Preceding Host History of Conjugative Resistance Plasmids Affects Intra- and Interspecific Transfer Potential from Biofilm.

Conjugative plasmids can confer antimicrobial resistance (AMR) to their host bacterium. The plasmids disperse even between distantly related host species, rescuing the host from otherwise detrimental effects of antibiotics. Little is known about the role of these plasmids in the spread of AMR during antibiotic treatment. One unstudied question is whether the past evolutionary history of a plasmid in a particular species creates host specificity in its rescue potential or if interspecific coevolution can improve interspecific rescues. To study this, we coevolved the plasmid RP4 under three different host settings; solely Escherichia coli or Klebsiella pneumoniae, or alternating between both of them. The ability of evolved plasmids in bacterial biofilm to rescue susceptible planktonic host bacteria of either the same or different species during beta-lactam treatment was tested. The interspecific coevolution seemed to decrease rescue potential for the RP4 plasmid, while the K. pneumoniae evolved plasmid became more host specific. Large deletion in the region encoding the mating pair formation (Tra2) apparatus was detected in the plasmids evolved with K. pneumoniae. This adaptation resulted in the exapted evolution of resistance against a plasmid-dependent bacteriophage PRD1. Further, previous studies have suggested that mutations in this region completely abolish the plasmid's ability to conjugate; however, our study shows it is not essential for conjugation but rather affects the host-specific conjugation efficiency. Overall, the results suggest that previous evolutionary history can result in the separation of host-specific plasmid lineages that may be further amplified by unselected exaptations such as phage resistance. IMPORTANCE Antimicrobial resistance (AMR) is a major global public health threat which can rapidly spread in microbial communities via conjugative plasmids. Here, we advance with evolutionary rescue via conjugation in a more natural setting, namely, biofilm, and incorporate a broad-host range plasmid RP4 to test whether intra- and interspecific host histories affect its transfer potential. Escherichia coli and Klebsiella pneumoniae hosts were seen to elicit different evolutionary influences on the RP4 plasmid, leading to clear differences in the rescue potential and underlining the significant role of the plasmid-host interactions in the spread of AMR. We also contradicted previous reports that established certain conjugal transfer genes of RP4 as essential. This work enhances the understanding of how plasmid host range evolve in different host settings and further, the potential effects it may have on the horizontal spread of AMR in complex environments such as biofilms.

ICTV Virus Taxonomy Profile: Simuloviridae 2023.

The family Simuloviridae includes tailless icosahedral viruses with an internal lipid membrane. The capsid is constructed from two major capsid proteins, both with a single jelly-roll fold. The genome is a circular dsDNA molecule of 16-19 kb. All members infect halophilic archaea in the class Halobacteria (phylum Euryarchaeota) and are temperate viruses, their proviruses residing in host cells as extrachromosomal episomes. Once the lytic life cycle is triggered, production of virions causes cell lysis. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Simuloviridae, which is available at ictv.global/report/simuloviridae.

ICTV Virus Taxonomy Profile: Sphaerolipoviridae 2023.

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.

Plasmid Viability Depends on the Ecological Setting of Hosts within a Multiplasmid Community.

Plasmids are extrachromosomal genetic elements, some of which disperse horizontally between different strains and species of bacteria. They are 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 within these multiplasmid communities have remained unstudied. Here, we set to investigate the survival of 11 mobilizable or conjugative plasmids under five different conditions where the hosts had a differing ecological status in comparison to other bacteria in the system. The key incentive was to determine whether plasmid dynamics are reproducible and whether there are tradeoffs 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. Large multiresistance-conferring plasmids were unfit when the community also contained smaller plasmids with fewer resistance genes. This suggests that restraining the use of a few antibiotics can make bacterial communities sensitive to others. In general, the presence or absence of antibiotic selection and plasmid-free hosts (of various fitnesses) has a notable influence on which plasmids survive. These tradeoffs in different settings can help explain, for example, why some resistance plasmids have an advantage during a rapid proliferation of antibiotic-sensitive pathogens whereas others dominate in alternative situations. IMPORTANCE Conjugative and mobilizable plasmids are ubiquitous in bacterial systems. Several different plasmids can compete within a single bacterial community. We here show that the ecological setting of the host bacteria has a notable effect on the survival of individual plasmids. Selection for opportunistic genes such as antibiotic resistance genes and the presence of plasmid-free hosts can determine which plasmids survive in the system. Host bacteria appear to adapt specifically to a situation where there are multiple plasmids present instead of alleviating the plasmid-associated fitness costs of individual plasmids. Plasmids providing antibiotic resistance survived under all conditions even if there was a constant migration of higher-fitness plasmid-free hosts and no selection via antibiotics. This study is one of the first to observe the behavior of multiple genetically different plasmids as a part of a single system.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains.

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.

Indirect Selection against Antibiotic Resistance via Specialized Plasmid-Dependent Bacteriophages.

Antibiotic resistance genes of important Gram-negative bacterial pathogens are residing in mobile genetic elements such as conjugative plasmids. These elements rapidly disperse between cells when antibiotics are present and hence our continuous use of antimicrobials selects for elements that often harbor multiple resistance genes. Plasmid-dependent (or male-specific or, in some cases, pilus-dependent) bacteriophages are bacterial viruses that infect specifically bacteria that carry certain plasmids. The introduction of these specialized phages into a plasmid-abundant bacterial community has many beneficial effects from an anthropocentric viewpoint: the majority of the plasmids are lost while the remaining plasmids acquire mutations that make them untransferable between pathogens. Recently, bacteriophage-based therapies have become a more acceptable choice to treat multi-resistant bacterial infections. Accordingly, there is a possibility to utilize these specialized phages, which are not dependent on any particular pathogenic species or strain but rather on the resistance-providing elements, in order to improve or enlengthen the lifespan of conventional antibiotic approaches. Here, we take a snapshot of the current knowledge of plasmid-dependent bacteriophages.

Beta-Lactam Sensitive Bacteria Can Acquire ESBL-Resistance via Conjugation after Long-Term Exposure to Lethal Antibiotic Concentration.

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.

Silent rain: does the atmosphere-mediated connectivity between microbiomes influence bacterial evolutionary rates?

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 provide a capsid-free vector for oncolytic adenoviral DNA delivery.

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.

Midbiotics: conjugative plasmids for genetic engineering of natural gut flora.

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.

Targeting antibiotic resistant bacteria with phage reduces bacterial density in an insect host.

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 with in 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 of in vitro inferences.

Dual-stressor selection alters eco-evolutionary dynamics in experimental communities.

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.

Black Queen Evolution and Trophic Interactions Determine Plasmid Survival after the Disruption of the Conjugation Network.

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.

Extending the hosts of Tectiviridae into four additional genera of Gram-positive bacteria and more diverse Bacillus species.

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.

Sublethal streptomycin concentrations and lytic bacteriophage together promote resistance evolution.

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, as well 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'.

Genomic evolution of bacterial populations under coselection by antibiotics and phage.

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.

Scoping the effectiveness and evolutionary obstacles in using plasmid-dependent phages to fight antibiotic resistance.

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.