Dielectrophoresis-based purification of antibiotic-treated bacterial subpopulations.

Persistence of bacteria during antibiotic therapy is a widespread phenomenon, of particular importance in refractory mycobacterial infections such as leprosy and tuberculosis. Persistence is characterized by the phenotypic tolerance of a subpopulation of bacterial cells to antibiotics. Characterization of these "persister" cells is often difficult due to the transient, non-heritable nature of the phenotype and due to the presence of contaminating material from non-persisting cells, which usually comprise the larger fraction. In this study, we use 3D carbon-electrode arrays for dielectrophoresis-based separation of intact cells from damaged cells, revealed by differential staining with propidium iodide, and we use this procedure to purify intact cells from cultures of Mycobacterium smegmatis treated with isoniazid, a frontline anti-tuberculosis drug. The method presented in this study could be used for rapid label-free enrichment of intact persister cells from antibiotic-treated cultures while preserving the metastable persister phenotype. This approach would facilitate the downstream analysis of low-frequency subpopulations of cells using conventional omics techniques, such as transcriptomic and proteomic analysis.

[1]  G. Markx,et al.  Construction by dielectrophoresis of microbial aggregates for the study of bacterial cell dormancy. , 2010, Biomicrofluidics.

[2]  R. Pethig.  Review article-dielectrophoresis: status of the theory, technology, and applications. , 2010, Biomicrofluidics.

[3]  Frederick F Becker,et al.  Microsample preparation by dielectrophoresis: isolation of malaria. , 2002, Lab on a chip.

[4]  J. Mckinney,et al.  A single-cell perspective on non-growing but metabolically active (NGMA) bacteria. , 2013, Current topics in microbiology and immunology.

[5]  Jan Michiels,et al.  Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. , 2011, Journal of medical microbiology.

[6]  Michael P Hughes,et al.  Dielectrophoretic assay of bacterial resistance to antibiotics. , 2003, Physics in medicine and biology.

[7]  Stanislas Leibler,et al.  Dynamic Persistence of Antibiotic-Stressed Mycobacteria , 2013, Science.

[8]  Rodrigo Martinez-Duarte,et al.  Microfabrication technologies in dielectrophoresis applications—A review , 2012, Electrophoresis.

[9]  Magnus Willander,et al.  Trapping single molecules by dielectrophoresis. , 2005, Physical review letters.

[10]  Richard Welford.  E. H. A. , 1900 .

[11]  Liming Ying,et al.  Surface conductivity of biological macromolecules measured by nanopipette dielectrophoresis. , 2007, Physical review letters.

[12]  T. Jones,et al.  Basic theory of dielectrophoresis and electrorotation , 2003, IEEE Engineering in Medicine and Biology Magazine.

[13]  J. Mckinney,et al.  Microbial phenotypic heterogeneity and antibiotic tolerance. , 2007, Current opinion in microbiology.

[14]  J. Crain,et al.  Dielectrophoretic manipulation of ribosomal RNA. , 2011, Biomicrofluidics.

[15]  D. Mitchison.  Prevention of drug resistance by combined drug treatment of tuberculosis. , 2012, Handbook of experimental pharmacology.

[16]  Gerard H. Markx,et al.  Dielectric measurement of cell death , 2008 .

[17]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[18]  A. Errachid,et al.  Dielectrophoresis as a tool to characterize and differentiate isogenic mutants of Escherichia coli. , 2006, Biophysical journal.

[19]  I. Peitz,et al.  Single-cell bacteria growth monitoring by automated DEP-facilitated image analysis. , 2010, Lab on a chip.

[20]  P. Renaud,et al.  Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis. , 2013, Biosensors & bioelectronics.

[21]  Junya Suehiro,et al.  Selective detection of viable bacteria using dielectrophoretic impedance measurement method , 2003 .

[22]  Zachary Gagnon,et al.  Aligning fast alternating current electroosmotic flow fields and characteristic frequencies with dielectrophoretic traps to achieve rapid bacteria detection , 2005, Electrophoresis.

[23]  Gerard H Markx,et al.  Separation by dielectrophoresis of dormant and nondormant bacterial cells of Mycobacterium smegmatis. , 2010, Biomicrofluidics.

[24]  M. Rito‐Palomares,et al.  Protein manipulation with insulator-based dielectrophoresis and direct current electric fields. , 2008, Journal of chromatography. A.

[25]  Hannah Stower.  Molecular evolution: Adaptation by loss of function , 2013, Nature Reviews Genetics.

[26]  J. Mckinney,et al.  Malachite Green Interferes with Postantibiotic Recovery of Mycobacteria , 2012, Antimicrobial Agents and Chemotherapy.

[27]  J. Bigger.  TREATMENT OF STAPHYLOCOCCAL INFECTIONS WITH PENICILLIN BY INTERMITTENT STERILISATION , 1944 .

[28]  Rashid Bashir,et al.  Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes , 2008, Journal of biological engineering.

[29]  Catherine Vilchèze,et al.  The mechanism of isoniazid killing: clarity through the scope of genetics. , 2007, Annual review of microbiology.

[30]  Hsien-Chang Chang,et al.  Screening of antibiotic susceptibility to β-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis. , 2012, Analytical chemistry.

[31]  Junya Suehiro,et al.  Quantitative estimation of biological cell concentration suspended in aqueous medium by using dielectrophoretic impedance measurement method , 1999 .

[32]  Chia-Fu Chou,et al.  Electrodeless dielectrophoresis of single- and double-stranded DNA. , 2002, Biophysical journal.

[33]  Marc J Madou,et al.  A novel approach to dielectrophoresis using carbon electrodes , 2011, Electrophoresis.

[34]  Michael P Hughes,et al.  Dielectrophoresis-activated multiwell plate for label-free high-throughput drug assessment. , 2008, Analytical chemistry.

[35]  K. Takayama,et al.  Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis , 1972, Antimicrobial Agents and Chemotherapy.

[36]  A. Heck,et al.  Next-generation proteomics: towards an integrative view of proteome dynamics , 2012, Nature Reviews Genetics.

[37]  K. Takayama,et al.  Relationship Between the Uptake of Isoniazid and Its Action on In Vivo Mycolic Acid Synthesis in Mycobacterium tuberculosis , 1972, Antimicrobial Agents and Chemotherapy.

[38]  Hywel Morgan,et al.  Measuring the dielectric properties of herpes simplex virus type 1 virions with dielectrophoresis. , 2002, Biochimica et biophysica acta.

[39]  Antonio Juárez,et al.  On‐line separation of bacterial cells by carbon‐electrode dielectrophoresis , 2010, Electrophoresis.

[40]  Vojo Deretic,et al.  Mechanisms of action of isoniazid , 2006, Molecular microbiology.

[41]  Alexandra Ros,et al.  Dielectrophoresis of lambda‐DNA using 3D carbon electrodes , 2013, Electrophoresis.

[42]  R. Peters,et al.  British anti-lewisite; its use and therapeutic value in arsenical intoxications. , 1947, Lancet.

[43]  Ebru Özgür,et al.  Dielectrophoresis: Applications and future outlook in point of care , 2013, Electrophoresis.

[44]  B. Kirby,et al.  Automated dielectrophoretic characterization of Mycobacterium smegmatis. , 2011, Analytical chemistry.