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Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population

Scheler, Ott and Makuch, Karol and Debski, Pawel R. and Horka, Michal and Ruszczak, Artur and Pacocha, Natalia and Sozański, Krzysztof and Smolander, Olli-Pekka and Postek, Witold and Garstecki, Piotr (2020) Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population. Scientific Reports, 10 . Art. No. 3282. ISSN 2045-2322. PMCID PMC7039976. https://resolver.caltech.edu/CaltechAUTHORS:20200303-134259605

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Abstract

Since antibiotic resistance is a major threat to global health, recent observations that the traditional test of minimum inhibitory concentration (MIC) is not informative enough to guide effective antibiotic treatment are alarming. Bacterial heteroresistance, in which seemingly susceptible isogenic bacterial populations contain resistant sub-populations, underlies much of this challenge. To close this gap, here we developed a droplet-based digital MIC screen that constitutes a practical analytical platform for quantifying the single-cell distribution of phenotypic responses to antibiotics, as well as for measuring inoculum effect with high accuracy. We found that antibiotic efficacy is determined by the amount of antibiotic used per bacterial colony forming unit (CFU), not by the absolute antibiotic concentration, as shown by the treatment of beta-lactamase-carrying Escherichia coli with cefotaxime. We also noted that cells exhibited a pronounced clustering phenotype when exposed to near-inhibitory amounts of cefotaxime. Overall, our method facilitates research into the interplay between heteroresistance and antibiotic efficacy, as well as research into the origin and stimulation of heterogeneity by exposure to antibiotics. Due to the absolute bacteria quantification in this digital assay, our method provides a platform for developing reference MIC assays that are robust against inoculum-density variations.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/s41598-020-60381-zDOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039976PubMed CentralArticle
https://doi.org/10.1101/328393DOIDiscussion Paper
ORCID:
AuthorORCID
Scheler, Ott0000-0002-8428-1350
Makuch, Karol0000-0002-9769-7646
Smolander, Olli-Pekka0000-0002-6795-7734
Garstecki, Piotr0000-0001-9101-7163
Alternate Title:Antibiotic inhibition of bacteria growth in droplets reveals heteroresistance pattern at the single cell level
Additional Information:© 2020 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Received 15 September 2019; Accepted 07 February 2020; Published 24 February 2020. We are very grateful to Professor Jeff Gore from MIT for sharing the bacteria strain we used here. This research received support from the Foundation for Polish Science within the Team-Tech/2016-2/10 program. P.G. acknowledges support from the Polish National Science Centre based on decision number DEC-2014/12/W/NZ6/00454 (Symfonia). O.S. was supported by Estonian Research Council grant PUTJD589. O.S. and O-P.S. were supported by TTÜ development program 2016–2022, project code 2014–2020.4.01.16-0032. W.P. received funding through a doctoral scholarship from the Polish National Science Centre, scholarship code UMO-2018/28/T/ST4/00318. This project was partially performed in laboratories funded by NanoFun POIG.02.02.00-00-025/09. K.M. has received funding from the National Science Center, Poland (2016/21/D/ST3/00988). Author Contributions: O.S. conceived the study, designed the research, analysed data, interpreted the results, and was responsible for performing the experiments. K.M. was responsible for data analysis and interpreted the results. P.R.D. assisted in data analysis. M.H. assisted in data analysis and was responsible for writing LabView scripts. A.R. assisted in microbiology-related work. N.P. assisted in droplet microfluidic experiments. K.S. assisted with confocal microscopy. O-P.S. assisted data analysis. W.P. participated in research design. P.G. conceived the study, assisted in designing the research, and interpreted the results. O.S., K.M., and P.G. wrote the paper, with input from all co-authors. The authors declare no competing interests.
Funders:
Funding AgencyGrant Number
Foundation for Polish ScienceTeam-Tech/2016-2/10
National Science Centre (Poland)DEC-2014/12/W/NZ6/00454
Estonian Research CouncilPUTJD589
Tallinn University of Technology (TTÜ)2014-2020.4.01.16-0032
National Science Centre (Poland)UMO-2018/28/T/ST4/00318
NanoFunPOIG.02.02.00-00-025/09
National Science Centre (Poland)2016/21/D/ST3/00988
PubMed Central ID:PMC7039976
Record Number:CaltechAUTHORS:20200303-134259605
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200303-134259605
Official Citation:Scheler, O., Makuch, K., Debski, P.R. et al. Droplet-based digital antibiotic susceptibility screen reveals single-cell clonal heteroresistance in an isogenic bacterial population. Sci Rep 10, 3282 (2020). https://doi.org/10.1038/s41598-020-60381-z
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101684
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:04 Mar 2020 20:00
Last Modified:04 Mar 2020 20:00

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