Aminoglycoside‐Enabled Elucidation of Bacterial Persister Metabolism

Mehmet A. Orman1, Wendy W. K. Mok1, Mark P. Brynildsen1

1 Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 17.9
DOI:  10.1002/9780471729259.mc1709s36
Online Posting Date:  February, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Bacterial persisters are cells with an impressive, yet transient, tolerance toward extraordinary concentrations of antibiotics. Persisters are believed to impose a significant burden on the healthcare system because of their role in the proclivity of infections to relapse. During antibiotic challenge, these rare, phenotypic variants enter a dormant state where antibiotic primary targets are rendered inactive, allowing them to survive. Once the antibiotic is removed, persisters reawaken and resume growth, leading to repopulation of the environment. Metabolism plays a pivotal role in coordinating the entry, maintenance, and exit from the persister state. However, the low abundance, transient nature, and similarity of persisters to other cell types have prevented their isolation, which is needed for direct metabolic measurements. In this unit, we describe a technique known as the aminoglycoside (AG) potentiation assay, which can be used to rapidly and specifically measure the breadth of persister metabolism in heterogeneous populations. © 2015 by John Wiley & Sons, Inc.

Keywords: persisters; persister metabolism; aminoglycoside

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Persister Assay
  • Basic Protocol 2: Aminoglycoside Potentiation Assay
  • Basic Protocol 3: Competition Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Persister Assay

  Materials
  • Bacterial strain of interest (e.g., E. coli MG1655, ATCC #700926) from frozen 25% (v/v) glycerol stocks stored at −80°C
  • Bacterial medium appropriate to the strain of interest e.g., Luria‐Bertani (LB) medium (see recipe)
  • Antibiotic, e.g., 5 mg/ml ofloxacin (OFL; see recipe)
  • Phosphate‐buffered saline (PBS; Fisher Bioreagents)
  • Luria‐Bertani (LB) agar plates (see recipe)
  • 17 × 100–mm glass (Pyrex) or polypropylene (Fisher Scientfic) test tubes
  • 37°C incubator, with a rotary shaker
  • 500‐ml baffled flask (Pyrex)
  • Benchtop centrifuge
  • 20‐, 200‐, and 1000‐μl single channel (Gilson) and 5‐ to 50‐μl and 30‐ to 300‐μl multichannel (Bio‐One, Grenier) micropipettors and sterile pipet tips
  • 1.5‐ml microcentrifuge tubes, sterile
  • 96‐well round‐bottom plates (Bio‐One, Grenier)

Basic Protocol 2: Aminoglycoside Potentiation Assay

  Materials
  • 1 ml sample (e.g., E. coli MG1655) where persisters comprise the only remaining culturable cells ( protocol 1)
  • 1.25× M9 minimal salts (see recipe)
  • 1 M potassium cyanide (KCN; see recipe)
  • Luria‐Bertani (LB) agar plates (see recipe)
  • 10× antibiotic solutions (see recipe): aminoglycoside, e.g., 250 μg/ml kanamycin (KAN), and β‐lactam, e.g., 1 mg/ml ampicillin (AMP)
  • 600 mM carbon source stock solutions (see recipe)
  • Phosphate‐buffered saline (PBS; Fisher Bioreagents)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Benchtop centrifuge
  • 20‐, 200‐, and 1000‐μl single channel (Gilson) and 5‐ to 50‐μl and 30‐ to 300‐μl multichannel (Bio‐One, Grenier) micropipettors and sterile pipet tips
  • 96‐well round‐bottom plates
  • Sterile, gas‐permeable sealing membranes (e.g., Breathe‐Easy sealing membranes, Sigma‐Aldrich)

Basic Protocol 3: Competition Assay

  Materials
  • Cultures of persisters of wild‐type (WT; e.g., E. coli MG1655::CMR; created in‐house) and mutant (e.g., E. coli MG1655ΔgldAΔglpK::GENTR; the Keio collection, Baba et al., ) bacterial strains with antibiotic resistance markers ( protocol 1)
  • 10× antibiotic solutions (see recipe): 1 mg/ml amplicillin (AMP); 250 μg/ml kanamycin (KAN)
  • 1.25× M9 minimal salts (see recipe)
  • Luria‐Bertani (LB) agar plates without antibiotics (see recipe) and with antibiotics (see recipe), 25 μg/ml CM to select for WT (E. coli MG1655::CMR) persisters, and 10 μg/ml GENT to select for mutant E. coli MG1655ΔgldAΔglpK::GENTR persisters
  • Benchtop centrifuge
  • 20‐, 200‐, and 1000‐μl single channel (Gilson) and 5‐ to 50‐μl and 30‐ to 300‐μl multichannel (Bio‐One, Grenier) micropipettors and sterile pipet tips
  • Additional reagent and equipment for growing persister cells ( protocol 1) and carrying out the AG potentiation assay ( protocol 2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Allison, K.R. , Brynildsen, M.P. , and Collins, J.J. 2011. Metabolite‐enabled eradication of bacterial persisters by aminoglycosides. Nature 473:216‐220.
   Amato, S.M. , Orman, M.A. , and Brynildsen, M.P. 2013. Metabolic control of persister formation in Escherichia coli . Mol. Cell 50:475‐487.
   Amato, S.M. , Fazen, C.H. , Henry, T.C. , Mok, W.W. , Orman, M.A. , Sandvik, E.L. , Volzing, K.G. , and Brynildsen, M.P. 2014. The role of metabolism in bacterial persistence. Front. Microbiol. 5:70.
   Andrews, J.M. 2001. Determination of minimum inhibitory concentrations. J. Antimicrob. Chemother. 48:5‐16.
   Baba, T. , Ara, T. , Hasegawa, M. , Takai, Y. , Okumura, Y. , Baba, M. , Datsenko, K.A. , Tomita, M. , Wanner, B.L. , and Mori, H. 2006. Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: The Keio collection. Mol. Syst. Biol. 2:2006.0008.
   Balaban, N.Q. , Merrin, J. , Chait, R. , Kowalik, L. , and Leibler, S. 2004. Bacterial persistence as a phenotypic switch. Science 305:1622‐1625.
   Balaban, N.Q. , Gerdes, K. , Lewis, K. , and McKinney, J.D. 2013. A problem of persistence: Still more questions than answers? Nat. Rev. Microbiol. 11:587‐591.
   Bigger, J.W. 1944. The bactericidal action of penicillin on Staphylococcus pyogenes . Irish J. Med. Sci. 19:553‐568.
   Conlon, B.P. , Nakayasu, E.S. , Fleck, L.E. , LaFleur, M.D. , Isabella, V.M. , Coleman, K. , Leonard, S.N. , Smith, R.D. , Adkins, J.N. , and Lewis, K. 2013. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503:365‐370.
   Davis, B.D. 1987. Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev. 51:341‐350.
   Fauvart, M. , De Groote, V.N. , and Michiels, J. 2011. Role of persister cells in chronic infections: Clinical relevance and perspectives on anti‐persister therapies. J. Med. Microbiol. 60:699‐709.
   Iino, R. , Hayama, K. , Amezawa, H. , Sakakihara, S. , Kim, S.H. , Matsumono, Y. , Nishino, K. , Yamaguchi, A. , and Noji, H. 2012. A single‐cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array. Lab Chip 12:3923‐3929.
   Iino, R. , Matsumoto, Y. , Nishino, K. , Yamaguchi, A. , and Noji, H. 2013. Design of a large‐scale femtoliter droplet array for single‐cell analysis of drug‐tolerant and drug‐resistant bacteria. Front. Microbiol. 4:300.
   Keren, I. , Kaldalu, N. , Spoering, A. , Wang, Y. , and Lewis, K. 2004a. Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230:13‐18.
   Keren, I. , Shah, D. , Spoering, A. , Kaldalu, N. , and Lewis, K. 2004b. Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli . J. Bacteriol. 186:8172‐8180.
   Kim, J.S. , Heo, P. , Yang, T.J. , Lee, K.S. , Cho, D.H. , Kim, B.T. , Suh, J.H. , Lim, H.J. , Shin, D. , Kim, S.K. , and Kweon, D.H. 2011. Selective killing of bacterial persisters by a single chemical compound without affecting normal antibiotic‐sensitive cells. Antimicrob. Agents Chemother. 55:5380‐5383.
   Lewis, K. 2007. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5:48‐56.
   Lewis, K. 2010. Persister cells. In Annual Review of Microbiology, Vol. 64, ( S. Gottesman and C.S. Harwood , eds.) pp. 357‐372. Annual Reviews, Palo Alto, Calif.
   Luidalepp, H. , Jõers, A. , Kaldalu, N. , and Tenson, T. 2011. Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J. Bacteriol. 193:3598‐3605.
   Orman, M.A. and Brynildsen, M.P. 2013a. Dormancy is not necessary or sufficient for bacterial persistence. Antimicrob. Agents Chemother. 57:3230‐3239.
   Orman, M.A. and Brynildsen, M.P. 2013b. Establishment of a method to rapidly assay bacterial persister metabolism. Antimicrob. Agents Chemother. 57:4398‐4409.
   Pan, J.C. , Bahar, A.A. , Syed, H. , and Ren, D.C. 2012. Reverting antibiotic tolerance of Pseudomonas aeruginosa PAO1 persister Cells by (Z)‐4‐bromo‐5‐(bromomethylene)‐3‐methylfuran‐2(5H)‐one. Plos One 7:e45778.
   Pan, J.C. , Xie, X. , Tian, W. , Bahar, A.A. , Lin, N. , Song, F.C. , An, J. , and Ren, D.C. 2013. (Z)‐4‐bromo‐5‐(bromomethylene)‐3‐methylfuran‐2(5H)‐one sensitizes Escherichia coli persister cells to antibiotics. Appl. Microbiol. Biotechnol. 97:9145‐9154.
   Roostalu, J. , Jõers, A. , Luidalepp, H. , Kaldalu, N. , and Tenson, T. 2008. Cell division in Escherichia coli cultures monitored at single cell resolution. BMC Microbiol. 8:68.
   Shah, D. , Zhang, Z. , Khodursky, A. , Kaldalu, N. , Kurg, K. , and Lewis, K. 2006. Persisters: A distinct physiological state of E. coli . BMC Microbiol. 6:53.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library