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Published August 2, 2018 | Published + Supplemental Material
Journal Article Open

RNA markers enable phenotypic test of antibiotic susceptibility in Neisseria gonorrhoeae after 10 minutes of ciprofloxacin exposure


Antimicrobial-resistant Neisseria gonorrhoeae is an urgent public-health threat, with continued worldwide incidents of infection and rising resistance to antimicrobials. Traditional culture-based methods for antibiotic susceptibility testing are unacceptably slow (1–2 days), resulting in the use of broad-spectrum antibiotics and the further development and spread of resistance. Critically needed is a rapid antibiotic susceptibility test (AST) that can guide treatment at the point-of-care. Rapid phenotypic approaches using quantification of DNA have been demonstrated for fast-growing organisms (e.g. E. coli) but are challenging for slower-growing pathogens such as N. gonorrhoeae. Here, we investigate the potential of RNA signatures to provide phenotypic responses to antibiotics in N. gonorrhoeae that are faster and greater in magnitude compared with DNA. Using RNA sequencing, we identified antibiotic-responsive transcripts. Significant shifts (>4-fold change) in transcript levels occurred within 5 min of antibiotic exposure. We designed assays for responsive transcripts with the highest abundances and fold changes, and validated gene expression using digital PCR. Using the top two markers (porB and rpmB) we correctly determined the antibiotic susceptibility and resistance of 49 clinical isolates after 10 min exposure to ciprofloxacin. RNA signatures are therefore promising as an approach on which to build rapid AST devices for N. gonorrhoeae at the point-of-care, which is critical for disease management, surveillance, and antibiotic stewardship efforts.

Additional Information

© 2018 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 11 April 2018; Accepted 11 July 2018; Published 02 August 2018. Data availability: Data on candidate markers for N. gonorrhoeae and sequences for the primers used in this study are provided in the Supplementary file available. The complete sequencing data generated during this study are available in the National Center for Biotechnology Information Sequence Read Archive repository under the study accession number SRP150785. This work was supported in part by a Burroughs Wellcome Fund Innovation in Regulatory Science Award, a Natural Sciences and Engineering Research Council of Canada (NSERC) fellowship [PGSD3-438474-2013] (to T.K.), an NIH National Research Service Award (NRSA) [5T32GM07616NSF] (to N.G.S.), and a grant from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine. Research reported in this publication was also supported by the Department of Health and Human Services (HHS) Office of the Assistant Secretary for Preparedness and Response (ASPR) and the Wellcome Trust under the CARB-X program (federal award number IDSEP160030-02); the content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of HHS Office of the ASPR. This work is funded in part by CARB-X as a collaboration between Talis Biomedical and Caltech. This work was also supported by the Millard & Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology and we acknowledge lab director Igor Antoshechkin for his assistance. We thank Dr. Olusegun O. Soge at the UW Neisseria Reference Laboratory, and the Clinical Microbiology Laboratory at UCLA for providing N. gonorrhoeae isolates. We also thank Natasha Shelby for contributions to writing and editing this manuscript. Author Contributions: T.K., N.G.S., and R.F.I. designed the study; T.K., N.G.S, J.T.B., and R.F.I. developed the methodology and performed the research; T.K., J.T.B., and R.F.I analyzed and interpreted the data; T.K. wrote the text for the manuscript, and all authors contributed to the final preparation of the submission. 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.

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August 19, 2023
August 19, 2023