Methods for Enrichment of Bacterial Persister Populations for Phenotypic Screens and Genomic Studies

Transient phenotypic adaptations in bacteria that enable survival at bactericidal antibiotic concentrations give rise to bacterial persistence. Naturally, the abundance of persister cells is very low (about 1 in 10⁵ cells) in actively growing bacterial populations. Therefore, in order to study bacterial persistence mechanisms for therapeutics development, persister cells need to be enriched from a larger culture. Here, we describe three enrichment methods for obtaining Burkholderia thailandensis persisters: (1) flow sorting for persisters from exponentially dividing cultures by fluorescent staining of bacterial cells with a translational membrane depolarization-specific DiBAC₄(3) dye, (2) antibiotic lysis of nonpersisters, and (3) culture aging to induce persister survival. We also describe herein the lysis of persister cells obtained by all three methods for downstream bacterial RNA extraction and transcriptomics analysis.