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.
Metrics
-
- 2,931
- views
-
- 328
- downloads
-
- 23
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
-
- Developmental Biology
- Genetics and Genomics
We present evidence implicating the BAF (BRG1/BRM Associated Factor) chromatin remodeler in meiotic sex chromosome inactivation (MSCI). By immunofluorescence (IF), the putative BAF DNA binding subunit, ARID1A (AT-rich Interaction Domain 1 a), appeared enriched on the male sex chromosomes during diplonema of meiosis I. Germ cells showing a Cre-induced loss of ARID1A arrested in pachynema and failed to repress sex-linked genes, indicating a defective MSCI. Mutant sex chromosomes displayed an abnormal presence of elongating RNA polymerase II coupled with an overall increase in chromatin accessibility detectable by ATAC-seq. We identified a role for ARID1A in promoting the preferential enrichment of the histone variant, H3.3, on the sex chromosomes, a known hallmark of MSCI. Without ARID1A, the sex chromosomes appeared depleted of H3.3 at levels resembling autosomes. Higher resolution analyses by CUT&RUN revealed shifts in sex-linked H3.3 associations from discrete intergenic sites and broader gene-body domains to promoters in response to the loss of ARID1A. Several sex-linked sites displayed ectopic H3.3 occupancy that did not co-localize with DMC1 (DNA meiotic recombinase 1). This observation suggests a requirement for ARID1A in DMC1 localization to the asynapsed sex chromatids. We conclude that ARID1A-directed H3.3 localization influences meiotic sex chromosome gene regulation and DNA repair.
-
- Genetics and Genomics
- Immunology and Inflammation
Systemic lupus erythematosus (SLE) is an autoimmune disease, the pathophysiology and genetic basis of which are incompletely understood. Using a forward genetic screen in multiplex families with SLE, we identified an association between SLE and compound heterozygous deleterious variants in the non-receptor tyrosine kinases (NRTKs) ACK1 and BRK. Experimental blockade of ACK1 or BRK increased circulating autoantibodies in vivo in mice and exacerbated glomerular IgG deposits in an SLE mouse model. Mechanistically, NRTKs regulate activation, migration, and proliferation of immune cells. We found that the patients’ ACK1 and BRK variants impair efferocytosis, the MERTK-mediated anti-inflammatory response to apoptotic cells, in human induced pluripotent stem cell (hiPSC)-derived macrophages, which may contribute to SLE pathogenesis. Overall, our data suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis in macrophages.
