How Spatial Structure Shapes Microbial Communities
Humans gravitate toward places where they will survive and be successful based on factors like job prospects, resource availability and proximity to family and friends. But people aren't the only ones whose livelihood is closely intertwined with spatial organization—microbes also develop complex spatial structures depending on nutrient availability, oxygen demands and pressure from external threats. Researchers are using emerging technologies to understand microbial spatial structure and probe deeper into the complex mechanisms that determine how microbes organize. In doing so, they hope to gain new insights into how spatial organization impacts and is impacted by factors such as virulence and antimicrobial resistance.
Developing Structure
Bacteria proliferate all around us—in aqueous environments such as oceans or creeks where they attach to small particles of organic matter, and on surfaces such as teeth. As small populations expand into larger ones, bacteria can cluster into aggregates, or clumps of cells that form in liquid environments or slough off from biofilms, potentially seeding new biofilms. These aggregates and biofilms may consist of a single species, or they may be composed of multiple genetically distinct members and different species. In contrast to planktonic growth in a well-mixed system, such as a culture tube in a shaker, these aggregates take on a physical 3-dimensional structure, and within that structure, members may be spatially divided due to cellular and environmental factors.
Nutrients Shape Microbial Organization
Every environment in which microbes are found provides a different set of nutrients to its microscopic denizens. Those nutrients can play a big role in the shape and makeup of the microbial aggregates and biofilms that form. Even single-species systems have been observed to develop spatial structure. Clonal populations of single species may develop specialized subpopulations that produce different enzymes and engage in distinct metabolic processes. Nutrient availability may also impact the overall size and shape of microbial communities.
In computational models, when E. coli colonies grown on a surface become dense, cells in the inner portion of the colony may be subject to decreased oxygen and glucose availability compared to the cells on the surface of the colony, resulting in variations in metabolism and growth dynamics between the different locations. Early during colony formation, when most cells have equal access to oxygen and glucose, the colony expands outward in all directions to form a hemisphere. As growth progresses, cells at the top of the colony lose access to the glucose on the plate, and their growth rates are predicted to slow. Over time, the cells at the edges of the colony in contact with the plate continue to grow, while upward expansion halts, resulting in a wide, flat appearance that aligns with laboratory observations.
Priority Effect Influences the Structure of Multi-Species Communities
Interactions become more complicated in multi-species systems. As these communities are established, who gets to the location of colonization first is an important factor in determining the eventual layout of the microbial community, a phenomenon known as priority effect.
For example, leaf surfaces are periodically dry and nutrient-poor, making them inhospitable environments for bacterial colonization. However, researchers found that bacteria can and do form colonies on leaf surfaces, and these colonies are often comprised of multiple species. Notably, the identity of the resident species—those that had arrived at and colonized the leaf first—impacted the growth success of immigrant species. Some resident species provided permissive environments in which new species could successfully establish themselves. Other residents competed with new species for nutrients and thus did not provide a beneficial environment in which the immigrant bacteria could grow. These priority effects can determine which microbes are capable of survival and proliferation in multi-species systems, laying the foundation for more complex interactions.
In the laboratory, researchers determined that if species arrive at the same time, the initial positioning of different members can determine the future spatial organization of the colony. At first, different cells may exist as small seed populations that arrived at their locations because of random distribution. In a permissive environment (i.e. one with sufficient nutrients and no antibiotics), these seed populations grow and expand into the space that is available to them. The organization of that community is largely determined by where the seed populations started out in relation to each other. As the community is established, factors such as growth rates, cell size and shape, the extracellular matrix and motility play a role in determining how microbes are organized.
As a group of cells grows in number, it may make room for new members by physically pushing aside neighboring cells, resulting in changes to the physical conformation of the larger colony. Faster-growing species may also expand into nutrient-dense regions before slower-growing species can get there, staking their claim on that space. Cells with high motility may move in an undirected manner, increasing heterogeneity of the community by moving around and mixing together, or in a directed manner that allows cells to migrate to locations with higher nutrient density, creating cellular clusters near necessary nutrients. Even a cell's shape can dictate where it will end up in a biofilm, with some research showing that cells may cluster in different regions of a biofilm depending on their morphology. In one study, mixing long and short cells together resulted in the formation of layered structures, with short cells located on top of longer ones.
The Role of Metabolites
Furthermore, the production and diffusion of metabolites can also influence where different species will be able to grow by generating more or less hospitable microenvironments. As an example of a nutrient-based interaction that occurs in marine species, Colwellia spp. synthesize a ligand that allows Roseovarius spp. to produce B12, a nutrient both species are independently unable to produce and must obtain from external sources. For this interaction to occur, these species must be close to one another.
While cooperative communities can exist, relationships between microbes can also be destructive. In the absence of necessary metabolites, some species may kill their neighbors and use the products of lysis for nourishment. These interactions occur in close proximity and can cause the lysed population size to shrink while others grow, capitalizing on the nutrients released during lysis. In the case mentioned above, when Roseovarius spp. and Colwellia spp. are grown in co-culture, B12 is released upon prophage induction and lysis of Roseovarius spp., allowing Colwellia to access the B12.
Understanding the Link Between Form and Function
As colonies of bacteria grow, it is important to ensure that growth isn’t going to cause detrimental effects, like crowding or nutrient limitation. One well-known mechanism by which bacteria communicate about how crowded the local space is getting is through quorum sensing. Bacteria use concentrations of quorum sensing molecules (autoinducers) to sense how crowded the environment is and regulate their gene expression accordingly. These dynamics come together when bacteria sense nearby competition or increasing population density and begin expressing genes that produce toxins and toxin delivery systems, such as type 6 secretion systems (T6SS). These toxins and T6SS can kill neighboring cells, shaping colonies’ spatial structure by removing some members from the community.
In addition to knowing when to go on the offensive, spatial structure can influence bacterial protection as well. While antibiotic resistance emerges at the individual level, larger social dynamics also play a role in the phenomenon. In a mixed population of antibiotic resistant and sensitive Enterococcus faecalis, spatial structure between the 2 subpopulations varied based on the starting ratios of each, as well as antibiotic concentration. When the ratio of starting subpopulations was equal, or antibiotic concentrations were low, the community appeared to be well-mixed. However, at higher antibiotic concentrations, or lower ratios of resistant:sensitive subpopulations, large clusters of resistant E. faecalis would appear, interspersed with smaller clusters of the sensitive subpopulation. The interactions between the populations were a mix of competition and protection, wherein the resistant population conferred protection to the sensitive population.
In a more complex case, competing species Pseudomonas aeruginosa and Klebsiella pneumoniae were grown independently and then together in the presence of cefotaxime, a β-lactam antibiotic. Alone, both species suffered reduced mobility and expansion on an agar plate. Together, however, the co-culture expanded beyond the range of each individual species. Upon further inspection, it was determined that motile P. aeruginosa dominated and spread, while immotile K. pneumoniae was relegated to the area in which it had started and became the minority species. K. pneumoniae degraded the antibiotic and thus facilitated the growth of P. aeruginosa by providing an antibiotic-free zone in which it could expand.
If tightly controlled laboratory experiments focused on 1 or 2 species can display this level of complexity, then the multi-species communities that are abundant in nature will pose an even greater challenge to untangle. These interactions become even more complicated in dynamic environments, such as a host’s gut. In order to study the multitudinous mechanisms that dictate how different bacteria cluster together and organize in space, researchers are turning to a slate of emerging and existing technologies.
Looking Closer
Microbial communities dictate the health of many systems. Understanding the spatial structure and related function of these communities may yield insights into how to promote or prevent certain interactions. To that end, both bench-based and computational analysis methods are helping uncover the mechanisms underlying microbial spatial organization.
It is often helpful to observe the structure of a microbial population while it is forming, as the early stages dictate what later stages of organization will look like. Microfluidics, in which microbes are grown in a small chamber through which liquid can be flowed under precise control, is a useful tool to observe biofilm formation. The effects of mechanical factors, such as shear stress, as well as molecular factors, like medium composition and the presence of antibiotics, can be investigated by altering the flow and fluid composition in these small devices.
Once structures like biofilms have been formed, microscopy can probe the spatial organization within. Using fluorescently tagged proteins, researchers can get a glimpse of how microbes are organized in space, and even how that organization changes over time.
In a recent example, researchers combined light sheet fluorescence microscopy and microfluidics to observe the dynamics of biofilm formation by S. aureus and P. aeruginosa when grown alone or together. In isolation, S. aureus formed a thicker biofilm layer than P. aeruginosa after 20 hours of growth. When cultured together at equal ratios, S. aureus proliferated faster initially, but by hour 20, its biofilms began to degrade as P. aeruginosa took over. After 40 hours of growth, the resultant mixed-species biofilm was thin and predominantly composed of P. aeruginosa. The microfluidics system allowed researchers to observe that S. aureus grown in medium conditioned during S. aureus and P. aeruginosa co-culture did not proliferate, even after the conditioned medium was replaced with fresh medium, indicating long-term inhibition by P. aeruginosa metabolites.
Mass spectrometry imaging, such as a technique called Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI), can be used to obtain molecular data directly from structured colonies like biofilms without a reliance on fluorescent reporters. The surfaces of biofilms are “scanned” with a laser beam pixel-by-pixel, and their molecular composition is determined based on the mass spectra generated. These molecular data can be combined with pixel coordinates, and even overlaid with optical images, to determine per-cell spectra and elucidate metabolic profiles of individual cells within the biofilm. While the resolution obtained by these techniques can be as small as 1-2 micrometers, they are largely limited to obtaining information about the surfaces of biofilms without probing deeper.
These methods can be strengthened using computational techniques, such as enhanced microscopic image analysis or multi-omic integration of modalities like MALDI-MSI. Mathematical modeling and simulations can also be useful to predict interactions in systems that may not be easy to probe using other tools.
Moving Forward
As methods advance, researchers are exploring combinations of techniques—such as using microfluidics in tandem with microscopy, or mathematical modeling to bolster experimental methods—to enhance their ability to form conclusions about the formation, growth and makeup of multicellular microbial communities. In doing so, they can gain increasingly advanced information on forces that shape, and are shaped by, microbial spatial structure, allowing for new and exciting insights into these microscopic communities.
Biofilms can impact antimicrobial efficacy, as well as the immune response, contributing to antimicrobial resistance and allowing the establishment of persistent or chronic infections. How can we combat them?
Author: Megan Hockman, Ph.D.
In This Issue
- Why Does Basic Science Research Matter?
- The Value of Curiosity-Driven Research: Mechanism Discovery With Glen McGugan
- Stuck on You: How Bacteria Migrate and Adhere to Their Hosts
- Cable Bacteria: Electric Marvels of the Microbial World
- Can't Sleep? Your Microbiome May Play a Role
- How Spatial Structure Shapes Microbial Communities
- Plasmids and the Spread of Antibiotic Resistance Genes
- From Mpox to Measles: Why Viruses Cause Lesions and Rashes
- Bringing Microbial Dark Matter Into the Light