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Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae

Schoepp, Nathan G. and Liaw, Eric J. and Winnett, Alexander and Savela, Emily S. and Garner, Omai B. and Ismagilov, Rustem F. (2020) Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae. PLoS Biology, 18 (3). Art. No. 3000652. ISSN 1545-7885. PMCID PMC7081982. https://resolver.caltech.edu/CaltechAUTHORS:20200320-065850359

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Abstract

The rise in carbapenem-resistant Enterobacteriaceae (CRE) infections has created a global health emergency, underlining the critical need to develop faster diagnostics to treat swiftly and correctly. Although rapid pathogen-identification (ID) tests are being developed, gold-standard antibiotic susceptibility testing (AST) remains unacceptably slow (1–2 d), and innovative approaches for rapid phenotypic ASTs for CREs are urgently needed. Motivated by this need, in this manuscript we tested the hypothesis that upon treatment with β-lactam antibiotics, susceptible Enterobacteriaceae isolates would become sufficiently permeabilized, making some of their DNA accessible to added polymerase and primers. Further, we hypothesized that this accessible DNA would be detectable directly by isothermal amplification methods that do not fully lyse bacterial cells. We build on these results to develop the polymerase-accessibility AST (pol-aAST), a new phenotypic approach for β-lactams, the major antibiotic class for gram-negative infections. We test isolates of the 3 causative pathogens of CRE infections using ceftriaxone (CRO), ertapenem (ETP), and meropenem (MEM) and demonstrate agreement with gold-standard AST. Importantly, pol-aAST correctly categorized resistant isolates that are undetectable by current genotypic methods (negative for β-lactamase genes or lacking predictive genotypes). We also test contrived and clinical urine samples. We show that the pol-aAST can be performed in 30 min sample-to-answer using contrived urine samples and has the potential to be performed directly on clinical urine specimens.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1371/journal.pbio.3000652DOIArticle
https://doi.org/10.1371/journal.pbio.3000652.s001DOIS1 Table
https://doi.org/10.1371/journal.pbio.3000652.s002DOIS2 Table
https://doi.org/10.1371/journal.pbio.3000652.s003DOIS3 Table
https://doi.org/10.1371/journal.pbio.3000652.s004DOIS4 Table
https://doi.org/10.1371/journal.pbio.3000652.s005DOIS5 Table
https://doi.org/10.1371/journal.pbio.3000652.s006DOIS6 Table
https://doi.org/10.1371/journal.pbio.3000652.s007DOIS1 Fig.
https://doi.org/10.1371/journal.pbio.3000652.s008DOIS1 Text
http://www.ncbi.nlm.nih.gov/pmc/articles/pmc7081982/PubMed CentralArticle
ORCID:
AuthorORCID
Schoepp, Nathan G.0000-0002-2406-3693
Liaw, Eric J.0000-0003-2244-8335
Winnett, Alexander0000-0002-7338-5605
Savela, Emily S.0000-0001-9614-4276
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© 2020 Schoepp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: July 3, 2019; Accepted: February 14, 2020; Published: March 19, 2020. We thank Sukantha Chandrasekaran, Shelley Miller, Romney Humphries, Marisol Trejo, Catherine Le, and Lyna Chheang at the UCLA Clinical Microbiology Laboratory for providing isolates and clinical urine samples and for discussion of gold-standard practices. We thank Jennifer Dien Bard at the Keck School of Medicine of USC for performing Cepheid Xpert Carba-R tests. We also thank Natasha Shelby for help with writing and editing this manuscript. Data Availability Statement: The authors declare that all the data supporting these findings are available within the article and its Supporting Information. This work was funded in part by the Defense Threat Reduction Agency (DTRA) award MCDC-18-01-01-007; an effort sponsored by the U.S. Government under Other Transaction number W15QKN-16-9-1002 between the MCDC, and the Government. The U.S. Government is authorized to reproduce and distribute reprints for Governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of the U.S. Government. This work was also supported by a Burroughs Wellcome Fund Innovation in Regulatory Science Award, an NIH National Research Service Award (NRSA) [5T32GM07616NSF] (to N.G.S.), NIH NIGMS Predoctoral Training Grants (GM008042) to A.W. and E.J.L., a grant from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine, and a fellowship (to E.S.S.) from Joan and Jerry Doren. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The technology described in this publication is the subject of a patent application filed by Caltech. R.F.I. has a financial interest in Talis Biomedical Corp. Author Contributions: Conceptualization: Nathan G. Schoepp, Eric J. Liaw, Emily S. Savela, Rustem F. Ismagilov. Data curation: Nathan G. Schoepp. Formal analysis: Nathan G. Schoepp, Alexander Winnett, Emily S. Savela. Funding acquisition: Nathan G. Schoepp, Alexander Winnett, Rustem F. Ismagilov. Investigation: Nathan G. Schoepp, Eric J. Liaw, Alexander Winnett. Methodology: Nathan G. Schoepp, Eric J. Liaw, Alexander Winnett, Emily S. Savela, Rustem F. Ismagilov. Project administration: Rustem F. Ismagilov. Resources: Omai B. Garner, Rustem F. Ismagilov. Supervision: Rustem F. Ismagilov. Validation: Nathan G. Schoepp, Alexander Winnett. Visualization: Nathan G. Schoepp. Writing – original draft: Nathan G. Schoepp, Eric J. Liaw. Writing – review & editing: Nathan G. Schoepp, Alexander Winnett, Emily S. Savela, Omai B. Garner, Rustem F. Ismagilov.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Defense Threat Reduction Agency (DTRA)MCDC-18-01-01-007
Defense Threat Reduction Agency (DTRA)W15QKN-16-9-1002
Burroughs Wellcome FundUNSPECIFIED
NIH Predoctoral Fellowship5T32GM07616NSF
NIH Predoctoral FellowshipGM008042
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Joan and Jerry DorenUNSPECIFIED
Issue or Number:3
PubMed Central ID:PMC7081982
Record Number:CaltechAUTHORS:20200320-065850359
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200320-065850359
Official Citation:Schoepp NG, Liaw EJ, Winnett A, Savela ES, Garner OB, Ismagilov RF (2020) Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae. PLoS Biol 18(3): e3000652. https://doi.org/10.1371/ journal.pbio.3000652
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:102007
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:20 Mar 2020 16:06
Last Modified:31 Mar 2020 18:18

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