Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR
- Cornell University, United States
- Robert Woods Johnson Medical School, United States
- Weill Cornell Medicine, United States
Abstract
The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.
Data availability
All fusion PCR amplicons are deposited in SRA under the accession number PRJNA701446. The metagenomic samples analyzed can be obtained through SRA with under the accession number PRJNA649316.
Article and author information
Author details
Funding
Centers for Disease Control and Prevention (OADS BAA 2016-N-17812)
- Michael J Satlin
- Ilana Brito
National Science Foundation (1661338)
- Ilana Brito
National Science Foundation (1650122)
- Ilana Brito
National Institutes of Health (K23 AI114994)
- Michael J Satlin
National Institutes of Health (1DP2HL141007-01)
- Ilana Brito
Pew Charitable Trusts
- Ilana Brito
Alfred P. Sloan Foundation
- Ilana Brito
David and Lucile Packard Foundation
- Ilana Brito
State University of New York
- Felicia N New
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: All human subjects research was approved by the Weill Cornell Medicine IRB (#1504016114) and Cornell University IRB (#1609006586). Informed consent and consent to publish were obtained from individuals receiving a hematopoietic stem cell transplant at NewYork-Presbyterian Hospital/Weill Cornell Medical Center. Serial stool samples were obtained from consenting patients. Consent documents and procedures were approved by the Institutional Review Boards at Weill Cornell Medical College (#1504016114) and Cornell University (#1609006586).
Copyright
© 2021, Diebold et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR
eLife 10:e66834.
https://doi.org/10.7554/eLife.66834
Further reading
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