Session S37: Microbial Communities III

Collective behaviors of bacteria in flow: Divergent roles for transport and microcolony morphology

Fluid flows are dominant features of many bacterial environments, yet interactions between fluid flow, surface association, and colonization factors remain largely underexplored. One specific example is infective endocarditis, in which bacteria preferentially colonize heart valves, areas with the greatest flow rates. The mechanisms underlying this important yet paradoxical behavior are unknown. We know bacteria interact on different length scales, from single cell gene expression to collective biofilm communities and introducing flow will allow us to further explore the emergent behaviors. We developed a microfluidic system to study flow effects on colonization of two endocarditis-inducing pathogens: Staphylococcus aureus MRSA and Enterococcus faecalis. We observe bacteria growth from single cells to microcolonies in two different flow rates: high flow is 10x larger than low flow. Both species demonstrate a counter-intuitive larger colonization in high flow compared to low flow. Our experimental and computational studies elucidate two different strategies leading to the preferential adherence in high flow: S. aureus is driven by signaling molecule transport while E. faecalis chains experience a mechanical response under flow. Our divergent mechanisms introduce an intriguing area of further study for bacteria in flow environments, particularly focusing on differnt colony morphologies (e.g. clusters, chains) and how those morphologies may provide advantages (or disadvantages) in complex environments.   

Mechanical properties of Staphylococcus aureus and Pseudomonas aeruginosa dual-species biofilms grown in chronic wound based models

Biofilms are frequently found in oral, respiratory, and chronic wound infections caused by various diseases. These biofilms are typically polymicrobial, and collaborative interactions among bacteria are linked to their persistence. One common pair of species that form polymicrobial biofilms in chronic wounds are Staphylococcus aureus and Pseudomonas aeruginosa. Multiple studies have demonstrated how these species synergize to enhance colonization and chronicity, defying effective treatments.

This study builds on previous work, investigating how wound bed environment aids in the formation of polymicrobial biofilms, affects their mechanical stability, and ultimately impacts debridement. We hypothesize that collagen, a primary component in wound extracellular matrix, mediates between S. aureus and P. aeruginosa allowing formation of more robust polymicrobial biofilms. Using microrheology and colony counting, we have observed that biofilms grown in cultures that include collagen and secondary wound proteins form polymicrobial biofilms when both bacteria are initially present in culture. Furthermore, these biofilms are more elastic and stiffer in comparison to single species biofilms grown in identical conditions. However, if collagen is absent or collagen is present without a secondary protein, a single species biofilm will form from an initial co‑culture.  These results indicate that the host environment plays an important role in mediating the formation of polymicrobial biofilms.   

Biophysical constraints on vertical biofilm growth

The development of surface-attached bacterial biofilms is one of the most common modes of bacterial growth in natural environments and experimental settings. While extensive research has sought to understand the radial growth of biofilms, fundamental questions remain about the vertical growth dynamics of biofilms. In a recent study [1], it was observed that colony height saturates on long time scales for a wide range of microorganisms. It was also observed that the vertical growth rate decreased as a slow linear function of biofilm height; surprisingly, nutrient depletion in the environment is not the cause of this slowing, and eventual cessation, of vertical growth. We have thus been performing experiments to determine why vertical growth slows. Using interferometry and confocal imaging techniques, we investigate how different factors such as cell death and viscous relaxation affect biofilm heights. Inspired by recent work [2], we have been investigating the role of starvation within the biofilm, i.e., the behavior of cells too far from the agar surface for nutrients to reach them. Our ongoing investigations aim to unravel the interplay of different factors influencing biofilm growth. By shedding light on the mechanisms governing the vertical expansion and stability of these bacterial colonies, we hope to contribute to a deeper understanding of microbial communities in various environmental contexts and pave the way for potential applications in fields such as biotechnology and medicine.

[1] P. Bravo, et al., PNAS (2023)

[2] H.Kannan, P.Sun, et al., bioRxiv, doi.org/10.1101/2023.08.27.554977 (2023)   

Liquid channels within B. subtilis biofilms allow the escape of trapped clones and population rescue

Bacterial communities undergoing spatial expansions, typically exhibit a loss of genetic diversity due to gene surfing, a phenomenon by which only cells close to the expanding edge have a chance of contributing to the next generation. As a result, mutants that emerge behind the front remain trapped in the bulk of the population, even if they carry a significant growth advantage. While these findings are applicable to smooth, uniformly dense colonies, where cell spatial re-arrangement is limited, it is unclear whether they hold in bacterial communities with complex three-dimensional morphologies, such as wrinkly biofilms.

We employ an experimental procedure that enables a rapid switch of the chemical environment with minimal physical manipulation of the community to investigate the fate of trapped clones carrying a selective advantage in wrinkly B. subtilis biofilms. We find that these clones are able to use the channels underneath the wrinkles to traverse long distances and take over the expansion front. Moreover, we find that active motility is required to accomplish this task. Collectively, our findings reveal an intriguing novel role of wrinkles in enabling potentially advantageous mutants to propel themselves towards the leading edge of the expansion and rescue the population upon environmental change. Our results also point at motility, which has traditionally received little attention in communities on hard substrates, as a key mechanism for population rescue in biofilms with complex morphologies   

Effective bacterial aggregation is achieved by partial disassembly

Biofilm engineering is a promising field with many environmental, medical, industrial, and scientific applications, which requires understanding and control of biofilm properties. Here we study a system of suspended bacteria expressing blue-light-sensitive adhesive proteins on their membranes. Experiments show that the aggregation of the bacteria is more efficient and yields bigger clusters under pulsed-light-illumination than when they are exposed to constant blue light. To understand how this occurs, we used an individual-based-model of the bacteria as spherocylindrical particles with switchable adhesion. We chose the parameters of the adhesive forces by fitting to earlier experiments. With the decay rate of the adhesive force as the only fit parameter, we can quantitatively recreate the experimental results. We thus find that the dynamics of the aggregation are controlled by the interplay between the time scale of cluster disassembly and the length of the periods of darkness in between pulses of light.   

Physics of phenotypic segregation in a developing biofilm

Biofilm is an important bacterial lifestyle in which individual bacterial cells form surface-associated aggregates embedded in a polymeric matrix they secrete. The regulation of biofilm formation involves cell-cell communication that enables cells to make synchronous decisions. However, phenotypic heterogeneity can play a significant role in biofilm development. Using fluorescent biosensors and reporters, we reveal that a growing Vibrio cholerae biofilm exhibits high levels of heterogeneity in the intracellular cyclic diguanylate (c-di-GMP) concentration and expression of biofilm matrix genes. The intracellular c-di-GMP concentration correlates with biofilm matrix production, and cells with different c-di-GMP levels spatially segregate during biofilm development. We show that such spatial segregation arises from physical interactions associated with matrix production and is affected by the presence or absence of specific matrix components. Combining high-temporal-resolution tracking and single-cell gene expression, we reveal the physical principles behind phenotypic segregation in a developing biofilm and its implications on fitness.   

Determining biophysical mechanisms of phenotypic segregation in bacterial biofilms with agent-based models

Bacteria often exist as surface-adhered communities called biofilms that are embedded within extracellular matrices of polymeric substances. Experimental efforts to dissect the molecular processes underlying the biofilm life cycle have uncovered many signaling pathways in various species, but we lack a comprehensive understanding of how these pathways dictate the biofilm’s architecture. Here, we discuss the consequences of spatiotemporal heterogeneity in intracellular signaling on the architecture of biofilms formed by the model species Vibrio cholerae. Recent work has shown that growing V. cholerae biofilms exhibit cell-to-cell bimodality in the intracellular signaling of the key second messenger cyclic diguanylate (c-di-GMP), which governs the transition between planktonic and sessile phenotypes by regulating matrix component production. Moreover, cells with different c-di-GMP levels spatially segregate during biofilm growth, as cells with low c-di-GMP are pushed towards the periphery and give rise to a concentration of cells with high c-di-GMP in the biofilm’s core. Using agent-based models that integrate c-di-GMP signaling and its effects on cellular physiology with the mechanics of biofilm growth, we reveal the biophysical mechanisms underlying this spatial segregation. We discuss the broader consequences of cell-to-cell heterogeneity on biofilm architecture and the utility of agent-based modeling as a tool for investigating emergent properties of microbial communities.