- Tytuł:
- Droplet microfluidics for high-throughput analysis of antibiotic susceptibility in bacterial cells and populations
- Autorzy:
-
Garstecki, Piotr
Postek, Witold - Wydawca:
- American Chemical Society
- Cytata wydawnicza:
- Acc. Chem. Res. 2022, 55, 5, 605–615 Publication Date:February 4, 2022 https://doi.org/10.1021/acs.accounts.1c00729
- Opis:
-
Antibiotic-resistant bacteria are an increasing concern both in everyday life and specialized environments such as healthcare. As the rate of antibiotic-resistant infections rises, so do complications to health and the risk of disability and death. Urgent action is required regarding the discovery of new antibiotics and rapid diagnosis of the resistance profile of an infectious pathogen as well as a better understanding of population and single-cell distribution of the resistance level. High-throughput screening is the major affordance of droplet microfluidics. Droplet screens can be exploited both to look for combinations of drugs that could stop an infection of multidrug-resistant bacteria and to search for the source of resistance via directed-evolution experiments or the analysis of various responses to a drug by genetically identical bacteria. In droplet techniques that have been used in this way for over a decade, aqueous droplets containing antibiotics and bacteria are manipulated both within and outside of the microfluidic devices. The diagnostics problem was approached by producing a series of microfluidic systems with integrated dilution modules for automated preparation of antibiotic concentration gradients, achieving the speed that allowed for high-throughput combinatorial assays. We developed a method for automated emulsification of a series of samples that facilitated measuring the resistance levels of thousands of individual cells encapsulated in droplets and quantifying the inoculum effect, the dependence of resistance level on bacterial cell count. Screening of single cells encapsulated in droplets with varying antibiotic contents has revealed a distribution of resistance levels within populations of clonally identical cells. To be able to screen bacteria from clinical samples, a study of fluorescent dyes in droplets determined that a derivative of a popular viability marker is more suitable for droplet assays. We have developed a detection system that analyzes the growth or death state of bacteria with antibiotics for thousands of droplets per second by measuring the scattering of light hitting the droplets without labeling the cells or droplets. The droplet-based microchemostats enabled long-term evolution of resistance experiments, which will be integrated with high-throughput single-cell assays to better understand the mechanism of resistance acquisition and loss. These techniques underlie automated combinatorial screens of antibiotic resistance in single cells from clinical samples. We hope that this Account will inspire new droplet-based research on the antibiotic susceptibility of bacteria.
Foundation for Polish Science (POIR.04.04.00-00-16ED/18-00, START 069.2021 scholarship), NAWA Polskie Powroty, grant no. PPN/PPO/2019/1/00030/U/0001. National Science Centre, Poland, (2018/30/A/ST4/00036, Maestro 10) - Dostawca treści:
- Repozytorium Centrum Otwartej Nauki
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