AbstractIron 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. 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. Therefore 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.

Climate change is bringing unforeseen alterations to disturbance
regimes, exposing 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. We find 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.

AbstractMetal contamination poses both a direct threat to human health as well as an indirect threat through its potential to affect bacterial pathogens. Metals can not only co-select for antibiotic resistance, but also might affect pathogen virulence via increased siderophore production. Siderophores are extracellular compounds released to increase ferric iron uptake — a common limiting factor for pathogen growth within hosts – making them an important virulence factor. However, siderophores can also be positively selected for to detoxify non-ferrous metals, and consequently metal stress can potentially increase bacterial virulence. Anthropogenic methods to remediate environmental metal contamination commonly involve amendment with lime-containing materials, but whether this reduces in situ co-selection for antibiotic resistance and virulence remains unknown. Here, using microcosms containing metal-contaminated river water and sediment, we experimentally test whether metal remediation by liming reduces co-selection for these traits in the opportunistic pathogen Pseudomonas aeruginosa embedded within a natural microbial community. To test for the effects of environmental structure, which can 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, but this effect was reduced in the limed treatments. Evolved P. aeruginosa populations 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. We therefore demonstrate that although remediation via liming reduced the strength of selection for metal resistance mechanisms, it 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.Graphical abstract

Accumulation of plastics in the marine environment has widespread detrimental consequences for ecosystems and wildlife. Marine plastics are rapidly colonised by a wide diversity of bacteria, including human pathogens, posing potential risks to health. Here, we investigate the effect of polymer type, residence time and estuarine location on bacterial colonisation of common household plastics, including pathogenic bacteria. We submerged five main household plastic types: low-density PE (LDPE), high-density PE (HDPE), polypropylene (PP), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) at an estuarine site in Cornwall (U.K.) and tracked bacterial colonisation dynamics. Using both culture-dependent and culture-independent approaches, we found that bacteria rapidly colonised plastics irrespective of polymer type, reaching culturable densities of up to 1000 cells cm3 after 7 weeks. Community composition of the biofilms changed over time, but not among polymer types. The presence of pathogenic bacteria, quantified using the insect model Galleria mellonella, increased dramatically over a five-week period, with Galleria mortality increasing from 4% in week one to 65% in week five. No consistent differences in virulence were observed between polymer types. Pathogens isolated from plastic biofilms using Galleria enrichment included Serratia and Enterococcus species and they harboured a wide range of antimicrobial resistance genes. Our findings show that plastics in coastal waters are rapidly colonised by a wide diversity of bacteria independent of polymer type. Further, our results show that marine plastic biofilms become increasingly associated with virulent bacteria over time.

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.

Infections by bacterial pathogens are one of the biggest causes of human mortality each year globally. Increasingly they are being caused by bacteria that can survive in, and adapt to, non-host associated environments. It is therefore crucial we understand what may cause bacteria in these settings to both become more pathogenic and to successfully colonise new environments.
the aim of this thesis is to improve our understanding of the effect of metals on bacterial virulence, and the effect of pathogen invasion into a community. Anthropogenic pollution is increasing concentrations of metal ions to toxic levels in many environments around the world, and whilst their effect on antimicrobial resistance is known, their effect on virulence is not.
Copper selected for increased virulence in populations of the opportunistic pathogen Pseudomonas aeruginosa at environmentally relevant concentrations. This was largely due to copper selecting for increased siderophore production, which is both a metal detoxification mechanism, and a virulence factor. Conversely, copper decreased the virulence of multiple wastewater influent bacterial communities. It is most likely that this was due to changes in community composition, and decreases in community productivity. As these results demonstrated that copper can affect bacterial virulence, it was then tested whether liming, which is a common metal remediation method, can reduce this selective effect in metal polluted river water and sediments. Liming was found to have little effect on either virulence or antibiotic resistance, demonstrating that even remediated environments can remain as reservoirs for pathogens. Finally, pulse mortality events facilitated the invasion of P. aeruginosa into a bacterial community. Furthermore, it was shown that the combined effect of pulse mortality and invasion greatly reduced resident diversity. This demonstrates that disturbance events may increase the risk posed by bacterial pathogens.
These results demonstrate that ever-increasing metal pollution is likely to affect bacterial virulence, and that pathogen colonisation can detrimentally affect resident communities.

Infections by bacterial pathogens are one of the biggest causes of human mortality each year globally. Increasingly they are being caused by bacteria that can survive in, and adapt to, non-host associated environments. It is therefore crucial we understand what may cause bacteria in these settings to both become more pathogenic and to successfully colonise new environments.
the aim of this thesis is to improve our understanding of the effect of metals on bacterial virulence, and the effect of pathogen invasion into a community. Anthropogenic pollution is increasing concentrations of metal ions to toxic levels in many environments around the world, and whilst their effect on antimicrobial resistance is known, their effect on virulence is not.
Copper selected for increased virulence in populations of the opportunistic pathogen Pseudomonas aeruginosa at environmentally relevant concentrations. This was largely due to copper selecting for increased siderophore production, which is both a metal detoxification mechanism, and a virulence factor. Conversely, copper decreased the virulence of multiple wastewater influent bacterial communities. It is most likely that this was due to changes in community composition, and decreases in community productivity. As these results demonstrated that copper can affect bacterial virulence, it was then tested whether liming, which is a common metal remediation method, can reduce this selective effect in metal polluted river water and sediments. Liming was found to have little effect on either virulence or antibiotic resistance, demonstrating that even remediated environments can remain as reservoirs for pathogens. Finally, pulse mortality events facilitated the invasion of P. aeruginosa into a bacterial community. Furthermore, it was shown that the combined effect of pulse mortality and invasion greatly reduced resident diversity. This demonstrates that disturbance events may increase the risk posed by bacterial pathogens.
These results demonstrate that ever-increasing metal pollution is likely to affect bacterial virulence, and that pathogen colonisation can detrimentally affect resident communities.

Disturbances can play a major role in biological invasions: by destroying biomass, they alter habitat and resource abundances. Previous field studies suggest that disturbance-mediated invader success is a consequence of resource influxes, but the importance of other potential covarying causes, notably the opening up of habitats, have yet to be directly tested. Using experimental populations of the bacterium Pseudomonas fluorescens, we determined the relative importance of disturbance-mediated habitat opening and resource influxes, plus any interaction between them, for invader success of two ecologically distinct morphotypes. Resource addition increased invasibility, while habitat opening had little impact and did not interact with resource addition. Both invaders behaved similarly, despite occupying different ecological niches in the microcosms. Treatment also affected the composition of the resident population, which further affected invader success. Our results provide experimental support for the observation that resource input is a key mechanism through which disturbance increases invasibility.

Successful colonisations by invasive organisms are causing catastrophic changes to communities: altering their dynamics, reducing biodiversity and impeding ecosystem services. These ecological costs are only surpassed by habitat loss, and when combined with the huge associated economic costs, makes understanding how these events occur of growing importance. This study will focus on how disturbances, which change the availability of resources and habitat, may facilitate the establishment of novel species. First, we factorially separate resource influxes and habitat opening to test the mechanism by which disturbance increases invader success; using diversified populations of Pseudomonas fluorescens. We homogenised communities to open habitat and added nutrients to increase resources. Resource influxes were key in successful establishment, habitat opening had little affect and no interaction was found. Secondly, we expanded upon this by testing if resource abundance interacts with disturbance frequency; hypothesising when more resources are available disturbance-induced influxes, and thus invader success, would be greater. To do this communities of P. fluorescens were disturbed at different frequencies in three resource concentrations and invaded multiple times. We found disturbance and resources to interact: manipulating the mortality-growth rate balance, and thus success, of the invader. Resources also interacted with evolved biodiversity to effect invasion resistance. We finish by testing disturbance and resource effects on a stably coexisting 5-species bacterial community, using a 5x5 factorial design. Disturbance and resource both manipulated the variation in fitness between species: impacting biodiversity. Interactions were only found at high-disturbance-high-resources. This highlights the suitability of this system for future disturbance-resource studies on stably coexisting systems, including future invasion work.
In conclusion, we show disturbance, through adding resources, to be a key factor in invasion success: the extent to which being strongly affected by resource abundance. We also find disturbance and resource changes are likely to impact the stability of communities.