The identification of Pseudomonas aeruginosa persisters using flow cytometry

Abstract

Pseudomonas aeruginosa persisters are a rare and poorly characterized subpopulation of cells that are responsible for many recurrent infections. The lack of knowledge on the mechanisms that lead to persister cell development is mainly a result of the difficulty in isolating and characterizing this rare population. Flow cytometry is an ideal method for identifying such subpopulations because it allows for high-content single-cell analysis. However, there are fewer established protocols for bacterial flow cytometry compared to mammalian cell work. Herein, we describe and propose a flow cytometry protocol to identify and isolate P. aeruginosa persister cells. Additionally, we show that the percentage of potential persister cells increases with increasing antibiotic concentrations above the MIC.

The (p)ppGpp synthetase Rsh promotes rifampicin tolerant persister cell formation in Brucella abortus by regulating the type II toxin-antitoxin module mbcTA

“…The latter are one of the major culprits of chronic infections ( Bigger, 1944 ; Nicolau and Lewis, 2022 ) and are linked to long-term antibiotic treatments that are often required to cure long-lasting infections. Persistence has been observed in diverse species, including P. aeruginosa ( Grandy et al, 2022 ), E. coli ( Chen et al, 2022 ), Salmonella ( Hill et al, 2021 ), M. tuberculosis ( Sarathy and Dartois, 2020 ), and S. aureus ( Vasudevan et al, 2022 ). Several lines of evidence point to partial bacteria generate high persister cells in clinical patients with chronic infectious symptoms ( Mulcahy et al, 2010 ; Bartell et al, 2020 ).…”

Microbial Musings – Winter 2022

“…Finally, they assemble a table of pathogens and clinically relevant antibiotics and argue that these are the combinations that should be used to learn about persisters in more clinical settings. Reflecting on a key methodological step in the study of persisters, namely the isolation of surviving sub-populations, a related Methods papers was also published in this quarter outlining a flow-cytometry protocol to identify Pseudomonas aeruginosa persister cells [11]. The protocol, from Shannon Grandy, Renee Raudonis and Zhenyu Cheng at Dalhousie University, Canada, presents their analysis of the best dyes to use with different strains of P. aeruginosa for the identification and isolation of antibiotic-induced persister cells.…”