Abstract
Persister cells are difficult to study owing to their transient nature and their usually small number in bacterial populations. In the past, numerous attempts have been made to elucidate persistence mechanisms. However, because of the challenges involved in studying persisters and the clear redundancy in mechanisms underlying their generation, our knowledge of molecular pathways to persistence remains incomplete. Here, we describe how to use experimental evolution with cyclic antibiotic treatments to generate mutants with an increased persister level in stationary phase, ranging from the initial ancestral level up to 100 %. This method will help to unravel molecular pathways to persistence, and opens up a myriad of new possibilities in persister research, such as the convenient study of nearly pure persister cultures and the possibility to investigate the role of time and environmental aspects in the evolution of persistence.
*Authors contributed equally.
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References
Cohen NR, Lobritz MA, Collins JJ (2013) Microbial persistence and the road to drug resistance. Cell Host Microbe 13:632–642
Balaban NQ, Merrin J, Chait R et al (2004) Bacterial persistence as a phenotypic switch. Science 305:1622–1625
Lewis K (2010) Persister cells. Annu Rev Microbiol 64:357–372
Fauvart M, De Groote VN, Michiels J (2011) Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J Med Microbiol 60:699–709
Manuel J, Zhanel GG, de Kievit T (2010) Cadaverine suppresses persistence to carboxypenicillins in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 54:5173–5179
Li Y, Zhang Y (2007) PhoU is a persistence switch involved in persister formation and tolerance to multiple antibiotics and stresses in Escherichia coli. Antimicrob Agents Chemother 51:2092–2099
Girgis HS, Harris K, Tavazoie S (2012) Large mutational target size for rapid emergence of bacterial persistence. Proc Natl Acad Sci U S A 109:12740–12745
De Groote VN, Verstraeten N, Fauvart M et al (2009) Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening. FEMS Microbiol Lett 297:73–79
Leung V, Levesque CM, Lévesque CM (2012) A stress-inducible quorum-sensing peptide mediates the formation of persister cells with noninherited multidrug tolerance. J Bacteriol 194:2265–2274
Hu Y, Coates ARM (2005) Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli. FEMS Microbiol Lett 243:117–124
Dhar N, McKinney JD (2010) Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice. Proc Natl Acad Sci USA 107:12275–12280
Ma C, Sim S, Shi W et al (2010) Energy production genes sucB and ubiF are involved in persister survival and tolerance to multiple antibiotics and stresses in Escherichia coli. FEMS Microbiol Lett 303:33–40
Hansen S, Lewis K, Vulić M (2008) Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother 52:2718–2726
Spoering AL, Vulic M, Lewis K et al (2006) GlpD and PlsB participate in persister cell formation in Escherichia coli. J Bacteriol 188:5136–5144
Germain E, Castro-Roa D, Zenkin N et al (2013) Molecular mechanism of bacterial persistence by HipA. Mol Cell 52:248–254
Shah D, Zhang Z, Khodursky A et al (2006) Persisters: a distinct physiological state of E. coli. BMC Microbiol 6:53
Keren I, Minami S, Rubin E et al (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2:e00100–e00111
Keren I, Shah D, Spoering A et al (2004) Specialized persister cells and the mechanism of multidrug tolerance in Escherichia coli. J Bacteriol 186:8172–8180
Van Acker H, Sass A, Bazzini S et al (2013) Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species. PLoS One 8:e58943
Kint CI, Verstraeten N, Fauvart M et al (2012) New-found fundamentals of bacterial persistence. Trends Microbiol 20:577–585
Amato SM, Fazen CH, Henry TC et al (2014) The role of metabolism in bacterial persistence. Front Microbiol 5:70
Balaban NQ, Gerdes K, Lewis K et al (2013) A problem of persistence: still more questions than answers? Nat Rev Microbiol 11:587–591
Blaby IK, Lyons BJ, Wroclawska-Hughes E et al (2012) Experimental evolution of a facultative thermophile from a mesophilic ancestor. Appl Environ Microbiol 78:144–155
Barrick JE, Yu DS, Yoon SH et al (2009) Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature 461:1243–1247
Lafleur MD, Qi Q, Lewis K (2010) Patients with long-term oral carriage harbor high-persister mutants of Candida albicans. Antimicrob Agents Chemother 54:39–44
Mulcahy LR, Burns JL, Lory S et al (2010) Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J Bacteriol 192:6191–6199
Fridman O, Goldberg A, Ronin I et al (2014) Optimization of lag time underlies antibiotic tolerance in evolved bacterial populations. Nature 513:418–421
Yu J, Xiao J, Ren X et al (2006) Probing gene expression in live cells, one protein molecule at a time. Science 311:1600–1603
Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48(Suppl 1):5–16
Wiegand I, Hilpert K, Hancock REW (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–175
Liebens V, Defraine V, Van der Leyden A et al (2014) A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance in Pseudomonas aeruginosa. Pathog Dis 71:39–54
Drlica K (2003) The mutant selection window and antimicrobial resistance. J Antimicrob Chemother 52:11–17
Barrick JE, Lenski RE (2013) Genome dynamics during experimental evolution. Nat Rev Genet 14:827–839
Acknowledgements
The authors are fellows of the Research Foundation—Flanders (FWO) and the Agency for Innovation by Science and Technology (IWT). The research was further supported by grants from the KU Leuven Research Council (PF/10/010; IDO/09/010) and the IAP-BELSPO initiative.
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Van den Bergh, B., Michiels, J.E., Michiels, J. (2016). Experimental Evolution of Escherichia coli Persister Levels Using Cyclic Antibiotic Treatments. In: Michiels, J., Fauvart, M. (eds) Bacterial Persistence. Methods in Molecular Biology, vol 1333. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2854-5_12
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DOI: https://doi.org/10.1007/978-1-4939-2854-5_12
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