Abstract
Antibiotic resistance is a major challenge for modern medicine, and there is a dire need to refresh the antibiotic development pipeline to treat infections that are resistant to currently available drugs. Peptide-based antimicrobials represent a promising source of novel anti-infectives, but their development is severely impeded due to the lack of suitable techniques to accurately quantify their antimicrobial efficacy. A major problem involves the heterogeneity of cellular phenotypes in response to these peptides, even within a clonal population of bacteria. There is thus a need to develop single-cell resolution assays to quantify drug efficacy for these novel therapeutics. We present here a detailed microfluidics-microscopy protocol for testing the efficacy of peptide-based antimicrobials on hundreds to thousands of individual bacteria in well-defined microenvironments. This enables the study of cell-to-cell differences in drug response within a clonal population. It is a highly versatile tool, which can be used to quantify drug efficacy, including the number of individual survivors at defined drug doses; it even enables the potential exploration of the molecular mechanisms of action of the drug, which are often unknown in the early stages of drug development. We present here protocols for working with Escherichia coli, but organisms of different geometric shapes and sizes may also be tested with suitable modifications of the microfluidic device.
Key words
- Peptide-based antimicrobials
- Microfluidics
- Single-cell analysis
- Phenotypic heterogeneity
- Persisters
- Viable-but-nonculturable cells
- Antibiotic susceptibility
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References
Andrews JM (2001) Determination of minimum inhibitory concentrations. J Antimicrob Chemother 48:5–16
Taylor PC, Schoenknecht FD, Sherris JC, Linner EC (1983) Determination of minimum bactericidal concentrations of oxacillin for Staphylococcus aureus: influence and significance of technical factors. Antimicrob Agents Chemother 23:142–150
Mouton JW, Meletiadis J, Voss A, Turnidge J (2018) Variation of MIC measurements: the contribution of strain and laboratory variability to measurement precision. J Antimicrob Chemother 73:2374–2379
Mouton JW, Muller AE, Canton R et al (2018) MIC-based dose adjustment: facts and fables. J Antimicrob Chemother 73:564–568
Kavanagh A, Ramu S, Gong Y et al (2019) Effects of microplate type and broth additives on microdilution MIC susceptibility assays. Antimicrob Agents Chemother 63:e01760–e01718
Jepson AK, Schwarz-Linek J, Ryan L et al (2016) What is the ‘minimum inhibitory concentration’ (MIC) of pexiganan acting on Escherichia coli?—a cautionary case study. In: Leake M (ed) Biophysics of infection. Advances in experimental medicine and biology, vol 915. Springer, Cham
Snoussi M, Talledo JP, Del Rosario N-A et al (2018) Heterogeneous absorption of antimicrobial peptide LL37 in Escherichia coli cells enhances population survivability. eLife 7:e38174
O’Neill J (2016) Tackling drug-resistant infections globally: final report and recommendations, The Review on Antimicrobial Resistance
Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol 6:194
Hancock REW, Nijnik A, Philpott DJ (2012) Modulating immunity as a therapy for bacterial infections. Nat Rev Microbiol 10:243–254
Hancock REW (1997) Peptide antibiotics. Lancet 349:418–422
Grönberg A, Mahlapuu M, Ståhle M et al (2014) Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers: a randomized, placebo-controlled clinical trial. Wound Repair Regen 22:613–621
Nguyen LT, Haney EF, Vogel HJ (2011) The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol 29:464–472
Ackermann M (2015) A functional perspective on phenotypic heterogeneity in microorganisms. Nat Rev Microbiol 13:497–508
Balaban NQ, Gerdes K, Lewis K, McKinney JD (2013) A problem of persistence: still more questions than answers? Nat Rev Microbiol 11:587–591
Lewis K (2010) Persister Cells. Annu Rev Microbiol 64:357–372
Balaban NQ, Merrin J, Chait R et al (2004) Bacterial persistence as a phenotypic switch. Science 305:1622–1625
Ayrapetyan M, Williams TC, Oliver JD (2015) Bridging the gap between viable but non-culturable and antibiotic persistent bacteria. Trends Microbiol 23:7–13
Gossett DR, Weaver WM, MacH AJ et al (2010) Label-free cell separation and sorting in microfluidic systems. Anal Bioanal Chem 397:3249–3267
Tan AP, Dudani JS, Arshi A et al (2014) Continuous-flow cytomorphological staining and analysis. Lab Chip 14:522–531
Guo MT, Rotem A, Heyman JA, Weitz DA (2012) Droplet microfluidics for high-throughput biological assays. Lab Chip 12:2146–2155
Wang P, Robert L, Pelletier J et al (2010) Robust growth of Escherichia coli. Curr Biol 20:1099–1103
Taheri-Araghi S, Bradde S, Sauls JT et al (2015) Cell-size control and homeostasis in bacteria. Curr Biol 25:385–391
Bergmiller T, Andersson AMC, Tomasek K et al (2017) Biased partitioning of the multidrug efflux pump AcrAB-TolC underlies long-lived phenotypic heterogeneity. Science 356:311–315
Chait R, Ruess J, Bergmiller T et al (2017) Shaping bacterial population behavior through computer-interfaced control of individual cells. Nat Commun 8:1535
Lord ND, Norman TM, Yuan R et al (2019) Stochastic antagonism between two proteins governs a bacterial cell fate switch. Science 366:116–120
Łapińska U, Glover G, Capilla-Lasheras P et al (2019) Bacterial ageing in the absence of external stressors. Philos Trans R Soc B 374:20180442
Tanouchi Y, Pai A, Park H et al (2015) A noisy linear map underlies oscillations in cell size and gene expression in bacteria. Nature 523:357–360
Cama J, Voliotis M, Metz J et al (2020) Single-cell microfluidics facilitates the rapid quantification of antibiotic accumulation in Gram-negative bacteria, Lab on a Chip, https://doi.org/10.1039/D0LC00242A
Bamford RA, Smith A, Metz J et al (2017) Investigating the physiology of viable but non-culturable bacteria by microfluidics and time-lapse microscopy. BMC Biol 15:121
Smith A, Metz J, Pagliara S (2019) MMHelper: an automated framework for the analysis of microscopy images acquired with the mother machine. Sci Rep 9:10123
Kepiro IE, Marzuoli I, Hammond K et al (2020) Engineering chirally blind protein pseudocapsids into antibacterial persisters, ACS Nano 14(2):1609–1622
Stuurman N, Amdodaj N, Vale R (2007) μManager: open source software for light microscope imaging. Micros Today 15:42–43
Kaiser M, Jug F, Julou T et al (2018) Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software. Nat Commun 9:212
Acknowledgments
J.C. was supported by a Wellcome Trust Institutional Strategic Support Award (204909/Z/16/Z) to the University of Exeter. S.P. was supported by a MRC Proximity to Discovery EXCITEME2 grant (MCPC17189), a Royal Society Research Grant (RG180007), a Wellcome Trust Strategic Seed Corn Fund (WT097835/Z/11/Z), and a Marie Skłodowska-Curie grant (H2020-MSCA-ITN-2015-675752). We thank the Jun laboratory for providing us with an epoxy copy of their mother machine mold.
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Cama, J., Pagliara, S. (2021). Microfluidic Single-Cell Phenotyping of the Activity of Peptide-Based Antimicrobials. In: Ryadnov, M. (eds) Polypeptide Materials. Methods in Molecular Biology, vol 2208. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0928-6_16
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DOI: https://doi.org/10.1007/978-1-0716-0928-6_16
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