1887

Microbiology Society logo

Volume 168, Issue 10

The identification of persisters using flow cytometry Open Access

Abstract

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 persister cells. Additionally, we show that the percentage of potential persister cells increases with increasing antibiotic concentrations above the MIC.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotic , flow cytometry , persisters , Pseudomonas aeruginosa and viability dye
Funding
This study was supported by the:
  • Nova Scotia Graduate Student Award
    • Principle Award Recipient: ShannenGrandy
  • Cystic Fibrosis Canada Martha Morton Early Career Investigator Award
    • Principle Award Recipient: ZhenyuCheng
  • Research Nova Scotia, Scotia Scholars Award
    • Principle Award Recipient: ShannenGrandy
© 2022 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001252
2022-10-26
2024-05-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/168/10/mic001252.html?itemId=/content/journal/micro/10.1099/mic.0.001252&mimeType=html&fmt=ahah

References

  1. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 2000; 406:959–964 [View Article] [PubMed]
     [Google Scholar]
  2. Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000; 2:1051–1060 [View Article] [PubMed]
     [Google Scholar]
  3. Davies JC. Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence. Paediatr Respir Rev 2002; 3:128–134 [View Article] [PubMed]
     [Google Scholar]
  4. Sordé R, Pahissa A, Rello J. Management of refractory Pseudomonas aeruginosa infection in cystic fibrosis. Infect Drug Resist 2011; 4:31–41 [View Article] [PubMed]
     [Google Scholar]
  5. Pang Z, Raudonis R, Glick BR, Lin T-J, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019; 37:177–192 [View Article]
     [Google Scholar]
  6. Drenkard E. Antimicrobial resistance of Pseudomonas aeruginosa biofilms. Microbes Infect 2003; 5:1213–1219 [View Article]
     [Google Scholar]
  7. Stewart PS. Mechanisms of antibiotic resistance in bacterial biofilms. Int J Med Microbiol 2002; 292:107–113 [View Article]
     [Google Scholar]
  8. Mulcahy LR, Burns JL, Lory S, Lewis K. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 2010; 192:6191–6199 [View Article] [PubMed]
     [Google Scholar]
  9. Balaban NQ, Helaine S, Lewis K, Ackermann M, Aldridge B et al. Definitions and guidelines for research on antibiotic persistence. Nat Rev Microbiol 2019; 17:441–448 [View Article] [PubMed]
     [Google Scholar]
  10. Mlynarcik P, Kolar M. Starvation- and antibiotics-induced formation of persister cells in Pseudomonas aeruginosa. Biomed Pap 2017; 161:58–67 [View Article]
     [Google Scholar]
  11. Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O et al. Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science 2011; 334:982–986 [View Article]
     [Google Scholar]
  12. Möker N, Dean CR, Tao J. Pseudomonas aeruginosa increases formation of multidrug-tolerant persister cells in response to quorum-sensing signaling molecules. J Bacteriol 2010; 192:1946–1955 [View Article] [PubMed]
     [Google Scholar]
  13. Grassi L, Di Luca M, Maisetta G, Rinaldi AC, Esin S et al. Generation of persister cells of Pseudomonas aeruginosa and Staphylococcus aureus by chemical treatment and evaluation of their susceptibility to membrane-targeting agents. Front Microbiol 2017; 8:8 [View Article]
     [Google Scholar]
  14. Ambriz-Aviña V, Contreras-Garduño JA, Pedraza-Reyes M. Applications of flow cytometry to characterize bacterial physiological responses. Biomed Res Int 2014; 2014:1–14 [View Article]
     [Google Scholar]
  15. Rundberg Nilsson A, Bryder D, Pronk CJH. Frequency determination of rare populations by flow cytometry: a hematopoietic stem cell perspective. Cytometry A 2013; 83:721–727 [View Article]
     [Google Scholar]
  16. Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 2008; 3:163–175 [View Article] [PubMed]
     [Google Scholar]
  17. Knudsen GM, Ng Y, Gram L. Survival of bactericidal antibiotic treatment by a persister subpopulation of Listeria monocytogenes. Appl Environ Microbiol 2013; 79:7390–7397 [View Article] [PubMed]
     [Google Scholar]
  18. Vinckx T, Wei Q, Matthijs S, Cornelis P. The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin. Microbiology 2010; 156:678–686 [View Article]
     [Google Scholar]
  19. McDougald D, Rice SA, Weichart D, Kjelleberg S. Nonculturability: adaptation or debilitation?. FEMS Microbiology Ecology 1991; 25:1–9 [View Article]
     [Google Scholar]
  20. Nader WF, Nebe CT, Nebe G, Dastani A, Birr C. eds Rapid Methods and Automation in Microbiology and Immunology Berlin, Heidelberg: Springer; 1991 [View Article]
     [Google Scholar]
  21. Nyström T. Not quite dead enough: on bacterial life, culturability, senescence, and death. Arch Microbiol 2001; 176:159–164 [View Article] [PubMed]
     [Google Scholar]
  22. Ramamurthy T, Ghosh A, Pazhani GP, Shinoda S. Current perspectives on viable but non-culturable (VBNC) pathogenic bacteria. Front Public Health 2014; 2:103 [View Article]
     [Google Scholar]
  23. Lopez PA, Hulspas R. Special issue on enhancement of reproducibility and rigor. Cytometry A 2020; 97:105–106 [View Article]
     [Google Scholar]
  24. Pfister G, Toor SM, Sasidharan Nair V, Elkord E. An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS?. J Immunol Methods 2020; 487:112902 [View Article]
     [Google Scholar]
  25. Ryan K, Rose RE, Jones DR, Lopez PA. Sheath fluid impacts the depletion of cellular metabolites in cells afflicted by sorting induced cellular stress (SICS). Cytometry A 2021; 99:921–929 [View Article]
     [Google Scholar]
  26. Lebaron P, Catala P, Parthuisot N. Effectiveness of SYTOX Green stain for bacterial viability assessment. Appl Environ Microbiol 1998; 64:2697–2700 [View Article] [PubMed]
     [Google Scholar]
  27. Suller MTE, Lloyd D. Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry. Cytometry 1999; 35:235–241 [View Article]
     [Google Scholar]
  28. Subedi D, Vijay AK, Kohli GS, Rice SA, Willcox M. Comparative genomics of clinical strains of Pseudomonas aeruginosa strains isolated from different geographic sites. Sci Rep 2018; 8:15668 [View Article]
     [Google Scholar]
  29. Weiser R, Green AE, Bull MJ, Cunningham-Oakes E, Jolley KA et al. Not all Pseudomonas aeruginosa are equal: strains from industrial sources possess uniquely large multireplicon genomes. Microb Genom 2019; 5:e00276 [View Article]
     [Google Scholar]
  30. Owlia P, Nosrati R, Alaghehbandan R, Lari AR. Antimicrobial susceptibility differences among mucoid and non-mucoid Pseudomonas aeruginosa isolates. GMS Hyg Infect Control 2014; 9:Doc13 [View Article]
     [Google Scholar]
  31. Dörr T, Vulić M, Lewis K. Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS Biol 2010; 8:e1000317 [View Article]
     [Google Scholar]
  32. Dwyer DJ, Kohanski MA, Collins JJ. Role of reactive oxygen species in antibiotic action and resistance. Curr Opin Microbiol 2009; 12:482–489 [View Article] [PubMed]
     [Google Scholar]
  33. Khakimova M, Ahlgren HG, Harrison JJ, English AM, Nguyen D. The stringent response controls catalases in Pseudomonas aeruginosa and is required for hydrogen peroxide and antibiotic tolerance. J Bacteriol 2013; 195:2011–2020 [View Article] [PubMed]
     [Google Scholar]
  34. Maisonneuve E, Gerdes K. Molecular mechanisms underlying bacterial persisters. Cell 2014; 157:539–548 [View Article] [PubMed]
     [Google Scholar]
  35. Rotem E, Loinger A, Ronin I, Levin-Reisman I, Gabay C et al. Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence. Proc Natl Acad Sci U S A 2010; 107:12541–12546 [View Article] [PubMed]
     [Google Scholar]
  36. Cornforth DM, Dees JL, Ibberson CB, Huse HK, Mathiesen IH et al. Pseudomonas aeruginosa transcriptome during human infection. Proc Natl Acad Sci U S A 2018; 115:E5125–E5134 [View Article] [PubMed]
     [Google Scholar]
  37. Bartell JA, Cameron DR, Mojsoska B, Haagensen JAJ, Pressler T et al. Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment. PLoS Pathog 2020; 16:e1009112 [View Article]
     [Google Scholar]
  38. Hazan R, Maura D, Que YA, Rahme LG. Assessing pseudomonas aeruginosa persister/antibiotic tolerant cells. Methods Mol Biol 2014; 1149:699–707 [View Article]
     [Google Scholar]
  39. Amor KB, Breeuwer P, Verbaarschot P, Rombouts FM, Akkermans ADL et al. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress. Appl Environ Microbiol 2002; 68:5209–5216 [View Article]
     [Google Scholar]
  40. Müller S, Nebe-von-Caron G. Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol Rev 2010; 34:554–587 [View Article] [PubMed]
     [Google Scholar]
  41. Caron GN, Stephens P, Badley RA. Assessment of bacterial viability status by flow cytometry and single cell sorting. J Appl Microbiol 1998; 84:988–998 [View Article] [PubMed]
     [Google Scholar]
  42. Waring MJ. Complex formation between ethidium bromide and nucleic acids. J Mol Biol 1965; 13:269–282 [View Article] [PubMed]
     [Google Scholar]
  43. Kubista M, Akerman B, Nordén B. Characterization of interaction between DNA and 4’,6-diamidino-2-phenylindole by optical spectroscopy. Biochemistry 1987; 26:4545–4553 [View Article] [PubMed]
     [Google Scholar]
  44. Wolfgang MC, Kulasekara BR, Liang X, Boyd D, Wu K et al. Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2003; 100:8484–8489 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001252
Loading
The identification of Pseudomonas aeruginosa persisters using flow cytometry
Microbiology 168, 001252 (2022); https://doi.org/10.1099/mic.0.001252
/content/journal/micro/10.1099/mic.0.001252
/content/journal/micro/10.1099/mic.0.001252
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error