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. 2014:1149:699-707.
doi: 10.1007/978-1-4939-0473-0_54.

Assessing Pseudomonas aeruginosa Persister/antibiotic tolerant cells

Ronen Hazan  1 , Damien Maura, Yok Ai Que, Laurence G Rahme
Affiliations

Assessing Pseudomonas aeruginosa Persister/antibiotic tolerant cells

Ronen Hazan et al. Methods Mol Biol. 2014.

Abstract

Bacterial persistence, which is observed in a broad range of microbial species, is the capacity of a bacterial cell subpopulation called "persisters" to tolerate exposure to normally lethal concentrations of bactericidal antibiotics. This ability, which is not due to antibiotic-resistant mutants, has been implicated in antibiotic treatment failures and may account for latent, chronic, and relapsing infections. Antibiotic tolerant/Persister (AT/P) cells have been notoriously difficult to study due to their low frequency and transient nature. This chapter describes the main methods used to isolate and study Pseudomonas aeruginosa AT/P cells and discusses new technologies that may ease research of P. aeruginosa persisters in the near future.

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Figures

Fig. 1
Fig. 1
Antibiotic tolerant cell assessment using the CFU counts method. The killing curve of P. aeruginosa strain PA14 exponential-phase cells exposed to a lethal concentration of the bactericidal antibiotic meropenem indicates that the majority of cells died quickly, showing a sharp drop-off in survival kinetics within 24 h, while a small fraction of cells (~10–6) survived the treatment even after 48 h of antibiotic exposure. This surviving fraction of cells reflects the number of antibiotic tolerant cells. PA14 cells were exposed to 10 mg/L meropenem for 48 h
Fig. 2
Fig. 2
Antibiotic tolerant cell assessment using the SGT method. Comparative assessment of the persister cell fraction between two strains subjected to a 24 h treatment with meropenem (10 mg/L) at 37 °C (no meropenem added to normalizers). Following a 1:500 dilution, the growth kinetics of normalizers and treated samples were recorded. (a) Using OD600nm=0.15, the ΔSGT values were calculated as the difference between treated and normalizer SGTs. The ΔΔSGT values were calculated as the difference between ΔSGTs of each strain compared to that of the calibrator. (b) For the SGT method, the log2-fold change was calculated as –ΔΔSGT (empty bars). For CFU counting, normalizers and treated cells were serially diluted
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