Top-down and bottom-up cohesiveness in microbial community coalescence.

Microbial communities frequently invade one another as a whole, a phenomenon known as community coalescence. Despite its potential importance for the assembly, dynamics, and stability of microbial consortia, as well as its prospective utility for microbiome engineering, our understanding of the processes that govern it is still very limited. Theory has suggested that microbial communities may exhibit cohesiveness in the face of invasions emerging from collective metabolic interactions across microbes and their environment. This cohesiveness may lead to correlated invasional outcomes, where the fate of a given taxon is determined by that of other members of its community-a hypothesis known as ecological coselection. Here, we have performed over 100 invasion and coalescence experiments with microbial communities of various origins assembled in two different synthetic environments. We show that the dominant members of the primary communities can recruit their rarer partners during coalescence (top-down coselection) and also be recruited by them (bottom-up coselection). With the aid of a consumer-resource model, we found that the emergence of top-down or bottom-up cohesiveness is modulated by the structure of the underlying cross-feeding networks that sustain the coalesced communities. The model also predicts that these two forms of ecological coselection cannot co-occur under our conditions, and we have experimentally confirmed that one can be strong only when the other is weak. Our results provide direct evidence that collective invasions can be expected to produce ecological coselection as a result of cross-feeding interactions at the community level.

Ureolytic Biomineralization Reduces Proteus mirabilis Biofilm Susceptibility to Ciprofloxacin.

Ureolytic biomineralization induced by urease-producing bacteria, particularly Proteus mirabilis, is responsible for the formation of urinary tract calculi and the encrustation of indwelling urinary catheters. Such microbial biofilms are challenging to eradicate and contribute to the persistence of catheter-associated urinary tract infections, but the mechanisms responsible for this recalcitrance remain obscure. In this study, we characterized the susceptibility of wild-type (ure+) and urease-negative (ure-) P. mirabilis biofilms to killing by ciprofloxacin. Ure+ biofilms produced fine biomineral precipitates that were homogeneously distributed within the biofilm biomass in artificial urine, while ure- biofilms did not produce biomineral deposits under identical growth conditions. Following exposure to ciprofloxacin, ure+ biofilms showed greater survival (less killing) than ure- biofilms, indicating that biomineralization protected biofilm-resident cells against the antimicrobial. To evaluate the mechanism responsible for this recalcitrance, we observed and quantified the transport of Cy5-conjugated ciprofloxacin into the biofilm by video confocal microscopy. These observations revealed that the reduced susceptibility of ure+ biofilms resulted from hindered delivery of ciprofloxacin into biomineralized regions of the biofilm. Further, biomineralization enhanced retention of viable cells on the surface following antimicrobial exposure. These findings together show that ureolytic biomineralization induced by P. mirabilis metabolism strongly regulates antimicrobial susceptibility by reducing internal solute transport and increasing biofilm stability.

Swimming Motility Reduces Deposition to Silica Surfaces.

The transport and fate of bacteria in porous media is influenced by physicochemical and biological properties. This study investigated the effect of swimming motility on the attachment of cells to silica surfaces through comprehensive analysis of cell deposition in model porous media. Distinct motilities were quantified for different strains using global and cluster-based statistical analyses of microscopic images taken under no-flow condition. The wild-type, flagellated strain DJ showed strong swimming as a result of the actively swimming subpopulation whose average speed was 25.6 µm/s; the impaired swimming of strain DJ77 was attributed to the lower average speed of 17.4 µm/s in its actively swimming subpopulation; and both the nonflagellated JZ52 and chemically treated DJ cells were nonmotile. The approach and deposition of these bacterial cells were analyzed in porous media setups, including single-collector radial stagnation point flow cells (RSPF) and two-dimensional multiple-collector micromodels under well-defined hydrodynamic conditions. In RSPF experiments, both swimming and nonmotile cells moved with the flow when at a distance ≥20 µm above the collector surface. Closer to the surface, DJ cells showed both horizontal and vertical movement, limiting their contact with the surface, while chemically treated DJ cells moved with the flow to reach the surface. These results explain how wild-type swimming reduces attachment. In agreement, the deposition in micromodels was also lowest for DJ compared with those for DJ77 and JZ52. Wild-type swimming specifically reduced deposition on the upstream surfaces of the micromodel collectors. Conducted under environmentally relevant hydrodynamic conditions, the results suggest that swimming motility is an important characteristic for bacterial deposition and transport in the environment.

Flagella-mediated differences in deposition dynamics for Azotobacter vinelandii in porous media.

A multiscale approach was designed to study the effects of flagella on deposition dynamics of Azotobacter vinelandii in porous media, independent of motility. In a radial stagnation point flow cell (RSPF), the deposition rate of a flagellated strain with limited motility, DJ77, was higher than that of a nonflagellated (Fla(-)) strain on quartz. In contrast, Fla(-) strain deposition exceeded that of DJ77 in two-dimensional silicon microfluidic models (micromodels) and in columns packed with glass beads. Both micromodel and column experiments showed decreasing deposition over time, suggesting that approaching cells were blocked from deposition by previously deposited cells. Modeling results showed that blocking became effective for DJ77 strain at lower ionic strengths (1 mM and 10 mM), while for the Fla(-) strain, blocking was similar at all ionic strengths. In late stages of micromodel experiments, ripening effects were also observed, and these appeared earlier for the Fla(-) strain. In RSPF and column experiments, deposition of the flagellated strain was influenced by ionic strength, while ionic strength dependence was not observed for the Fla(-) strain. The observations in all three setups suggested flagella affect deposition dynamics and, in particular, result in greater sensitivity to ionic strength.

Metabolic similarity and the predictability of microbial community assembly.

When microbial communities form, their composition is shaped by selective pressures imposed by the environment. Can we predict which communities will assemble under different environmental conditions? Here, we hypothesize that quantitative similarities in metabolic traits across metabolically similar environments lead to predictable similarities in community composition. To that end, we measured the growth rate and by-product profile of a library of proteobacterial strains in a large number of single nutrient environments. We found that growth rates and secretion profiles were positively correlated across environments when the supplied substrate was metabolically similar. By analyzing hundreds of in-vitro communities experimentally assembled in an array of different synthetic environments, we then show that metabolically similar substrates select for taxonomically similar communities. These findings lead us to propose and then validate a comparative approach for quantitatively predicting the effects of novel substrates on the composition of complex microbial consortia.

Functional attractors in microbial community assembly.

For microbiome biology to become a more predictive science, we must identify which descriptive features of microbial communities are reproducible and predictable, which are not, and why. We address this question by experimentally studying parallelism and convergence in microbial community assembly in replicate glucose-limited habitats. Here, we show that the previously observed family-level convergence in these habitats reflects a reproducible metabolic organization, where the ratio of the dominant metabolic groups can be explained from a simple resource-partitioning model. In turn, taxonomic divergence among replicate communities arises from multistability in population dynamics. Multistability can also lead to alternative functional states in closed ecosystems but not in metacommunities. Our findings empirically illustrate how the evolutionary conservation of quantitative metabolic traits, multistability, and the inherent stochasticity of population dynamics, may all conspire to generate the patterns of reproducibility and variability at different levels of organization that are commonplace in microbial community assembly.

The meta-gut: community coalescence of animal gut and environmental microbiomes.

All animals carry specialized microbiomes, and their gut microbiota are continuously released into the environment through excretion of waste. Here we propose the meta-gut as a novel conceptual framework that addresses the ability of the gut microbiome released from an animal to function outside the host and alter biogeochemical processes mediated by microbes. We demonstrate this dynamic in the hippopotamus (hippo) and the pools they inhabit. We used natural field gradients and experimental approaches to examine fecal and pool water microbial communities and aquatic biogeochemistry across a range of hippo inputs. Sequencing using 16S RNA methods revealed community coalescence between hippo gut microbiomes and the active microbial communities in hippo pools that received high inputs of hippo feces. The shared microbiome between the hippo gut and the waters into which they excrete constitutes a meta-gut system that could influence the biogeochemistry of recipient ecosystems and provide a reservoir of gut microbiomes that could influence other hosts. We propose that meta-gut dynamics may also occur where other animal species congregate in high densities, particularly in aquatic environments.

Adsorption of extracellular chromosomal DNA and its effects on natural transformation of Azotobacter vinelandii.

To better understand the influence of environmental conditions on the adsorption of extracellular chromosomal DNA and its availability for natural transformation, the amount and conformation of adsorbed DNA were monitored under different conditions in parallel with transformation assays using the soil bacterium Azotobacter vinelandii. DNA adsorption was monitored using the technique of quartz crystal microbalance with dissipation (QCM-D). Both silica and natural organic matter (NOM) surfaces were evaluated in solutions containing either 100 mM NaCl or 1 mM CaCl(2). The QCM-D data suggest that DNA adsorbed to silica surfaces has a more compact and rigid conformation in Ca(2+) solution than in Na(+) solution and that the reverse is true when DNA is adsorbed to NOM surfaces. While the amounts of DNA adsorbed on a silica surface were similar for Ca(2+) and Na(+) solutions, the amount of DNA adsorbed on an NOM-coated surface was higher in Ca(2+) solution than in Na(+) solution. Transformation frequencies for dissolved DNA and DNA adsorbed to silica and to NOM were 6 x 10(-5), 5 x 10(-5), and 2.5 x 10(-4), respectively. For NOM-coated surfaces, transformation frequencies from individual experiments were 2- to 50-fold higher in the presence of Ca(2+) than in the presence of Na(+). The results suggest that groundwater hardness (i.e., Ca(2+) concentration) will affect the amount of extracellular DNA adsorbed to the soil surface but that neither adsorption nor changes in the conformation of the adsorbed DNA will have a strong effect on the frequency of natural transformation of A. vinelandii.

Evaluation of bacterial run and tumble motility parameters through trajectory analysis.

In this paper, a method for extraction of the behavior parameters of bacterial migration based on the run and tumble conceptual model is described. The methodology is applied to the microscopic images representing the motile movement of flagellated Azotobacter vinelandii. The bacterial cells are considered to change direction during both runs and tumbles as is evident from the movement trajectories. An unsupervised cluster analysis was performed to fractionate each bacterial trajectory into run and tumble segments, and then the distribution of parameters for each mode were extracted by fitting mathematical distributions best representing the data. A Gaussian copula was used to model the autocorrelation in swimming velocity. For both run and tumble modes, Gamma distribution was found to fit the marginal velocity best, and Logistic distribution was found to represent better the deviation angle than other distributions considered. For the transition rate distribution, log-logistic distribution and log-normal distribution, respectively, was found to do a better job than the traditionally agreed exponential distribution. A model was then developed to mimic the motility behavior of bacteria at the presence of flow. The model was applied to evaluate its ability to describe observed patterns of bacterial deposition on surfaces in a micro-model experiment with an approach velocity of 200 µm/s. It was found that the model can qualitatively reproduce the attachment results of the micro-model setting.

Emergent simplicity in microbial community assembly.

A major unresolved question in microbiome research is whether the complex taxonomic architectures observed in surveys of natural communities can be explained and predicted by fundamental, quantitative principles. Bridging theory and experiment is hampered by the multiplicity of ecological processes that simultaneously affect community assembly in natural ecosystems. We addressed this challenge by monitoring the assembly of hundreds of soil- and plant-derived microbiomes in well-controlled minimal synthetic media. Both the community-level function and the coarse-grained taxonomy of the resulting communities are highly predictable and governed by nutrient availability, despite substantial species variability. By generalizing classical ecological models to include widespread nonspecific cross-feeding, we show that these features are all emergent properties of the assembly of large microbial communities, explaining their ubiquity in natural microbiomes.

Microbial diversity in an intensively managed landscape is structured by landscape connectivity.

Intensively managed land increases the rate of nutrient and particle transport within a basin, but the impact of these changes on microbial community assembly patterns at the basin scale is not yet understood. The objective of this study was to investigate how landscape connectivity and dispersal impacts microbial diversity in an agricultural-dominated watershed. We characterized soil, sediment and water microbial communities along the Upper Sangamon River basin in Illinois-a 3600 km2 watershed strongly influenced by human activity, especially landscape modification and extensive fertilization for agriculture. We employed statistical and network analyses to reveal the microbial community structure and interactions in the critical zone (water, soil and sediment media). Using a Bayesian source tracking approach, we predicted microbial community connectivity within and between the environments. We identified strong connectivity within environments (up to 85.4 ± 13.3% of sequences in downstream water samples sourced from upstream samples, and 44.7 ± 26.6% in soil and sediment samples), but negligible connectivity across environments, which indicates that microbial dispersal was successful within but not between environments. Species sorting based on sample media type and environmental parameters was the dominant driver of community dissimilarity. Finally, we constructed operational taxonomic unit association networks for each environment and identified a number of co-occurrence relationships that were shared between habitats, suggesting that these are likely to be ecologically significant.

Biomineralization strongly modulates the formation of Proteus mirabilis and Pseudomonas aeruginosa dual-species biofilms.

Proteus mirabilis and Pseudomonas aeruginosa are common pathogens that often form biofilms together in catheter-associated urinary tract infections (CAUTI). However, the interactions between these two species in biofilms are largely unknown. P. mirabilis induces ureolytic biomineralization that substantially modifies key biofilm properties including morphology, persistence, and recalcitrance to antimicrobial therapy. These processes are well known to complicate CAUTI, but the consequences for colonization and persistence of P. mirabilis in polymicrobial biofilms have not been explored. Here we characterized the role of biomineralization in regulating the development of P. mirabilis and P. aeruginosa dual-species biofilms. Time-series observations revealed that the dominance of P. mirabilis was synchronized with mineral formation in the biofilm. After 24 hours of development, the dual-species biofilm was dominated by P. mirabilis, and the distribution of P. mirabilis biomass was strongly correlated with the mineral fraction of the biofilm. Conversely, dual-species growth without biomineralization yielded strikingly different patterns in the biofilm, with P. aeruginosa dominating the biofilm biomass. These results show that biomineralization is responsible for the increased success of P. mirabilis in the polymicrobial biofilm. Since biofilm biomineralization commonly occurs in diverse clinical, natural and engineered systems, these findings imply that biomineralization could broadly influence the microbial ecology of multispecies biofilms.

Methods for characterizing the co-development of biofilm and habitat heterogeneity.

Biofilms are surface-attached microbial communities that have complex structures and produce significant spatial heterogeneities. Biofilm development is strongly regulated by the surrounding flow and nutritional environment. Biofilm growth also increases the heterogeneity of the local microenvironment by generating complex flow fields and solute transport patterns. To investigate the development of heterogeneity in biofilms and interactions between biofilms and their local micro-habitat, we grew mono-species biofilms of Pseudomonas aeruginosa and dual-species biofilms of P. aeruginosa and Escherichia coli under nutritional gradients in a microfluidic flow cell. We provide detailed protocols for creating nutrient gradients within the flow cell and for growing and visualizing biofilm development under these conditions. We also present protocols for a series of optical methods to quantify spatial patterns in biofilm structure, flow distributions over biofilms, and mass transport around and within biofilm colonies. These methods support comprehensive investigations of the co-development of biofilm and habitat heterogeneity.

Bayesian process-identification in bacteria transport in porous media.

A Bayesian parameter estimation approach is developed for the estimation of joint probability distribution functions for colloid and bacterial fate and transport model parameters describing breakthrough curves (BTCs) obtained through porous media column studies, and is applied to data involving different ionic strength solutions to fit models of differing complexity. Our approach focuses on the simultaneous fitting of a number of BTCs representing different conditions, and it provides a measure of the goodness of model structure, namely Deviance Information Criteria (DIC). Comparison of DIC per model fit enables the evaluation of the significance of various processes through step-wise increases in complexity due to the addition of process model components. We use the method to investigate the transport of both flagellated and non-flagellated strains of Azotobacter vinelandii in a simulated porous media under three ionic strengths. Three different model structures are considered: one without a detachment process and with Langmuirian blocking function, one with detachment, and one with detachment and a second-order blocking function based on random sequential adsorption. First, the model was applied separately to each single BTC. Next, the model was applied comprehensively to the experiments under various ionic strengths, whereas some transport parameters including dispersivity, detachment coefficient, the fraction of cells undergoing irreversible attachment, and the coefficient of the second-order blocking term were assumed to be the same under different ionic strengths. In most cases, including detachment substantially improved the DIC as expected, whereas using the second-order blocking improved DIC for most of the cases when the method was applied to separate BTCs but not when the method was applied collectively to the three BTCs obtained under various ionic strengths. Also, comparing the outcomes of the separate applications of the parameter estimation algorithm versus the collective application indicates that the uncertainty associated with the estimated parameters is substantially smaller when the collective approach is used and also that the estimated parameters are more consistent with the expectations based on the underlying physical processes.

Interactions between dissolved natural organic matter and adsorbed DNA and their effect on natural transformation of Azotobacter vinelandii.

To better understand gene transfer in the soil environment, the interactions between dissolved natural organic matter (NOM) and chromosomal or plasmid DNA adsorbed to silica surfaces were investigated. The rates of NOM adsorption onto silica surfaces coated with DNA were measured by quartz crystal microbalance (QCM) and showed a positive correlation with carboxylate group density for both soil and aquatic NOM in solutions containing either 1mM Ca(2+) or Mg(2+). Increasing total dissolved organic carbon (DOC) concentrations of the NOM solution also resulted in an increase in the adsorption rates, likely due to divalent cation complexation with NOM carboxylate groups and the phosphate backbones of the DNA. The results from Fourier transform infrared spectroscopy (FTIR) for dissolved DNA and DNA adsorbed on silica beads also suggest that adsorption may result from divalent cation complexation with the DNA's phosphate backbone. The interactions, between DNA and NOM, however, did not influence natural transformation of Azotobacter vinelandii by DNA. These results suggest that DNA adsorbed to NOM-coated silica or otherwise complexed with NOM remains available for natural transformation in the environment.