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Edited by Ralph R. Isberg, Tufts University School of Medicine, Boston, MA, and approved October 12, 2020 (received for review March 30, 2020)
Significance
Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant bacterial pathogens designated as urgent threats by the US Centers for Disease Control and Prevention. Resistance to carbapenems and other antibiotics is usually carried on diverse large, low-copy-number plasmids. Interfering with the replication of such resistance plasmids could potentially restore antibiotic susceptibility. Therefore, we designed a high-throughput screen to identify compounds with anti-CRE plasmid activity. We identified several compounds which by blocking plasmid replication and/or evicting a representative IncFIA CRE plasmid potentiated carbapenem activity, thereby providing proof of principle for our approach. Our findings underscore the potential of antiplasmid therapeutics to restore treatment options for highly drug-resistant pathogens and validate a high-throughput screening approach to identify these agents.
Abstract
Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant pathogens for which new treatments are desperately needed. Carbapenemases and other types of antibiotic resistance genes are carried almost exclusively on large, low-copy-number plasmids (pCRE). Accordingly, small molecules that efficiently evict pCRE plasmids should restore much-needed treatment options. We therefore designed a high-throughput screen to identify such compounds. A synthetic plasmid was constructed containing the plasmid replication machinery from a representative Escherichia coli CRE isolate as well as a fluorescent reporter gene to easily monitor plasmid maintenance. The synthetic plasmid was then introduced into an E. coli K12 tolC host. We used this screening strain to test a library of over 12,000 known bioactive agents for molecules that selectively reduce plasmid levels relative to effects on bacterial growth. From 366 screen hits we further validated the antiplasmid activity of kasugamycin, an aminoglycoside; CGS 15943, a nucleoside analog; and Ro 90-7501, a bibenzimidazole. All three compounds exhibited significant antiplasmid activity including up to complete suppression of plasmid replication and/or plasmid eviction in multiple orthogonal readouts and potentiated activity of the carbapenem, meropenem, against a strain carrying the large, pCRE plasmid from which we constructed the synthetic screening plasmid. Additionally, we found kasugamycin and CGS 15943 blocked plasmid replication, respectively, by inhibiting expression or function of the plasmid replication initiation protein, RepE. In summary, we validated our approach to identify compounds that alter plasmid maintenance, confer resensitization to antimicrobials, and have specific mechanisms of action.
- plasmid
- multidrug resistance
- carbapenem-resistant Enterobacteriaceae
- antimicrobial
- high-throughput screening
Footnotes
- ↵1To whom correspondence may be addressed. Email: jekirby{at}bidmc.harvard.edu.
Author contributions: K.E.Z. and J.E.K. designed research; K.E.Z. performed research; K.E.Z. and J.E.K. analyzed data; and K.E.Z. and J.E.K. wrote the paper.
This article is a PNAS Direct Submission.
Competing interest statement: The authors declare a competing interest (as defined by PNAS policy). This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award number R33AI119114 to J.E.K. K.E.Z. was supported in part by a National Institute of Allergy and Infectious Diseases training grant (T32AI007061). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The HP D300 digital dispenser and TECAN M1000 were provided for our use by TECAN (Morrisville, NC). Tecan had no role in study design, data collection/interpretation, manuscript preparation, or decision to publish.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2005948117/-/DCSupplemental.
Data Availability.
All study data are included in the paper, SI Appendix, Datasets S1–S6, and GenBank accession number MW057772.
Published under the PNAS license.
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Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach
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