Dietary fibre supplementation enhances radiotherapy tumour control and alleviates intestinal radiation toxicity.

BACKGROUND: Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts. RESULT: Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2% cellulose and raised intratumoural CD8+ cells. Post-irradiation, tumour control positively correlated with Lachnospiraceae family abundance. Psyllium plus resistant starch radiosensitised the tumours, positively correlating with Bacteroides genus abundance and increased caecal isoferulic acid levels, associated with a favourable response in terms of tumour control. Psyllium plus inulin mitigated the acute radiation injury caused by 14 Gy. Psyllium plus inulin increased caecal acetate, butyrate and propionate levels, and psyllium alone and psyllium plus resistant starch increased acetate levels. Human gut microbiota profiles at the phylum level were generally more like mouse 0.2% cellulose profiles than high fibre profiles. CONCLUSION: These supplements may be useful in combination with radiotherapy in patients with pelvic malignancy. Video Abstract.

Microbiome diversity protects against pathogens by nutrient blocking.

The human gut microbiome plays an important role in resisting colonization of the host by pathogens, but we lack the ability to predict which communities will be protective. We studied how human gut bacteria influence colonization of two major bacterial pathogens, both in vitro and in gnotobiotic mice. Whereas single species alone had negligible effects, colonization resistance greatly increased with community diversity. Moreover, this community-level resistance rested critically upon certain species being present. We explained these ecological patterns through the collective ability of resistant communities to consume nutrients that overlap with those used by the pathogen. Furthermore, we applied our findings to successfully predict communities that resist a novel target strain. Our work provides a reason why microbiome diversity is beneficial and suggests a route for the rational design of pathogen-resistant communities.

The evolution of short- and long-range weapons for bacterial competition.

Bacteria possess a diverse range of mechanisms for inhibiting competitors, including bacteriocins, tailocins, type VI secretion systems and contact-dependent inhibition (CDI). Why bacteria have evolved such a wide array of weapon systems remains a mystery. Here we develop an agent-based model to compare short-range weapons that require cell-cell contact, with long-range weapons that rely on diffusion. Our model predicts that contact weapons are useful when an attacking strain is outnumbered, facilitating invasion and establishment. By contrast, ranged weapons tend to be effective only when attackers are abundant. We test our predictions with the opportunistic pathogen Pseudomonas aeruginosa, which naturally carries multiple weapons, including CDI and diffusing tailocins. As predicted, short-range CDI can function at low and high frequencies, while long-range tailocins require high frequency and cell density to function effectively. Head-to-head competition experiments with the two weapon types further support our predictions: a tailocin attacker defeats CDI only when it is numerically dominant, but then we find it can be devastating. Finally, we show that the two weapons work well together when one strain employs both. We conclude that short- and long-range weapons serve different functions and allow bacteria to fight both as individuals and as a group.

Chaos in a bacterial stress response.

Cellular responses to environmental changes are often highly heterogeneous and exhibit seemingly random dynamics. The astonishing insight of chaos theory is that such unpredictable patterns can, in principle, arise without the need for any random processes, i.e., purely deterministically without noise. However, while chaos is well understood in mathematics and physics, its role in cell biology remains unclear because the complexity and noisiness of biological systems make testing difficult. Here, we show that chaos explains the heterogeneous response of Escherichia coli cells to oxidative stress. We developed a theoretical model of the gene expression dynamics and demonstrate that chaotic behavior arises from rapid molecular feedbacks that are coupled with cell growth dynamics and cell-cell interactions. Based on theoretical predictions, we then designed single-cell experiments to show we can shift gene expression from periodic oscillations to chaos on demand. Our work suggests that chaotic gene regulation can be employed by cell populations to generate strong and variable responses to changing environments.

Reactivity of complex communities can be more important than stability.

Understanding stability-whether a community will eventually return to its original state after a perturbation-is a major focus in the study of various complex systems, particularly complex ecosystems. Here, we challenge this focus, showing that short-term dynamics can be a better predictor of outcomes for complex ecosystems. Using random matrix theory, we study how complex ecosystems behave immediately after small perturbations. Our analyses show that many communities are expected to be 'reactive', whereby some perturbations will be amplified initially and generate a response that is directly opposite to that predicted by typical stability measures. In particular, we find reactivity is prevalent for complex communities of mixed interactions and for structured communities, which are both expected to be common in nature. Finally, we show that reactivity can be a better predictor of extinction risk than stability, particularly when communities face frequent perturbations, as is increasingly common. Our results suggest that, alongside stability, reactivity is a fundamental measure for assessing ecosystem health.

Time delays modulate the stability of complex ecosystems.

What drives the stability, or instability, of complex ecosystems? This question sits at the heart of community ecology and has motivated a large body of theoretical work exploring how community properties shape ecosystem dynamics. However, the overwhelming majority of current theory assumes that species interactions are instantaneous, meaning that changes in the abundance of one species will lead to immediate changes in the abundances of its partners. In practice, time delays in how species respond to one another are widespread across ecological contexts, yet the impact of these delays on ecosystems remains unclear. Here we derive a new body of theory to comprehensively study the impact of time delays on ecological stability. We find that time delays are important for ecosystem stability. Large delays are typically destabilizing but, surprisingly, short delays can substantially increase community stability. Moreover, in stark contrast to delay-free systems, delays dictate that communities with more abundant species can be less stable than ones with less abundant species. Finally, we show that delays fundamentally shift how species interactions impact ecosystem stability, with communities of mixed interaction types becoming the most stable class of ecosystem. Our work demonstrates that time delays can be critical for the stability of complex ecosystems.

Bacterial defences: mechanisms, evolution and antimicrobial resistance.

Throughout their evolutionary history, bacteria have faced diverse threats from other microorganisms, including competing bacteria, bacteriophages and predators. In response to these threats, they have evolved sophisticated defence mechanisms that today also protect bacteria against antibiotics and other therapies. In this Review, we explore the protective strategies of bacteria, including the mechanisms, evolution and clinical implications of these ancient defences. We also review the countermeasures that attackers have evolved to overcome bacterial defences. We argue that understanding how bacteria defend themselves in nature is important for the development of new therapies and for minimizing resistance evolution.

Collective protection against the type VI secretion system in bacteria.

Bacteria commonly face attacks from other strains using the type VI secretion system (T6SS), which acts like a molecular speargun to stab and intoxicate competitors. Here we show how bacteria can work together to collectively defend themselves against these attacks. This project began with an outreach activity: while developing an online computer game of bacterial warfare, we noticed that one strategist ("Slimy") that made extracellular polymeric substances (EPS) was able to resist attacks from another strategist that employed the T6SS ("Stabby"). This observation motivated us to model this scenario more formally, using dedicated agent-based simulations. The model predicts that EPS production can serve as a collective defence mechanism, which protects both producing cells and neighbouring cells that do not make EPS. We then tested our model with a synthetic community that contains a T6SS-wielding attacker (Acinetobacter baylyi), and two T6SS-sensitive target strains (Escherichia coli) that either secrete EPS, or not. As predicted by our modelling, we find that the production of EPS leads to collective protection against T6SS attacks, where EPS producers protect each other and nearby non-producers. We identify two processes that explain this protection: EPS sharing between cells and a second general mechanism whereby groups of resistant cells shield susceptible cells, which we call "flank protection". Our work shows how EPS-producing bacteria can work together to defend themselves from the type VI secretion system.

Phenotypic heterogeneity in the bacterial oxidative stress response is driven by cell-cell interactions.

Genetically identical bacterial cells commonly display different phenotypes. This phenotypic heterogeneity is well known for stress responses, where it is often explained as bet hedging against unpredictable environmental threats. Here, we explore phenotypic heterogeneity in a major stress response of Escherichia coli and find it has a fundamentally different basis. We characterize the response of cells exposed to hydrogen peroxide (H2O2) stress in a microfluidic device under constant growth conditions. A machine-learning model reveals that phenotypic heterogeneity arises from a precise and rapid feedback between each cell and its immediate environment. Moreover, we find that the heterogeneity rests upon cell-cell interaction, whereby cells shield each other from H2O2 via their individual stress responses. Our work shows how phenotypic heterogeneity in bacterial stress responses can emerge from short-range cell-cell interactions and result in a collective phenotype that protects a large proportion of the population.

3D printing of microbial communities: A new platform for understanding and engineering microbiomes.

3D printing has emerged as a powerful way to produce complex materials on-demand. These printing technologies are now being applied in microbiology, with many recent examples where microbes and matrices are co-printed to create bespoke living materials. Here, we propose a new paradigm for microbial printing. In addition to its importance for materials, we argue that printing can be used to understand and engineer microbiome communities, analogous to its use in human tissue engineering. Many microbes naturally live in diverse, spatially structured communities that are challenging to study and manipulate. 3D printing offers an exciting new solution to these challenges, as it can precisely arrange microbes in 3D space, allowing one to build custom microbial communities for a wide range of purposes in research, medicine, and industry.

Suicidal chemotaxis in bacteria.

Bacteria commonly live in surface-associated communities where steep gradients of antibiotics and other chemical compounds can occur. While many bacterial species move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here, we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of several antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that bacteria use pili-based ('twitching') motility to navigate towards antibiotics. Our analyses suggest that this behaviour is driven by a general response to the effects of antibiotics on cells. Migrating bacteria reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration within hours and remain highly motile. However, isolating cells - using fluid-walled microfluidic devices - reveals that these bacteria are terminal and unable to reproduce. Despite moving towards their death, migrating cells are capable of entering a suicidal program to release bacteriocins that kill other bacteria. This behaviour suggests that the cells are responding to antibiotics as if they come from a competing colony growing nearby, inducing them to invade and attack. As a result, clinical antibiotics have the potential to lure bacteria to their death.

The evolution of spectrum in antibiotics and bacteriocins.

A key property of many antibiotics is that they will kill or inhibit a diverse range of microbial species. This broad-spectrum of activity has its evolutionary roots in ecological competition, whereby bacteria and other microbes use antibiotics to suppress other strains and species. However, many bacteria also use narrow-spectrum toxins, such as bacteriocins, that principally target conspecifics. Why has such a diversity in spectrum evolved? Here, we develop an evolutionary model to understand antimicrobial spectrum. Our first model recapitulates the intuition that broad-spectrum is best, because it enables a microbe to kill a wider diversity of competitors. However, this model neglects an important property of antimicrobials: They are commonly bound, sequestered, or degraded by the cells they target. Incorporating this toxin loss reveals a major advantage to narrow-spectrum toxins: They target the strongest ecological competitor and avoid being used up on less important species. Why then would broad-spectrum toxins ever evolve? Our model predicts that broad-spectrum toxins will be favored by natural selection if a strain is highly abundant and can overpower both its key competitor and other species. We test this prediction by compiling and analyzing a database of the regulation and spectrum of toxins used in inter-bacterial competition. This analysis reveals a strong association between broad-spectrum toxins and density-dependent regulation, indicating that they are indeed used when strains are abundant. Our work provides a rationale for why bacteria commonly evolve narrow-spectrum toxins such as bacteriocins and suggests that the evolution of antibiotics proper is a signature of ecological dominance.

Biofilm cultivation facilitates coexistence and adaptive evolution in an industrial bacterial community.

The majority of ecological, industrial and medical impacts of bacteria result from diverse communities containing multiple species. This diversity presents a significant challenge as co-cultivation of multiple bacterial species frequently leads to species being outcompeted and, with this, the possibility to manipulate, evolve and improve bacterial communities is lost. Ecological theory predicts that a solution to this problem will be to grow species in structured environments, which reduces the likelihood of competitive exclusion. Here, we explored the ability of cultivation in a structured environment to facilitate coexistence, evolution, and adaptation in an industrially important community: Lactococcus lactis and Leuconostoc mesenteroides frequently used as dairy starter cultures. As commonly occurs, passaging of these two species together in a liquid culture model led to the loss of one species in 6 of 20 lineages (30%). By contrast, when we co-cultured the two species as biofilms on beads, a stable coexistence was observed in all lineages studied for over 100 generations. Moreover, we show that the co-culture drove evolution of new high-yield variants, which compared to the ancestor grew more slowly, yielded more cells and had enhanced capability of biofilm formation. Importantly, we also show that these high-yield biofilm strains did not evolve when each species was passaged in monoculture in the biofilm model. Therefore, both co-culture and the biofilm model were conditional for these high-yield strains to evolve. Our study underlines the power of ecological thinking-namely, the importance of structured environments for coexistence-to facilitate cultivation, evolution, and adaptation of industrially important bacterial communities.

Host control and the evolution of cooperation in host microbiomes.

Humans, and many other species, are host to diverse symbionts. It is often suggested that the mutual benefits of host-microbe relationships can alone explain cooperative evolution. Here, we evaluate this hypothesis with evolutionary modelling. Our model predicts that mutual benefits are insufficient to drive cooperation in systems like the human microbiome, because of competition between symbionts. However, cooperation can emerge if hosts can exert control over symbionts, so long as there are constraints that limit symbiont counter evolution. We test our model with genomic data of two bacterial traits monitored by animal immune systems. In both cases, bacteria have evolved as predicted under host control, tending to lose flagella and maintain butyrate production when host-associated. Moreover, an analysis of bacteria that retain flagella supports the evolution of host control, via toll-like receptor 5, which limits symbiont counter evolution. Our work puts host control mechanisms, including the immune system, at the centre of microbiome evolution.

Pleiotropic constraints promote the evolution of cooperation in cellular groups.

The evolution of cooperation in cellular groups is threatened by lineages of cheaters that proliferate at the expense of the group. These cell lineages occur within microbial communities, and multicellular organisms in the form of tumours and cancer. In contrast to an earlier study, here we show how the evolution of pleiotropic genetic architectures-which link the expression of cooperative and private traits-can protect against cheater lineages and allow cooperation to evolve. We develop an age-structured model of cellular groups and show that cooperation breaks down more slowly within groups that tie expression to a private trait than in groups that do not. We then show that this results in group selection for pleiotropy, which strongly promotes cooperation by limiting the emergence of cheater lineages. These results predict that pleiotropy will rapidly evolve, so long as groups persist long enough for cheater lineages to threaten cooperation. Our results hold when pleiotropic links can be undermined by mutations, when pleiotropy is itself costly, and in mixed-genotype groups such as those that occur in microbes. Finally, we consider features of multicellular organisms-a germ line and delayed reproductive maturity-and show that pleiotropy is again predicted to be important for maintaining cooperation. The study of cancer in multicellular organisms provides the best evidence for pleiotropic constraints, where abberant cell proliferation is linked to apoptosis, senescence, and terminal differentiation. Alongside development from a single cell, we propose that the evolution of pleiotropic constraints has been critical for cooperation in many cellular groups.

Reconfigurable Microfluidic Circuits for Isolating and Retrieving Cells of Interest.

Microfluidic devices are widely used in many fields of biology, but a key limitation is that cells are typically surrounded by solid walls, making it hard to access those that exhibit a specific phenotype for further study. Here, we provide a general and flexible solution to this problem that exploits the remarkable properties of microfluidic circuits with fluid walls─transparent interfaces between culture media and an immiscible fluorocarbon that are easily pierced with pipets. We provide two proofs of concept in which specific cell subpopulations are isolated and recovered: (i) murine macrophages chemotaxing toward complement component 5a and (ii) bacteria (Pseudomonas aeruginosa) in developing biofilms that migrate toward antibiotics. We build circuits in minutes on standard Petri dishes, add cells, pump in laminar streams so molecular diffusion creates attractant gradients, acquire time-lapse images, and isolate desired subpopulations in real time by building fluid walls around migrating cells with an accuracy of tens of micrometers using 3D printed adaptors that convert conventional microscopes into wall-building machines. Our method allows live cells of interest to be easily extracted from microfluidic devices for downstream analyses.

Bacterial species rarely work together.

Competition is prevalent and could be harnessed as an alternative to antibiotics.

The evolution of strategy in bacterial warfare via the regulation of bacteriocins and antibiotics.

Bacteria inhibit and kill one another with a diverse array of compounds, including bacteriocins and antibiotics. These attacks are highly regulated, but we lack a clear understanding of the evolutionary logic underlying this regulation. Here, we combine a detailed dynamic model of bacterial competition with evolutionary game theory to study the rules of bacterial warfare. We model a large range of possible combat strategies based upon the molecular biology of bacterial regulatory networks. Our model predicts that regulated strategies, which use quorum sensing or stress responses to regulate toxin production, will readily evolve as they outcompete constitutive toxin production. Amongst regulated strategies, we show that a particularly successful strategy is to upregulate toxin production in response to an incoming competitor's toxin, which can be achieved via stress responses that detect cell damage (competition sensing). Mirroring classical game theory, our work suggests a fundamental advantage to reciprocation. However, in contrast to classical results, we argue that reciprocation in bacteria serves not to promote peaceful outcomes but to enable efficient and effective attacks.

Ecological rules for the assembly of microbiome communities.

Humans and many other hosts establish a diverse community of beneficial microbes anew each generation. The order and identity of incoming symbionts is critical for health, but what determines the success of the assembly process remains poorly understood. Here we develop ecological theory to identify factors important for microbial community assembly. Our method maps out all feasible pathways for the assembly of a given microbiome-with analogies to the mutational maps underlying fitness landscapes in evolutionary biology. Building these "assembly maps" reveals a tradeoff at the heart of the assembly process. Ecological dependencies between members of the microbiota make assembly predictable-and can provide metabolic benefits to the host-but these dependencies may also create barriers to assembly. This effect occurs because interdependent species can fail to establish when each relies on the other to colonize first. We support our predictions with published data from the assembly of the preterm infant microbiota, where we find that ecological dependence is associated with a predictable order of arrival. Our models also suggest that hosts can overcome barriers to assembly via mechanisms that either promote the uptake of multiple symbiont species in one step or feed early colonizers. This predicted importance of host feeding is supported by published data on the impacts of breast milk in the assembly of the human microbiome. We conclude that both microbe to microbe and host to microbe interactions are important for the trajectory of microbiome assembly.

Droplet printing reveals the importance of micron-scale structure for bacterial ecology.

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology. However, a test of this hypothesis requires manipulation at the fine scales at which spatial structure naturally occurs. Here we develop a droplet-based printing method to arrange bacterial genotypes across a sub-millimetre array. We print strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins. Our experiments reveal that toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge where susceptible strains can persist in large numbers. Moving to competitions between toxin producers reveals that spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between competing strains to understand how ecological refuges form, which shows that cells closest to a toxin producer mop up the toxin and protect their clonemates. Our work provides a method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in ecology.