Quantitative dielectrophoretic tracking for characterization and separation of persistent subpopulations of
Yi-Hsuan Su1, Mikiyas Tsegaye, Walter Varhue
1Department of Electrical & Computer Engineering, University of Virginia, Charlottesville, VA 22904, USA. nswami@virginia.edu.
Abstract:
Microbial persistence to antibiotics is attributed to subpopulations with phenotypic variations that cause a spread of susceptibility levels, leading to the recurrence of infections and stability of biofilms. Herein, persistent oocyst subpopulations identified by animal infectivity and excystation assays during the disinfection of Cryptosporidium parvum, a water-borne pathogen capable of causing enteric infections at ultra-low doses, are separated and characterized by quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz). To enable the simultaneous and facile dielectrophoretic tracking of individual oocysts, insulator constrictions in a microfluidic channel are utilized to spatially modulate the localized field over the extent needed for defining oocyst trajectories and for obtaining high-resolution displacement versus time measurements under both, positive and negative dielectrophoresis. In this manner, by obviating the need for averaging dielectrophoretic data over a large collection region, the force response is more sensitive to differences in electrophysiology from sub-population fractions. Hence, the electrophysiology of sensitive and persistent oocysts after heat and silver nanoparticle treatments can be quantified by correlating the force response at low frequencies (<100 kHz) to the integrity of the oocyst wall and at high frequencies (0.4-1 MHz) to the sporozoites in the oocyst. This label-free method can characterize heterogeneous microbial samples with subpopulations of phenotypically different alterations, for quantifying the intensity of alteration and fraction with a particular alteration type.
Insights
Researchers developed a novel dielectrophoretic method to distinguish antibiotic-resistant microbial subpopulations. This technique precisely tracks individual Cryptosporidium parvum oocysts, aiding in understanding infection recurrence and biofilm stability.
Area of Science:
- Microbiology
- Biophysics
- Environmental Science
Background:
- Microbial antibiotic persistence stems from phenotypic variations within subpopulations, leading to treatment failure and recurrent infections.
- Cryptosporidium parvum, a water-borne pathogen, poses significant health risks due to its ability to cause enteric infections even at low doses.
- Understanding and characterizing persistent microbial subpopulations is crucial for developing effective disinfection and treatment strategies.
Purpose of the Study:
- To develop and validate a label-free dielectrophoretic method for separating and characterizing phenotypically distinct microbial subpopulations.
- To investigate the electrophysiological properties of persistent versus sensitive Cryptosporidium parvum oocysts.
- To quantify alterations in microbial subpopulations after specific treatments.
Main Methods:
- Utilized microfluidic channels with insulator constrictions for simultaneous, high-resolution dielectrophoretic tracking of individual oocysts.
- Applied quantitative dielectrophoretic tracking over a wide frequency range (10 kHz-10 MHz) to analyze oocyst trajectories and force responses.
- Correlated low-frequency responses to oocyst wall integrity and high-frequency responses to sporozoite status.
Main Results:
- Successfully separated and characterized persistent and sensitive Cryptosporidium parvum oocyst subpopulations based on their dielectrophoretic behavior.
- Demonstrated that dielectrophoretic force response is sensitive to electrophysiological differences between subpopulations, obviating the need for averaging.
- Quantified the electrophysiological changes in oocysts after heat and silver nanoparticle treatments.
Conclusions:
- The developed label-free dielectrophoretic method offers a sensitive approach to characterize heterogeneous microbial samples.
- This technique enables the quantification of phenotypic alterations and the fraction of subpopulations exhibiting specific changes.
- The findings contribute to a better understanding of microbial persistence and the development of targeted disinfection strategies for water-borne pathogens.
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