs, most notably the twofold cost of sex. Yet, sex has been suggested to be favourable in the presence of proliferating intragenomic parasites given that sexual recombination provides a mechanism to confine the accumulation of deleterious mutations. Kraaijeveld et al. compared recently the accumulation of transposons in sexually and asexually reproducing lines of the same species, the parasitoid wasp Leptopilina clavipes. They discovered that within asexually reproducing wasps, the number of gypsy-like retrotransposons was increased fourfold, whereas other retrotransposons were not. Interestingly, gypsy-like retrotransposons are closely related to retroviruses. Endogenous retroviruses are retroviruses that have integrated to the germ line cells and are inherited thereafter vertically. They can also replicate within the genome similarly to retrotransposons as well as form virus particles and infect previously uninfected cells. This highlights the possibility that endogenous retroviruses could play a role in the evolution of sexual reproduction. Here, we show with an individual-based computational model that a virus epidemic within a previously parasite-free asexual population may establish a new intragenomic parasite to the population. Moreover and in contrast to other transposons, the possibility of endogenous viruses to maintain a virus epidemic and simultaneously provide resistance to individuals carrying active endogenous viruses selects for the presence of active intragenomic parasites in the population despite their deleterious effects. Our results suggest that the viral nature of certain intragenomic parasites should be taken into account when sex and its benefits are being considered.

The emergence of pathogenic bacteria resistant to multiple antibiotics is a serious worldwide public health concern. Whenever antibiotics are applied, the genes encoding for antibiotic resistance are selected for within bacterial populations. This has led to the prevalence of conjugative plasmids that carry resistance genes and can transfer themselves between diverse bacterial groups. In this study, we investigated whether it is feasible to attempt to prevent the spread of antibiotic resistances with a lytic bacteriophage, which can replicate in a wide range of gram‐negative bacteria harbouring conjugative drug resistance–conferring plasmids. The counter‐selection against the plasmid was shown to be effective, reducing the frequency of multidrug‐resistant bacteria that formed via horizontal transfer by several orders of magnitude. This was true also in the presence of an antibiotic against which the plasmid provided resistance. Majority of the multiresistant bacteria subjected to phage selection also lost their conjugation capability. Overall this study suggests that, while we are obligated to maintain the selection for the spread of the drug resistances, the ‘fight evolution with evolution’ approach could help us even out the outcome to our favour. peerReviewed

Our biosphere is abundant with unique and small genes for which no homologs are known. These genes, often referred to as orphans or ORFans, are commonly found in bacteriophage genomes but their origins remain unclear. We discovered five novel tectivirus-like genetic elements by screening more than five-hundred Bacillus strains. A highly variable region (HVR) of these viruses was shown to harbor ORFans in most of these otherwise well-conserved bacteriophages. Previous studies demonstrated that mutations close to this region dramatically alter bacteriophage gene regulation, suggesting that the acquisition of those ORFans may provide a source of genetic diversity that is then subject to genetic selection during bacteriophage evolution. © 2012 Institut Pasteur.

Sub-minimum inhibiting concentrations (sub-MICs) of antibiotics frequently occur in natural environments owing to wide-spread antibiotic leakage by human action. Even though the concentrations are very low, these sub-MICs have recently been shown to alter bacterial populations by selecting for antibiotic resistance and increasing the rate of adaptive evolution. However, studies are lacking on how these effects reverberate into key ecological interactions, such as bacteria-phage interactions. Previously, co-selection of bacteria by phages and antibiotic concentrations exceeding MICs has been hypothesized to decrease the rate of resistance evolution because of fitness costs associated with resistance mutations. By contrast, here we show that sub-MICs of the antibiotic streptomycin (Sm) increased the rate of phage resistance evolution, aswell as causing extinction of the phage. Notably, Sm and the phage in combination also enhanced the evolution of Sm resistance compared with Sm alone. These observations demonstrate the potential of sub-MICs of antibiotics to impact key ecological interactions in microbial communities with evolutionary outcomes that can radically differ from those associated with high concentrations. Our findings also contribute to the understanding of ecological and evolutionary factors essential for the management of the antibiotic resistance problem. This article is part of the themed issue ‘Human influences on evolution, and the ecological and societal consequences’. © 2016 The Author(s) Published by the Royal Society. All rights reserved.

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogenSerratia marcescens. A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.

Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase (ESBL) Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus (VRE) and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus (MRSA) strains was found to be very difficult.

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 bothEscherichia coliandSalmonella entericain 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.

β-Lactams are a commonly used class of bactericidal antibiotics. The number of β-lactam-resistant pathogens is constantly increasing in hospitals around the world. Interestingly, most of the β-lactam-resistant bacteria carry mobile genetic elements, such as conjugative plasmids, that render the pathogen resistant. These elements mediate their own transfer from one bacterium to another, producing new resistant strains via horizontal gene transfer. Here we investigated whether it is possible that transfer of the resistance element from another bacterium may evolutionarily rescue a susceptible bacterium exposed to a lethal concentration of the β-lactam ampicillin. Indeed, the rescuing occurs even at very high, clinically significant antibiotic levels, suggesting that pathogens may acquire the resistance 'on the fly' from commensal bacteria during treatment.

Viruses SH1 and P23-77, infecting archaeal Haloarcula species and bacterial Thermus species, respectively, were recently designated to form a novel viral lineage. In this study, the lineage is expanded to archaeal Halomicrobium and bacterial Meiothermus species by analysis of five genome-integrated elements that share the core genes with these viruses.

The very early forms of life probably comprised ribozyme-like agents that were able to catalyze reactions and serve as templates for their own replication. The early evolution has also been suggested to occur mainly horizontally between proto-cells or inorganic compartments rather than vertically from parent cell to their dividing siblings. In order to study the evolutionary dynamics of such a community a rule-based computing system entitled as PrimordialEvo was developed. The system simulates a three dimensional matrix of compartments in which replicators, resource collectors and various other actors thrive. Horizontal movement between compartments may be due to genetically induced vesicle formation or random drift. Analysis of the simulation experiments suggests that active sharing of innovations between compartments is important for the overall reproductive success of life. The capability of natural selection to favor genes in the system was also tested, and, for example, the frequency of anti-parasites was observed to increase when parasites were allowed to emerge.

Viruses are the dominant form of genetically reproducing entities on Earth. Yet, viruses are mostly neglected in the context of astrobiology due to their non-living nature. In this discussion it is considered whether viruses are likely to be transferred within bacterial endospores to other planetary bodies through lithopanspermia. Interestingly, it seems possible that ecosystems of panspermial origin might yield biospheres in which viruses are absent. The evolutionary pathway of life in these systems might differ significantly from the path observed on Earth. We hypothesize that the difference in the two potential ways for the emergence of life, those being panspermial or local origin, on any certain planet could be clarified by analyzing the diversity of virosphere on the planet. The use of viruses as putative biomarkers is also discussed and studied.

Viruses and cells co-evolve due to the parasitic nature of viruses. Yet there are no models suggesting how the unicellular organisms and their viruses might co-evolve structurally. Here, in this study, we plunge into this unexplored field from a wide perspective and try to describe some of the intriguing ways in which viruses may have shaped the cellular life forms on the ancient Earth. At first we propose a scenario where viruses act as a driving force in the emergence of bacterial cell walls by providing favorable intermediates for the otherwise improbable steps in the cell wall generation. We also discuss the role of viruses in the evolution of cell surface components such as receptors and second membranes. Finally we focus on hypothetical proto-viruses, the selfish abusers of the RNA-world, in explaining some of the very early stages in the origin and evolution of life. Proto-viruses may be responsible for creating the first true cells in order to support their selfish needs. In this model we also suggest a logical pathway to explaining the emergence of modern viruses.

Prokaryotes harbor a variety of genetic replicators, including plasmids, viruses, and chromosomes, each having differing effects on the phenotype of the hosting cell. Here, we propose a classification for replicators of bacteria and archaea on the basis of their horizontal-transfer potential and the type of relationships (mutualistic, symbiotic, commensal, or parasitic) that they have with the host cell vehicle. Horizontal movement of replicators can be either active or passive, reflecting whether or not the replicator encodes the means to mediate its own transfer from one cell to another. Some replicators also have an infectious extracellular state, thus separating viruses from other mobile elements. From the perspective of the cell vehicle, the different types of replicators form a continuum from genuinely mutualistic to completely parasitic replicators. This classification provides a general framework for dissecting prokaryotic systems into evolutionarily meaningful components.

International audience; A new family of viruses named Sphaerolipoviridae has been proposed recently. It comprises icosahedral, tailless haloarchaeal viruses with an internal lipid membrane located between the protein capsid and the dsDNA genome. The proposed family Sphaerolipoviridae was divided into two genera: Alphasphaerolipovirus, including Haloarcula hispanica viruses SH1, PH1 and HHIV-2, and Betasphaerolipovirus, including Natrinema virus SNJ1. Here, we propose to expand the family Sphaerolipoviridae to include a group of bacteriophages infecting extreme thermophilic Thermus thermophilus and sharing a number of structural and genomic properties with archaeal sphaerolipoviruses. This new group comprises two members, lytic phage P23-77 and temperate phage IN93, as well as putative members P23-72 and P23-65H. In addition, several related proviruses have been discovered as integrated elements in bacterial genomes of the families Thermus and Meiothermus. Morphology of the virus particles and the overall capsid architecture of these bacteriophages resembles that of archaeal members of the Sphaerolipoviridae, including an unusual capsid arrangement in a T = 28 dextro lattice. Alpha- and betasphaerolipoviruses share with P23-77-like bacteriophages a conserved block of core genes that encode a putative genome-packaging ATPase and the two major capsid proteins (MCPs). The recently determined X-ray structure of the small and large MCPs of P23-77 revealed a single beta-barrel (jelly-roll) fold that is superimposable with the cryo-EM density maps of the SH1 capsomers. Given the common features of these viruses, we propose to include the so far unclassified P23-77-like bacteriophages into a new genus, "Gammasphaerolipovirus", within the family Sphaerolipoviridae.

Bacteriophage therapy, the use of viruses that infect bacteria as antimicrobials, has been championed as a promising alternative to conventional antibiotics. Although in the laboratory bacterial resistance against phages arises rapidly, resistance so far has been an only minor problem for the effectiveness of phage therapy. Resistance to antibiotics, however, has become a major issue after decades of extensive use. Should we expect similar problems after long-term use of phages as antimicrobials? Like antibiotics, phages are often noted to be drivers of bacterial evolution. Should we expect phage-treated pathogens to develop a general resistance to phages over time, a resistance against which only, for example, hypothetically co-evolved phages might be infective? Here we argue that the global infection patterns of phages suggest that this is not necessarily a concern as environmental phages often can infect bacteria with which those phages lack any recent co-evolutionary history.

Prokaryotic biosphere is vastly diverse in many respects. Any given bacterial cell may harbor in different combinations viruses, plasmids, transposons, and other genetic elements along with their chromosome(s). These agents interact in complex environments in various ways causing multitude of phenotypic effects on their hosting cells. In this discussion I perform a dissection for a bacterial cell in order to simplify the diversity into components that may help approach the ocean of details in evolving microbial worlds. The cell itself is separated from all the genetic replicators that use the cell vehicle for preservation and propagation. I introduce a classification that groups different replicators according to their horizontal movement potential between cells and according to their effects on the fitness of their present host cells. The classification is used to discuss and improve the means by which we approach general evolutionary tendencies in microbial communities. Moreover, the classification is utilized as a tool to help formulating evolutionary hypotheses and to discuss emerging bacterial pathogens as well as to promote understanding on the average phenotypes of different replicators in general. It is also discussed that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve to have horizontally moving replicators of various types.

Cellular life is immersed into an ocean of viruses. Virosphere forms the shadow of this cell-based tree of life: completely dependent on the tree for existence, yet, the tree is equally unable to escape its ever evolving companion. How important role has the shadow played in the evolution of life? Is it a mere ethereal partner or a constitutive factor? In this chapter four puzzles in virus research are taken under the scope in order to probe some of the intriguing ways by which viruses can help us understand life on Earth. These puzzles consider the origin of genetic information in viruses, viruses as symbiotic partners, the structural diversity of viruses and the role of viruses in the origin of cellular life. More than providing answers, this introduction exemplifies how viruses can be approached from various angles and how each of the angles can open up new ways to appreciate their potential contributions to life.

The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.