High-resolution lineage tracing of biofilm evolution
Abstract
Biofilms are a major form of microbial life in which multiple metabolically diverse bacterial species form densely populated communities. Limited diffusion within biofilms leads to the formation of a spatially structured, heterogeneous environment that constitutes a collection of different ecological niches. Unlike the homogeneous, well-mixed environment of planktonic cultures that, eventually, erases microbial diversity, the multi-niche biofilm environment sets a stage for evolution of the inter-species interactions between multiple microbial species inhabiting the biofilm. These emerging social interactions not only shape fitness of the individual species, but, crucially, also determine the unique properties of the entire microbial biofilm community, including its biodiversity, productivity, long-term persistence, and resistance to antimicrobial compounds. Recent experimental evolutionary studies have identified multiple mutations responsible for the facilitation of the inter-species interactions. However, despite the great importance of these studies, we still lack the full understanding of biofilm evolution. This is because evolution is a dynamic process, and uncovering the evolutionary dynamics of the emergence of inter-species interactions is as important to predicting the outcome of biofilm evolution as uncovering the mutations themselves. Indeed, population size, structure and composition, rates at which each mutation enters the population, and ever-changing mean population fitness, all conspire to determine the fate of an individual mutation entering a biofilm community. Due to the large population sizes of bacterial species inhabiting biofilms (~10^7Ð10^14), numerous low-frequency small-effect mutations tend to invade and spread simultaneously and drive the evolutionary dynamics already at the initial stages of biofilm evolution. Thus, capturing the dynamics and genetic complexity underlying the evolution of social interactions in biofilms requires the ability to follow evolutionary trajectories of numerous individual lineages, most of which occur at extremely low frequency (less than 10^-6), and do so in parallel for multiple species and over multiple generations. Since well-established whole genome sequencing techniques fall short of fulfilling these requirements, here we propose to overcome this problem by i) developing a high-resolution chromosomal barcoding of the individual cells comprising the mixed-species microbial biofilm community and subjecting it to laboratory evolution in the presence of antimicrobial compounds; and ii) developing a CRISPR/Cas-based barcode-guided system to isolate individual lineages of interest with high precision and efficiency along the evolutionary trajectories. This approach will allow us to reconstruct the evolutionary dynamics underlying the emergence of social interactions in biofilms exposed to environmental stresses in exquisite detail and to identify and characterize the mutations that shape the evolutionary trajectories of unique individual lineages. Specifically, we plan to barcode reproducible mixed-species biofilms comprised of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumonia, and follow their evolutionary dynamics upon exposure to Chemical Agent Resistant Coating (CARC) antimicrobial compounds. The proposed research will advance our understanding of the evolutionary and ecological mechanisms that shape the emergence of multi-species interactions within spatially heterogeneous microbial communities. In particular, it will illuminate the fundamental principles that drive the formation, proliferation, sustenance and robustness of microbial biofilms, will explain and predict bacterial adaptation under environmental stresses, and will help to devise successful strategies to combat biofilm-associated infections.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 25, 2021
- Source ID
- W911NF2110200
Entities
People
- Shimon Bershtein
Organizations
- Army Contracting Command
- Ben-Gurion University of the Negev
- United States Army