TY - CHAP
T1 - Analyzing persister physiology with fluorescence-activated cell sorting
AU - Orman, Mehmet A.
AU - Henry, Theresa C.
AU - Decoste, Christina J.
AU - Brynildsen, Mark P.
N1 - Publisher Copyright:
© Springer Science+Business Media New York 2016.
PY - 2016
Y1 - 2016
N2 - Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.
AB - Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.
KW - Antibiotic
KW - Fluorescence-activated cell sorting (FACS)
KW - Persister
KW - Phenotypic heterogeneity
KW - Redox sensor green (RSG)
KW - Viable but non-culturable cell (VBNC)
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U2 - 10.1007/978-1-4939-2854-5_8
DO - 10.1007/978-1-4939-2854-5_8
M3 - Chapter
C2 - 26468102
AN - SCOPUS:84945207667
T3 - Methods in Molecular Biology
SP - 83
EP - 100
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -