Biology subjects

Buckling, A.

Publications and source records attributed to Buckling, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Antimicrobial resistance in a warmer world: selection and transfer of kanamycin resistance is temperature dependent in E. coli.

Climate warming consistently correlates with increased antimicrobial resistance (AMR). Understanding the biological mechanisms underpinning this relationship is important for both prediction and mitigation of the future burden of antibiotic resistance in a warmer world, especially given AMR can spread through both selection and horizontal gene transfer. Here, we experimentally measure how temperature alters susceptibility to kanamycin, selection and spread of resistance in Escherichia coli MG1655 carrying the natural plasmids R1 and RP4. We predict that susceptibility should be lowest - and plasmid transfer highest - at maximal growth rate. Thermal performance of bacterial growth peaked at 40{degrees}C and we detected a small plasmid cost - for RP4, but not R1 - in the absence of kanamycin. The thermal performance of plasmid transfer did not mirror that of bacterial growth, instead, R1 transfer peaked at 35{degrees}C with a much narrower thermal tolerance. Susceptibility to kanamycin decreased with increasing growth rate until 31{degrees}C, before increasing again as temperatures approached and exceeded optimal growth temperature. Increasing susceptibility coupled with high growth rates meant that selection was strongest at the highest measured temperature, but the shape of the relationship changed with kanamycin concentration. In mixed populations of resistant and susceptible bacteria, we found that AMR spread most where selection was strongest, irrespective of plasmid transfer. Our findings suggest increased AMR at higher temperatures is predominantly driven by higher growth rates and increased susceptibility, resulting in stronger selection. Our study provides a framework to uncover generalities across antibiotic classes, strains, species, and plasmids, laying the foundation for improved prediction of AMR selection in the environment.

microbiology↗

bioRxiv2026

Disturbance regime changes leave long-lasting legacies on a microbial community's composition and function

Mortality-inducing disturbances are important, ubiquitous drivers of community composition and function. Importantly, human activities and climate change are increasingly altering disturbance regimes. Most disturbance studies focus on the effects of current disturbance regimes, rarely considering those of historical regimes. However, recent theoretical work predicts that historical regimes can leave persistent legacies, modulating the communitys response to novel disturbances and invasive species. Here, we complement this theoretical approach using a model bacterial system that experienced disturbance regimes for [~]120 generations, followed by novel regimes and invasions for another [~]120 generations. Our results show persistent effects of historical legacies on disturbance-diversity relationships. Furthermore, some combinations of past and novel regimes promote invasion with increasing resident diversity, while others prevent it; legacies may explain conflicting diversity-invasibility relationships. These findings demonstrate the importance of historical legacies in disturbance-prone ecosystems, and underscore the challenges in predicting future community responses to disturbance regime changes.

ecology↗

bioRxiv2026

Sign and strength of pairwise interactions in natural isolates depend on environment type.

Bacterial interactions-whether positive or negative - are crucial for the functioning of microbial communities. Though bacterial interactions are mainly expected to be negative, the sign and strength of interactions are predicted to be context dependent, with interactions typically being more positive in more stressful and nutrient-poor conditions. However, systematic studies investigating how the environment affects interactions between multiple taxa are lacking. Here, we determine if interactions between a panel of natural soil isolates change in response to the environment in which they are grown, with two different artificial media used (one simple and one complex) and a more ecologically relevant soil wash. To maximise natural variation in interactions, we collected multiple isolates from multiple sites: co-occurring (sympatric) isolates were predicted to show more negative interactions than allopatric isolates because of greater overlap in resource use. Pairwise interactions were in general negative, but more negative when grown in a complex lab-derived medium (Tryptic Soy Broth). Mutually beneficial interactions were most common in a simple resource medium (M9 minimal media) and exploitative interactions were most frequent in a soil broth. These patterns were independent of whether species originated from the same or a different site. The study supports the prediction that nutrient rich environments promote more negative interactions, and that measuring interactions of soil isolates in standard lab media is likely to misrepresent interactions occurring in natural environments.

microbiology↗

bioRxiv2026

SELECT 2.0: Refined and open access SELection Endpoints in Communities of bacTeria (SELECT) method to determine concentrations of antibiotics that may select for antimicrobial resistance in the environment

Antimicrobial resistance (AMR) is a significant and growing threat to human, plant and animal health, the global economy, and food security. The One Health approach to AMR recognises the role of the environment in the evolution, emergence, and dissemination of AMR. In part, this is due to anthropogenic pollution that releases AMR organisms alongside cocktails of compounds that may select for AMR in situ, which then pose an exposure risk to humans and animals. This has spurred growing interest from cross-sectoral stakeholders in environmental risk assessment (ERA) of antibiotics, with regards to their selective potential. Many different experimental and modelling approaches have been used to determine the lowest concentration of an antibiotic that may select for AMR. Debates continue regarding which individual approach, if any, may be best for determining concentrations of antibiotics that may select for AMR, for ERA purposes. This paper contributes to this ongoing discourse by refining and using a previously published method SELECT (SELection Endpoints in Communities of bacTeria) to rapidly generate predicted no effect concentrations for resistance (PNECRs) for 32 antibiotics on the premise that reduction in growth of complex community of bacteria correlates with selection for AMR resistance genes. The database of PNECRs of antibiotics presented here is the largest generated using a single experimental, empirical approach that will aid future efforts towards creating a standardised test. PNECR data were used to conduct ERAs using measured environmental concentrations of antibiotics to rank antibiotics by potential selection risk in different environments. The experimental approach and statistical code have been made open access, with online tutorials available to facilitate other laboratories using the SELECT 2.0 method. Finally, we discuss the limitations of this approach and how these could be addressed in future studies.

microbiology↗

bioRxiv2026

Bacterial phylogeny explains variation in virulence but not phage efficacy in vivo

The continuing emergence of antimicrobial resistance (AMR) poses one of the most urgent threats to public health in the 21st century, causing [~]1.2 million deaths per year. A promising alternative to traditional antimicrobials is phage therapy, which has proved effective in the treatment of antimicrobial-resistant infections, including methicillin-resistant Staphylococcus aureus (MRSA). However, considerable variation in the outcome of phage therapy remains, with phage that show high efficacy in vitro often showing little to no effect when applied in vivo. To allow for the efficient clinical use of phage, it is vital to understand how both bacterial virulence and phage efficacy vary in vivo and determine how well in vitro and in vivo measures of phage efficacy correlate. Here, we infected 4,968 Galleria mellonella larvae with 64 phylogenetically diverse Staphylococcaceae isolates, both with and without co-inoculation of the bacteriophage ISP, and recorded mortality and melanisation over 24 hours. We found that bacterial virulence varied among Staphylococcaceae strains, and that a large proportion of this variation could be explained by the evolutionary relationships between bacteria. These results indicate that pathogen phylogeny may be a useful tool for explaining variation in both the severity of clinical infections and the virulence of novel emerging pathogens. The addition of phage significantly improved the survival of G. mellonella larvae, with an average 15.2% increase in endpoint survival and 10.1% reduction in endpoint melanisation across Staphylococcaceae strains. Phage efficacy in vivo showed phylogenetic repeatability, but no detectable phylogenetic heritability across bacteria species, in contrast to previous in vitro studies of phage infections. Concurrently, we found no evidence of a correlation between in vivo and in vitro measures of phage efficacy across bacterial isolates, demonstrating that environment plays a major role in determining the outcomes of bacteria-phage interactions. This highlights that caution should be used when extrapolating from in vitro measures of phage efficacy to select phages for therapeutic uses.

microbiology↗

bioRxiv2025

A Novel Method to Simultaneously Estimate Bacterial Respiration and Growth from Oxygen Dynamics

Bacterial growth and respiration are fundamental metabolic processes that affect how energy is used and impact carbon sequestration at the ecosystem scale. However, these traits are usually quantified independently, growth is quantified with endpoint biomass measurements while respiration is quantified by monitoring oxygen or carbon dioxide. Because the two physiological traits are collected at different temporal and volumetric scales (hours-to-days for growth versus minutes-to-hours for respiration), reconciling them is challenging and often introduces scale-mismatch bias, obscuring causal links between metabolism and environmental drivers. In this study, we develop a novel method for quantifying the rates of bacterial growth and respiration concurrently from a single dissolved oxygen time series. Our approach introduces a model that couples exponential biomass growth with biomass-specific respiration, enabling the simultaneous inference of both rates in real time. We applied our high throughput method to 15 bacterial strains isolated from natural environments. Our approach yielded growth estimates in close agreement with measurements based on popular methods using optical density or flow cytometry (R2 > 0.9) with no evidence of taxon-specific bias. We also tested our approach in quantifying the effects of temperature on respiration, growth and carbon use-efficiency in Pseudomonas sp. Our method yielded typical unimodal thermal response curves for growth and respiration where rates were highest at moderate temperatures, while carbon-use efficiency increased from cooler temperatures, peaked around the thermal optimum ([~]30-35 {degrees}C), and declined at high temperature. By quantifying respiration and growth simultaneously and in high throughput, our approach effectively enables measurement of microbial metabolic strategies and adaptations to stress. It offers a non-invasive and scalable tool for high throughput phenotyping and studies of environmental perturbations, enabling a new class of trait-based microbial ecology that links cellular physiology to broader ecosystem function.

microbiology↗

bioRxiv2025

Community complexity does not weaken pairwise coevolution in a soil bacterial community

Exploitative interactions, such as predator-prey and host-parasite interactions, are ubiquitous in microbial communities. These interactions shape community density and composition, imposing strong selection on members to evolve countermeasures reducing the negative impacts of exploitation. Exploitative coevolution is often studied between species pairs in isolation, which may over-estimate the strength and relevance of pairwise coevolution. Here we studied how community context influences coevolution between Pseudomonas fluorescens (exploited) and Variovorax sp. (exploiter). We evolved these species in pairwise coculture and embedded within a five-species community to investigate evolved changes in pairwise interactions. We found evidence for asymmetrical coevolution: Variovorax evolved more rapidly than Pseudomonas, leading to increased exploitation through time, while Pseudomonas evolved increased tolerance to Variovorax with time lag. The pairwise coevolutionary dynamics were not affected by the presence of other community members. Understanding how coevolutionary patterns change with increasing community complexity can have important implications for community persistence and function.

evolutionary biology↗

bioRxiv2025

Antagonism between bacteriophages and macrophages decreases efficacy of a phage cocktail and increases bacteriophage resistance

Phage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis, but treatment success is very variable. Evolution of bacterial resistance to bacteriophages and bacteriophage counter-resistance (coevolution) during therapy may explain some of this variation, the dynamics of which may be affected by interactions with the patients immune system. Here, we examine how a pathogenic bacterium, Pseudomonas aeruginosa coevolves with two clinically relevant bacteriophages (14-1 and PNM) when in the presence of macrophages (RAW 264.7 cell line). We show macrophages reduced the rate by which bacteria were killed by bacteriophages, likely by reducing bacteria-bacteriophage contact rates. Over evolutionary time-scales, macrophages increased the proportion of bacteriophage resistant bacteria compared to where macrophages were absent. These differences in resistance rates were likely driven by the early advantage in density offered by macrophages to bacteria, and exclusion of PNM from the bacteriophage cocktail which otherwise increased in frequency in the absence of macrophages. Consequently, macrophages significantly altered the short- and long-term efficacy of a bacteriophage cocktail. In line with a growing body of work, our results suggest that the patients immune system can reduce the efficacy of phage therapy, potentially driving variable outcomes in therapy success in patients. Significance statementPhage therapy, the use of viruses that infect bacteria (bacteriophages), is a promising complement to antibiotics during the antimicrobial resistance crisis. However, treatment success is very variable. One, often overlooked, variable is the immune system and how this influences bacteriophage efficacy, and how bacteria evolve resistance to bacteriophages. We find that macrophages reduce the rate by which bacteria are killed by bacteriophages. Resistance to bacteriophages also increased in the presence of macrophages, showing macrophages affect the short- and long-term efficacy of phage therapy. These results highlight the importance of the immune system in phage therapy, and the need for more research in this area.

evolutionary biology↗

bioRxiv2025

Diversity Between but Not Within Microbial Communities Increases With the Diversity of Supplied Nutrients

Greater resource diversity within a habitat is typically expected to support greater biodiversity in the resident ecological community (-diversity). It may also make community composition less deterministic since a wider range of species have a chance establishing themselves and affecting subsequent community assembly processes. However, the relationship between microbial diversity and the diversity of resources exogenously supplied is complicated by cross-feeding between microbes, since much of the resource diversity encountered will be generated by the community. Here, we assembled microbial communities in simple growth media (where cross-feeding will be important) with 1 to 3 carbon sources and asked how increasing nutrient diversity affects diversity within and between communities. While there was (i) no clear difference in -diversity, increasing the number of carbon sources led to communities that varied (ii) more within and (iii) less between the different abiotic conditions. (i) is at odds with classical ecological theory, but fits predictions from a consumer-resource model that accounts for cross-feeding. (ii) shows an increase in the stochasticity of community assembly. (iii) demonstrates strong abiotic selection in the single carbon source communities. We then went on to show that this is leads to phylogenetic clustering (co-occurrence of more closely related strains).

microbiology↗

bioRxiv2025

Competition between plasmids conferring the same antimicrobial resistance leads to plasmid-host network specialisation

Plasmids have a key role in disseminating antimicrobial resistance (AMR). Previous work has shown that in the presence of antibiotics, plasmids conferring AMR will spread to a larger number and a more phylogenetically diverse group of host organisms. However, this pattern may be complicated when multiple plasmids confer the same resistance. Here, we analyse host-plasmid networks in experimental communities of three bacterial species cultured for six weeks with three incompatible plasmids (plasmids that cannot stably coexist within a single cell) conferring tetracycline resistance. The communities are cultured either without tetracycline, or with a low or high dose of the antibiotic. We find that while antibiotic selection leads to higher plasmid prevalence, it does not change the structure of host-plasmid networks. Under all conditions, the networks become highly specialised, as specific host-plasmid pairings emerge. This result is supported by a previously published mathematical model parameterised to match our experimental set-up. Our study reiterates the importance of fitness effects of plasmids in determining the structure of bacteria-plasmid networks. The spread of a given AMR plasmid is reduced when fitness differences are reduced due to other plasmids carrying AMR genes, leading to more specialised bacteria-plasmid networks.

microbiology↗

bioRxiv2025

Cheats can boost the success of a cooperative invader

Successful biological invasions are dependent on the invader being able to grow and reproduce in the new environment. One way that microbial invaders may facilitate this is to use cooperative public goods, such as metal-binding siderophores. However, siderophore production can be exploited by non-producing cheats who benefit from production without paying any associated costs. Here, we test the importance of cooperation for the success of Pseudomonas aeruginosa invading a 5-species microbial community. We do this by comparing the success of a siderophore-producing strain, a siderophore-deficient mutant strain and a 50:50 mixed population, both in environments with weak (copper absent) and strong (copper present) siderophore requirement. We found no effect of invader type on success when siderophores were less essential for growth, but large differences when they were selectively favoured. Here the producer-cheat mix had the greatest success, with both strains having near equal fitness and reaching high densities, whilst in isolation producers had intermediate success and cheats the lowest. Similarly, resident diversity only differed across invader treatments when copper was present. In conclusion, we show that the presence of cheats can provide a larger benefit for invasion success than pure cooperator populations, but only when public goods are particularly beneficial.

ecology↗

bioRxiv2025

Predicting clinical phage therapy outcomes in vitro: results using mixed versus single isolates from an MRSA case study

AimsIn phage therapy case studies, 1-3 bacteria isolates are typically tested against phages (phagogram). However, as bacteria populations differ in their susceptibility to phages and antibiotics, the strains selected may not represent how the infecting population will respond to treatment. Our aim was to assess whether the effects of phage on single or a mix of isolates in vitro show more comparable results to that observed during a clinical case study. Methods and ResultsThe patient presented with a methicillin resistant Staphylococcus aureus infection (MRSA). In this previously published case study, phage therapy alongside antibiotics rapidly cleared blood cultures of bacteria while localised regions, including the lungs, took longer to clear of bacteria. In this follow-up study, mixed isolates were more likely to persist than single isolates in vitro, more closely representing the lung, but not blood, infections. These results may reflect the different degrees of genetic diversity of the infecting bacteria in these sites. ConclusionsFor this patient, phage therapy predictions were significantly affected by whether we used mixed versus single isolates, but the predictive precision depended on the site of in vivo infection. Impact statementThis work is important for informing whether phage therapy design protocols give accurate insights into clinical outcomes.

pathology↗

bioRxiv2025

Deciphering the dynamics of Cyanobacteria-Phage in a natural lake: Insights from a decade-long investigation

Phages - viruses that infect bacteria - are often seen as key players in bacterial community dynamics and the ecosystem services those communities support. However, much of our understanding of phage-bacteria interactions comes from in vitro studies, which provide limited insights into how these interactions occur in natural environments. In this study, we used cyanobacterial blooms as a model microbial community to evaluate the potential role of phages in driving cyanobacterial populations. These blooms are characterized by massive and usually rapid accumulation of cyanobacterial biomass and occur worldwide, threatening aquatic systems. The frequency and intensity of cyanobacterial blooms are increasing over time, likely due to human activities, and, until now, the role of predators like phages has remained unclear. We used a deep amplicon sequencing approach targeting the g20 capsid gene to profile the cyanophage community in eutrophic Lake Champlain over time, comparing their dynamics and diversity with bacterial communities and examining their associations. We evaluated whether phages simply followed bacterial dynamics, with limited impact on bacterial composition, or instead whether they played an active role in drving bacterial community structure. We found that phages exhibited similar dynamics to their potential bacterial hosts and shared environmental niches. However, we also observed strong differences specific to the phage community, such as inter-annual variation and an increase in Shannon diversity over time. Phage-bacteria and phage-cyanobacteria co-variance uncovered potential interactions that resulted in strongly modular networks. However, while the network structure remained modular, the composition of the modules differed significantly between environmental and temporal conditions. Lastly, viral phylogeny partially explained phage-bacteria co-variance, but only for a small proportion of bacterial ASVs. Our results indicate that phage-bacterial interactions are partly genetically structured and vary within modules across environmental conditions.

ecology↗

bioRxiv2024

Bacteria-phage (co)evolution is constrained in a synthetic community across multiple bacteria-phage pairs

Bacteriophages can be important drivers of bacterial densities, and therefore microbial community composition and function. These ecological interactions are likely to be greatly affected by evolutionary dynamics, because bacteria can rapidly evolve resistance to phage while phage can reciprocally evolve to increase infectivity. Most studies to date have explored eco-evolutionary dynamics using isolated pairs of bacteria-phage but in nature, multiple bacteria and phages coexist and (co)evolve simultaneously. How coevolution plays out in this context is poorly understood. Here, we examine how three coexisting soil bacteria (Ochrobactrum sp., Pseudomonas sp., and Variovorax sp.) interact and evolve with three species-specific bacteriophages over eight weeks of experimental evolution, both as host-parasite pairs in isolation and as a mixed community. Across all species phage resistance evolution was inhibited in polyculture, with the most pronounced effect on Ochrobactrum. Between bacteria-phage pairs there were also substantial differences in the effect of phage on host densities, and evolutionary dynamics including whether pairs coevolved. These contrasts emphasise the difficulty in generalising from monoculture to polyculture, and between bacteria-phage pairs to wider systems. Future studies should consider how multiple bacteria and phage pairs interact simultaneously to better understand how coevolutionary dynamics happen in natural communities. ImportanceThis project is unique in examining evolutionary dynamics among coexisting bacteria and their phages, rather than focus on single focal species - this makes our work more applicable to natural contexts while still working with controlled synthetic communities. While it is commonly assumed that bacteria will evolve phage resistance and coevolve with phage, this may be uncommon in more complex communities due to reduced contact rates and/or reduced mutation rates. Furthermore, the contrast in population dynamics and ability to coevolve between bacteria-phage pairs highlights the need for more pairs to be studied. Over-reliance on model systems that are known to coevolve means we lack an understanding of how wider bacteria-phage pairs interact, and to what extent results can be generalised beyond these pairs.

evolutionary biology↗

bioRxiv2024

Estimates of community stability using the invasion criterion are robust across levels of invader species richness

A key feature of natural communities is that the species within them stably coexist. A common metric used to test community stability is measuring the ability of each species to recover from rare. Here, each species is assumed to have negative frequency dependent fitness and have a greater fitness relative to the other community members. A conceptual issue with measurements of relative invader fitness is that single species are invaded from rare. In natural communities, multiple species would likely decline following perpetuations e.g. antibiotic application, global warming, natural disasters. In our study, we compare previous estimates of community stability in a five species microbial community to experimental results in which multiple species are invaded from rare. Our results showed that single species invasions were broadly predictive of whole community stability when multiple species are invaded simultaneously. Precise values of relative invader fitness were less comparable, however being non-significantly different in most comparisons in 3/5 species. This work provides the first experimental test of the robustness of relative invader fitness metrics under multi-species invasion scenarios.

ecology↗

bioRxiv2024

Bacteriophage resistance increases bacterial productivity by shifting population growth dynamics in the soil bacterium Variovorax sp.

Lytic bacteriophages ( phages) can limit bacterial densities and shape community structure, either directly through lysis or indirectly through costs to resistance. However, phages have also been reported to have no, and in some cases even positive, effects on host densities. Here, we investigate the mechanisms behind an increase in host density in Variovorax sp. populations following a fixation of resistance that was maintained after phage extinction. Our results demonstrate that the density increase was a genetic trait coinciding with resistance emergence. Growth curves showed that phage resistance shifted population growth curves such that density was higher in the death-phase. This density-increasing effect of resistance had important implications for community structure with phage resistant Variovorax decreasing the density of a conspecific. That resistance to lytic phage can increase host densities has implications for wider ecology and phage therapy where lytic phages are presumed to have negative effects on their hosts.

microbiology↗

bioRxiv2023

Disturbances can facilitate prior invasions more than subsequent invasions in microbial communities

Invasions by microorganisms are commonly found to benefit from disturbance events. However, the importance of the relative timing and order of the invasion and disturbance for invader success remains uncertain. Here, by invading a stably coexisting community of five bacterial species at four different propagule pressures on eight separate occasions - four pre-disturbance and four post-disturbance - we experimentally tested the importance of timing and order for the invaders success. Furthermore, we quantified the impact of the invader on the composition of the resident community. Across a range of invader densities, both pre- and post-disturbance invader success was greatest the closer in time to the disturbance. While the greatest invasion success occurred when the invasion immediately followed the disturbance, invasion resistance rapidly recovered, such that the three subsequent invasions had negligible success. As a consequence, overall invader success was greatest pre-than post-disturbance. We found that invasion day as well as success significantly affected resident diversity, such that it was lowest in communities invaded immediately after the disturbance, but was overall lower in communities invaded pre-disturbance. Overall, these results demonstrate that invading immediately after a disturbance is highly beneficial for invader success and results in the biggest change to community composition. Importantly however, they also show that this window of opportunity is very brief, and that, on average, an invader will have a greater success and effect on community composition if it invades before a disturbance.

ecology↗

bioRxiv2023

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 virulence 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 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.

microbiology↗