Identification of Evolutionary Trajectories Associated with Antimicrobial Resistance Using Microfluidics

Abstract

In vitro experimental evolution of pathogens to antibiotics is commonly used for the identification of clinical biomarkers associated with antibiotic resistance. Microdroplet emulsions allow exquisite control of spatial structure, species complexity, and selection microenvironments for such studies. We investigated the use of monodisperse microdroplets in experimental evolution. Using Escherichia coli adaptation to doxycycline, we examined how changes in environmental conditions such as droplet size, starting lambda value, selection strength, and incubation method affected evolutionary outcomes. We also examined the extent to which emulsions could reveal potentially new evolutionary trajectories and dynamics associated with antimicrobial resistance. Interestingly, we identified both expected and unexpected evolutionary trajectories including large-scale chromosomal rearrangements and amplification that were not observed in suspension culture methods. As microdroplet emulsions are well-suited for automation and provide exceptional control of conditions, they can provide a high-throughput approach for biomarker identification as well as preclinical evaluation of lead compounds.

Microfluidic platform for spatially segregated experimental evolution studies with E. coli

“…One representative population of doxycycline-resistant E. coli evolved within microdroplets with the normal increase in selection strength is presented below in Table 1 . Specifically, evolved E. coli from whole-community samples have changes consistent with increased expression of genes, such as codA, cynR, acrR, tolB, marC, marR, setB, gabD, pstB, pstA, and rbsB and were consistent with earlier biomarker discovery and clinical observations ( Aly et al., 2012 ; Dunai et al., 2019 ; Seo et al., 2021 ; Toprak et al., 2012 ; Zhang et al., 2013 ). All doxycycline resistant E. coli in the population had adaptive mutations within marR while 22.2% carried an additional mutation in acrR .…”

“…Recently, microfluidic techniques have been applied as a strategy to provide well-controlled user-designed environments for the study of cellular consortia, synthetic biology and now, experimental evolution ( Alnahhas et al., 2019 ; Bachmann et al., 2013 ; Gupta et al., 2020 ; Hsu et al., 2019 ; Lawson et al., 2019 ; McDonald, 2019 ; van Tatenhove-Pel et al., 2021 ). Microdroplets-based approaches have the potential to provide a much broader range of experimental innovation that extends well beyond traditional batch serial transfer and bioreactor-based approaches ( Seo et al., 2021 ). Importantly, microdroplet encapsulation of cells allows the investigator to impose spatial structure into their experiments.…”

“…We also describe protocols for the propagation and serial passage of microbial populations across a range of selection schemes and potential spatial structures. For complete details on the use and execution of this protocol, please refer to Seo et al. (2021) .…”

Methyl Halide Transferase-Based Gas Reporters for Quantification of Filamentous Bacteria in Microdroplet Emulsions

“…S. venezuelae is a filamentous bacterium and its mature vegetative mycelium fragments are typically 0.9 to 1.8 μm in diameter and up to 150 μm in length 45 . To reduce the blocking of channels in microfluidic devices by mycelia, we used a droplet generator with 60 μm wide main channels and a 30 μm wide orifice at the flow-focusing point 6 . Firstly, multiple combinations of aqueous and oil phase flow rates were tested and the generation of emulsion microdroplets was observed (Fig 3A).…”

“…Application of microfluidic techniques in microbiology and environmental biology is a rapidly emerging field with substantial potential to provide new insights 1 2 . Recent studies have used microdroplet emulsions as ultrahigh-throughput approaches to experimental evolution of enzymes 3 4 and microbes 5 6 7 . Although the traditional serial transfer approach in flasks and bioreactors has been proven to be a powerful tool 8 9 for studies such as multicellularity 10 , antibiotic resistance 11 12 , and social behaviors 13 , it suffers from several significant limitations.…”