Critically evaluating the relative importance of phage in shaping microbial community composition.

The ubiquity of bacteriophages (phages) and the major evolutionary and ecological impacts they can have on their microbial hosts has resulted in phages often cited as key drivers shaping microbial community composition (the relative abundances of species). However, the evidence for the importance of phages is mixed. Here, we critically review the theory and data exploring the role of phages in communities, identifying the conditions when phages are likely to be important drivers of community composition. At ecological scales, we conclude that phages are often followers rather than drivers of microbial population and community dynamics. While phages can affect strain diversity within species, there is yet to be strong evidence suggesting that fluctuations in species' strains affects community composition.

CRISPR-Cas in Pseudomonas aeruginosa provides transient population-level immunity against high phage exposures.

The prokaryotic adaptive immune system, CRISPR-Cas (clustered regularly interspaced short palindromic repeats; CRISPR-associated), requires the acquisition of spacer sequences that target invading mobile genetic elements such as phages. Previous work has identified ecological variables that drive the evolution of CRISPR-based immunity of the model organism Pseudomonas aeruginosa PA14 against its phage DMS3vir, resulting in rapid phage extinction. However, it is unclear if and how stable such acquired immunity is within bacterial populations, and how this depends on the environment. Here, we examine the dynamics of CRISPR spacer acquisition and loss over a 30-day evolution experiment and identify conditions that tip the balance between long-term maintenance of immunity versus invasion of alternative resistance strategies that support phage persistence. Specifically, we find that both the initial phage dose and reinfection frequencies determine whether or not acquired CRISPR immunity is maintained in the long term, and whether or not phage can coexist with the bacteria. At the population genetics level, emergence and loss of CRISPR immunity are associated with high levels of spacer diversity that subsequently decline due to invasion of bacteria carrying pilus-associated mutations. Together, these results provide high resolution of the dynamics of CRISPR immunity acquisition and loss and demonstrate that the cumulative phage burden determines the effectiveness of CRISPR over ecologically relevant timeframes.

Host- plasmid network structure in wastewater is linked to antimicrobial resistance genes.

As mobile genetic elements, plasmids are central for our understanding of antimicrobial resistance spread in microbial communities. Plasmids can have varying fitness effects on their host bacteria, which will markedly impact their role as antimicrobial resistance vectors. Using a plasmid population model, we first show that beneficial plasmids interact with a higher number of hosts than costly plasmids when embedded in a community with multiple hosts and plasmids. We then analyse the network of a natural host-plasmid wastewater community from a Hi-C metagenomics dataset. As predicted by the model, we find that antimicrobial resistance encoding plasmids, which are likely to have positive fitness effects on their hosts in wastewater, interact with more bacterial taxa than non-antimicrobial resistance plasmids and are disproportionally important for connecting the entire network compared to non- antimicrobial resistance plasmids. This highlights the role of antimicrobials in restructuring host-plasmid networks by increasing the benefits of antimicrobial resistance carrying plasmids, which can have consequences for the spread of antimicrobial resistance genes through microbial networks. Furthermore, that antimicrobial resistance encoding plasmids are associated with a broader range of hosts implies that they will be more robust to turnover of bacterial strains.

Copper reduces the virulence of bacterial communities at environmentally relevant concentrations.

Increasing environmental concentrations of metals as a result of anthropogenic pollution are significantly changing many microbial communities. While there is evidence metal pollution can result in increased antibiotic resistance, the effects of metal pollution on the virulence of bacterial communities remains largely undetermined. Here, we experimentally test whether metal stress alters the virulence of bacterial communities. We do this by incubating three wastewater influent communities under different environmentally relevant copper concentrations for three days. We then quantify the virulence of the community phenotypically using the Galleria mellonella infection model, and test if differences are due to changes in the rate of biomass accumulation (productivity), copper resistance, or community composition (quantified using 16S amplicon sequencing). The virulence of the communities was found to be reduced by the highest copper concentration, but not to be affected by the lower concentration. As well as reduced virulence, communities exposed to the highest copper concentration were less diverse and had lower productivity. This work highlights that metal pollution may decrease virulence in bacterial communities, but at a cost to diversity and productivity.

Diversity loss from multiple interacting disturbances is regime-dependent.

Anthropogenic activities expose many ecosystems to multiple novel disturbances simultaneously. Despite this, how biodiversity responds to simultaneous disturbances remains unclear, with conflicting empirical results on their interactive effects. Here, we experimentally test how one disturbance (an invasive species) affects the diversity of a community over multiple levels of another disturbance regime (pulse mortality). Specifically, we invade stably coexisting bacterial communities under four different pulse frequencies, and compare their final resident diversity to uninvaded communities under the same pulse mortality regimes. Our experiment shows that the disturbances synergistically interact, such that the invader significantly reduces resident diversity at high pulse frequency, but not at low. This work therefore highlights the need to study simultaneous disturbance effects over multiple disturbance regimes as well as to carefully document unmanipulated disturbances, and may help explain the conflicting results seen in previous multiple-disturbance work.

Culturing the Plastisphere: comparing methods to isolate culturable bacteria colonising microplastics.

Microplastics quickly become colonised by diverse microbial communities, known as the Plastisphere. There is growing concern that microplastics may support the enrichment and spread of pathogenic or antimicrobial resistant microorganisms, although research to support the unique role of microplastics in comparison to control particles remains inconclusive. Limitations to this research include the microbiological methods available for isolating adhered microbes. Culture-based methods provide some of the most established, accessible and cost-effective microbiological protocols, which could be extremely useful in helping to address some of the remaining key questions in Plastisphere research. Previous works have successfully cultured bacteria from plastics, but these have not yet been reviewed, nor compared in efficiency. In this study, we compared four common biofilm extraction methods (swabbing, sonication, vortexing, sonication followed by vortexing) to extract and culture a mixed community of bacteria from both microplastic (polyethylene, polypropylene and polystyrene) and control (wood and glass) particles. Biofilm extraction efficiency and viability of bacterial suspension was determined by comparing CFU/mL of four different groups of bacteria. This was verified against optical density and 16S rRNA qPCR. Overall, we found that all tested methods were able to remove biofilms, but to varying efficiencies. Sonicating particles with glass beads for 15 min, followed by vortexing for a further minute, generated the highest yield and therefore greatest removal efficiency of culturable, biofilm-forming bacteria.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

Comparative experimental evolution reveals species-specific idiosyncrasies in marine phytoplankton adaptation to warming.

A number of experimental studies have demonstrated that phytoplankton can display rapid thermal adaptation in response to warmed environments. While these studies provide insight into the evolutionary responses of single species, they tend to employ different experimental techniques. Consequently, our ability to compare the potential for thermal adaptation across different, ecologically relevant, species remains limited. Here, we address this limitation by conducting simultaneous long-term warming experiments with the same experimental design on clonal isolates of three phylogenetically diverse species of marine phytoplankton; the cyanobacterium Synechococcus sp., the prasinophyte Ostreococcus tauri and the diatom Phaeodoactylum tricornutum. Over the same experimental time period, we observed differing levels of thermal adaptation in response to stressful supra-optimal temperatures. Synechococcus sp. displayed the greatest improvement in fitness (i.e., growth rate) and thermal tolerance (i.e., temperature limits of growth). Ostreococcus tauri was able to improve fitness and thermal tolerance, but to a lesser extent. Finally, Phaeodoactylum tricornutum showed no signs of adaptation. These findings could help us understand how the structure of phytoplankton communities may change in response to warming, and possible biogeochemical implications, as some species show relatively more rapid adaptive shifts in their thermal tolerance.

The effect of metal remediation on the virulence and antimicrobial resistance of the opportunistic pathogen Pseudomonas aeruginosa.

Anthropogenic metal pollution can result in co-selection for antibiotic resistance and potentially select for increased virulence in bacterial pathogens. Metal-polluted environments can select for the increased production of siderophore molecules to detoxify non-ferrous metals. However, these same molecules also aid the uptake of ferric iron, a limiting factor for within-host pathogen growth, and are consequently a virulence factor. Anthropogenic methods to remediate environmental metal contamination commonly involve amendment with lime-containing materials. However, whether this reduces in situ co-selection for antibiotic resistance and siderophore-mediated virulence remains unknown. Here, using microcosms containing non-sterile metal-contaminated river water and sediment, we test whether liming reduces co-selection for these pathogenicity traits in the opportunistic pathogen Pseudomonas aeruginosa. To account for the effect of environmental structure, which is known to impact siderophore production, microcosms were incubated under either static or shaking conditions. Evolved P. aeruginosa populations had greater fitness in the presence of toxic concentrations of copper than the ancestral strain and showed increased resistance to the clinically relevant antibiotics apramycin, cefotaxime and trimethoprim, regardless of lime addition or environmental structure. Although we found virulence to be significantly associated with siderophore production, neither virulence nor siderophore production significantly differed between the four treatments. Furthermore, liming did not mitigate metal-imposed selection for antibiotic resistance or virulence in P. aeruginosa. Consequently, metal-contaminated environments may select for antibiotic resistance and virulence traits even when treated with lime.

The host phylogeny determines viral infectivity and replication across Staphylococcus host species.

Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts.

The ecology of scale: impact of volume on coalescence and function in methanogenic communities.

Engineered ecosystems span multiple volume scales, from a nano-scale to thousands of cubic metres. Even the largest industrial systems are tested in pilot scale facilities. But does scale affect outcomes? Here we look at comparing different size laboratory anaerobic fermentors to see if and how the volume of the community affects the outcome of community coalescence (combining multiple communities) on community composition and function. Our results show that there is an effect of scale on biogas production. Furthermore, we see a link between community evenness and volume, with smaller scale communities having higher evenness. Despite those differences, the overall patterns of community coalescence are very similar at all scales, with coalescence leading to levels of biogas production comparable with that of the best-performing component community. The increase in biogas with increasing volume plateaus, suggesting there is a volume where productivity stays stable over large volumes. Our findings are reassuring for ecologists studying large ecosystems and industries operating pilot scale facilities, as they support the validity of pilot scale studies in this field.

Source-sink migration of natural enemies drives maladaptation of victim populations in sink habitats.

Natural enemies are critical drivers of species biogeography, and they may often limit the evolutionary adaptation and persistence of victim populations in sink habitats. Source-sink migration is also a major determinant of adaptation in sink habitats. Here, we specifically suggest that source-sink migration of enemies reduces evolutionary adaptation of victim populations in sink habitats. The underlying mechanisms may include depressed population size (which limits the supply of genetic variation) and enforced resistance evolution in victims (which shows a trade-off with growth performance). We experimentally tested this hypothesis using a model microbial system, bacterium Pseudomonas fluorescens (victim) and its lytic bacteriophage (enemy). The ancestral bacterial strain had lower growth performance at a cold temperature (10 °C, considered as sink habitat) than at its optimal temperature (28 °C, source habitat). Evolutionary adaptation took place in bacterial populations that evolved alone in the cold environment. When phages were present, no significant abiotic adaptation was observed. Crucially, phage immigration from source populations caused maladaptation, i.e., decreased growth performance relative to the ancestral genotype, although this was not the case when there was simultaneous immigration of phage and bacteria. Therefore, enemy-mediated intraspecific apparent competition could lead to prosperity in core habitats causing hardship in edge habitats.

Antagonistic Mobile Genetic Elements Can Counteract Each Other's Effects on Microbial Community Composition.

Bacteriophages ("phages") are hypothesized to be key drivers of bacterial population dynamics, driving microbial community composition, but empirical support for this is mixed. One reason why phages may have a less-than-expected impact on community composition is that many different phages and other mobile genetic elements (MGEs) interact with each bacterium. For instance, the same phage may have higher or lower costs to different bacterial strains or species. Assuming that resistance or susceptibility to MGE infection is not consistent across all MGEs, a simple prediction is that the net effect of MGEs on each bacterial taxon may converge with an increasing number of interactions with different MGEs. We formalized this prediction using in silico population dynamics simulations and then carried out experiments using three bacterial species, one generalist conjugative plasmid, and three species-specific phages. While the presence of only phages or only the plasmid altered community structure, these differential effects on community structure canceled out when both were together. The effects of MGEs were largely indirect and could not be explained by simple pairwise bipartite interactions (i.e., between each MGE and each bacterial species). Our results suggest that the effects of MGEs may be overestimated by studies that focus on a single MGE and not on interactions among multiple MGEs. IMPORTANCE While bacteriophages ("phages") are often cited as some of the key drivers of microbial diversity, evidence for this is greatly mixed. We demonstrate, in silico and experimentally, that the impact of phages, an example of a mobile genetic element (MGE), on community structure can diminish with increasing MGE diversity. This is because MGEs can have diverse effects on host fitness, and therefore as diversity increases, their individual effects cancel out, returning communities back to an MGE-free state. In addition, interactions in mixed-species and MGE communities could not be predicted from simple pairwise interactions, highlighting the difficulty in generalizing a MGE's effect from pairwise studies.

Copper selects for siderophore-mediated virulence in Pseudomonas aeruginosa.

BACKGROUND: Iron is essential for almost all bacterial pathogens and consequently it is actively withheld by their hosts. However, the production of extracellular siderophores enables iron sequestration by pathogens, increasing their virulence. Another function of siderophores is extracellular detoxification of non-ferrous metals. Here, we experimentally link the detoxification and virulence roles of siderophores by testing whether the opportunistic pathogen Pseudomonas aeruginosa displays greater virulence after exposure to copper. To do this, we incubated P. aeruginosa under different environmentally relevant copper regimes for either two or twelve days. Subsequent growth in a copper-free environment removed phenotypic effects, before we quantified pyoverdine production (the primary siderophore produced by P. aeruginosa), and virulence using the Galleria mellonella infection model. RESULTS: Copper selected for increased pyoverdine production, which was positively correlated with virulence. This effect increased with time, such that populations incubated with high copper for twelve days were the most virulent. Replication of the experiment with a non-pyoverdine producing strain of P. aeruginosa demonstrated that pyoverdine production was largely responsible for the change in virulence. CONCLUSIONS: We here show a direct link between metal stress and bacterial virulence, highlighting another dimension of the detrimental effects of metal pollution on human health.

Pulse and Press Disturbances Have Different Effects on Transient Community Dynamics.

AbstractDisturbances are important determinants of diversity, and the combination of their aspects (e.g., disturbance intensity, frequency) can result in complex diversity patterns. Here, we leverage an important approach to classifying disturbances in terms of temporal span to understand the implications for species coexistence: pulse disturbances are acute and discrete events, while press disturbances occur continuously through time. We incorporate the resultant mortality rates into a common framework involving disturbance frequency and intensity. Press disturbances can be encoded into models in two distinct ways, and we show that the appropriateness of each depends on the type of data available. Using this framework, we compare the effects of pulse versus press disturbance on both asymptotic and transient dynamics of a two-species Lotka-Volterra competition model to understand how they engage with equalizing mechanisms of coexistence. We show that press and pulse disturbances differ in transient behavior, though their asymptotic diversity patterns are similar. Our work shows that these differences depend on how the underlying disturbance aspects interact and that the two ways of characterizing press disturbances can lead to contrasting interpretations of disturbance-diversity relationships. Our work demonstrates how theoretical modeling can strategically guide and help the interpretation of empirical work.

Whole community invasions and the integration of novel ecosystems.

The impact of invasion by a single non-native species on the function and structure of ecological communities can be significant, and the effects can become more drastic-and harder to predict-when multiple species invade as a group. Here we modify a dynamic Boolean model of plant-pollinator community assembly to consider the invasion of native communities by multiple invasive species that are selected either randomly or such that the invaders constitute a stable community. We show that, compared to random invasion, whole community invasion leads to final stable communities (where the initial process of species turnover has given way to a static or near-static set of species in the community) including both native and non-native species that are larger, more likely to retain native species, and which experience smaller changes to the topological measures of nestedness and connectance. We consider the relationship between the prevalence of mutualistic interactions among native and invasive species in the final stable communities and demonstrate that mutualistic interactions may act as a buffer against significant disruptions to the native community.

Polymicrobial infections can select against Pseudomonas aeruginosa mutators because of quorum-sensing trade-offs.

Bacteria with increased mutation rates (mutators) are common in chronic infections and are associated with poorer clinical outcomes, especially in the case of Pseudomonas aeruginosa infecting cystic fibrosis (CF) patients. There is, however, considerable between-patient variation in both P. aeruginosa mutator frequency and the composition of co-infecting pathogen communities. We investigated whether community context might affect selection of mutators. Using an in vitro CF model community, we show that P. aeruginosa mutators were favoured in the absence of other species but not in their presence. This was because there were trade-offs between adaptation to the biotic and abiotic environments (for example, loss of quorum sensing and associated toxin production was beneficial in the latter but not the former in our in vitro model community) limiting the evolvability advantage of an elevated mutation rate. Consistent with a role of co-infecting pathogens selecting against P. aeruginosa mutators in vivo, we show that the mutation frequency of P. aeruginosa population was negatively correlated with the frequency and diversity of co-infecting bacteria in CF infections. Our results suggest that co-infecting taxa can select against P. aeruginosa mutators, which may have potentially beneficial clinical consequences.

Fitness effects of plasmids shape the structure of bacteria-plasmid interaction networks.

Antimicrobial resistance (AMR) genes are often carried on broad host range plasmids, and the spread of AMR within microbial communities will therefore depend on the structure of bacteria­plasmid networks. Empirical and theoretical studies of ecological interaction networks suggest that network structure differs between communities that are predominantly mutualistic versus antagonistic, with the former showing more generalized interactions (i.e., species interact with many others to a similar extent). This suggests that mutualistic bacteria­plasmid networks­where antibiotics are present and plasmids carry AMR genes­will be more generalized than antagonistic interactions, where plasmids do not confer benefits to their hosts. We first develop a simple theory to explain this link: fitness benefits of harboring a mutualistic symbiont promote the spread of the symbiont to other species. We find support for this theory using an experimental bacteria­symbiont (plasmid) community, where the same plasmid can be mutualistic or antagonistic depending on the presence of antibiotics. This short-term and parsimonious mechanism complements a longer-term mechanism (coevolution and stability) explaining the link between mutualistic and antagonistic interactions and network structure.

Disturbance-mediated invasions are dependent on community resource abundance.

Disturbances can facilitate biological invasions, with the associated increase in resource availability being a proposed cause. Here, we experimentally tested the interactive effects of disturbance regime (different frequencies of biomass removal at equal intensities) and resource abundance on invasion success using a factorial design containing five disturbance frequencies and three resource levels. We invaded populations of the bacterium Pseudomonas fluorescens with two ecologically different invader morphotypes: a fast-growing "colonizer" type and a slower growing "competitor" type. As resident populations were altered by the treatments, we additionally tested their effect on invader success. Disturbance frequency and resource abundance interacted to affect the success of both invaders, but this interaction differed between the invader types. The success of the colonizer type was positively affected by disturbance under high resources but negatively under low. However, disturbance negatively affected the success of the competitor type under high resource abundance but not under low or medium. Resident population changes did not alter invader success beyond direct treatment effects. We therefore demonstrate that the same disturbance regime can either be beneficial or detrimental for an invader depending on both community resource abundance and its life history. These results may help to explain some of the inconsistencies found in the disturbance-invasion literature.

Greater Phage Genotypic Diversity Constrains Arms-Race Coevolution.

Antagonistic coevolution between hosts and parasites, the reciprocal evolution of host resistance and parasite infectivity, has important implications in ecology and evolution. The dynamics of coevolution-notably whether host or parasite has an evolutionary advantage-is greatly affected by the relative amount of genetic variation in host resistance and parasite infectivity traits. While studies have manipulated genetic diversity during coevolution, such as by increasing mutation rates, it is unclear how starting genetic diversity affects host-parasite coevolution. Here, we (co)evolved the bacterium Pseudomonas fluorescens SBW25 and two bacteriophage genotypes of its lytic phage SBW25ɸ2 in isolation (one phage genotype) and together (two phage genotypes). Bacterial populations rapidly evolved phage resistance, and phage reciprocally increased their infectivity in response. When phage populations were evolved with bacteria in isolation, bacterial resistance and phage infectivity increased through time, indicative of arms-race coevolution. In contrast, when both phage genotypes were together, bacteria did not increase their resistance in response to increasing phage infectivity. This was likely due to bacteria being unable to evolve resistance to both phage via the same mutations. These results suggest that increasing initial parasite genotypic diversity can give parasites an evolutionary advantage that arrests long-term coevolution. This study has important implications for the applied use of phage in phage therapy and in understanding host-parasite dynamics in broader ecological and evolutionary theory.