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Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

Schoepp, Nathan G. and Khorosheva, Eugenia M. and Schlappi, Travis S. and Curtis, Matthew S. and Humphries, Romney M. and Hindler, Janet A. and Ismagilov, Rustem F. (2016) Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure. Angewandte Chemie International Edition in English, 55 (33). pp. 9557-9561. ISSN 0570-0833. PMCID PMC5215780. doi:10.1002/ange.201602763.

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Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

Item Type:Article
Related URLs:
URLURL TypeDescription CentralArticle
Schoepp, Nathan G.0000-0002-2406-3693
Khorosheva, Eugenia M.0000-0003-3620-4884
Schlappi, Travis S.0000-0001-6132-6459
Curtis, Matthew S.0000-0002-9662-3266
Humphries, Romney M.0000-0002-6568-156X
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:© 2016 WILEY-VCH Verlag GmbH & Co. Received: March 29, 2016. Revised: May 5, 2016. Version of Record online: 30 Jun 2016. This research was supported by DARPA Cooperative Agreement HR0011-11-2-0006, NIH Grant R01EB012946, and a grant from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine. We thank Shelley Miller for advice and experimental assistance. We thank Natasha Shelby for contributions to writing and editing this manuscript.
Errata:In the “Antibiotic exposure time course experiments” section of the “Experimental Section” of the Supporting Information for this Communication, “early logarithmic” should read “early stationary (OD600 3.1–6.9).” Also, the authors do not recommend the use of Epicentre QuickExtract RNA Extraction Solution for pathogens because they found that this solution does not reproducibly and completely lyse some clinical E. coli isolates, which could present a biohazard unless the extracts are handled at the appropriate biosafety level.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-11-2-0006
Joseph J. Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Subject Keywords:analytical methods · antibiotics · DNA replication · polymerase chain reaction · susceptibility
Issue or Number:33
PubMed Central ID:PMC5215780
Record Number:CaltechAUTHORS:20160705-083001025
Persistent URL:
Official Citation:N. G. Schoepp, E. M. Khorosheva, T. S. Schlappi, M. S. Curtis, R. M. Humphries, J. A. Hindler, R. F. Ismagilov, Angew. Chem. Int. Ed. 2016, 55, 9557.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:68822
Deposited By: Ruth Sustaita
Deposited On:05 Jul 2016 21:40
Last Modified:19 Apr 2023 20:56

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