Unraveling the Microbial Arms Race: Exploring Novel Resistance Dynamics in 3D Porous Media Between K-12 MG1655-GFP E. coli and T4 Bacteriophages

Abstract

E. coli, and T4 Bacteriophages (the viruses which infect them), co-exist ubiquitously in nature, and as a result, are in a constant flux of co-evolution, in which each population must adapt resistant mechanism to perpetuate. Bacteria morphodynamics as a result of phage predation are often studied in bulk, homogenously mixed liquid media, or on 2D surfaces, such as petri dishes. However, these typical in vitro methods of study do not accurately recapitulate the heterogeneously structured, and often confining, three dimensional environments in vivo in which E. coli and T4 Bacteriophages co-inhabit, such as living in living tissue, soils, and sediments, and visualization of bacteria-phage dynamics directly in vivo is difficult due to the opacity of the tissue and soils they inhabit. This collectively implies the need to establish in vitro models which better mimic the physical 3D structure of their in vivo counterparts. Prior work in the Datta Lab has utilized extrusion bioprinting of E. coli using Carbopol-based granular hydrogels to recapitulate such 3D environments, and have found that E. coli morphodynamics differs considerably than in traditional in vitro models, in that the cells of the colony are trapped within the pore spaces of the granular hydrogel, and furthermore that cells in the core of the colony are nutrient replete due to shielding from the outer cells. Subsequently, I hypothesized that using a similar in vitro model to observe and quantify E. coli morphodynamics due to phage predation would likewise deviate from current knowledge on bacteria-phage dynamics from experimentation carried out in well-mixed and 2D environments. Furthermore, taking into consideration the gaps in the field of bacteria- phage dynamics, in which it has yet to be clearly elucidated how initial phage concentration impacts timing and fluctuations of bacteria colony growth, decline, and maintenance, and that these dynamics are often not measured along a continuous time scale, the goal of my thesis was to (1) develop a novel protocol for observing bacteria- phage dynamics in 3D porous media and spatially structuring the E. coli in the hydrogel through extrusion bioprinting, and directly measuring areas of colony proliferation and death using timelapse confocal microscopy imaging, and (2) using this protocol to determine how differing initial phage concentrations impacts the patterns of growth, death, as well as possible instances of phage-resistant bacteria emerging. Three distinct phenotypes of growth, death and possible resistance emergence were found, based on initial phage concentration in which the highest initial concentration showed decline of the colony until ~24 hours, in which the population recovered thereafter, but at the same time, cell death continued to accumulate beyond 24 hours, while for the middle phage concentration, there was a general maintenance of the quantity of cellular growth and death, while the lowest phage concentration demonstrated slowed, but continuous growth of the colony throughout the experiment. In the proposed model to describe these unique behaviors, I suggest that the dynamics of phage predation are dependent on initial phage concentration, the proliferative state of the cell in which the phage is infecting, which is mediated by nutrient gradients, and furthermore position within the 3D colony. Future work aims to confirm the emergence of phage-resistant bacteria through genetic sequencing and imaging at single cell resolution, to more robustly characterize these dynamics for implications in improving the safety profile and efficacy of phage therapies and bioremediation in agriculture.