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Published on: February 26, 2019
Dissecting pOXA-48 fitness effects in clinical Enterobacterales using plasmid-wide CRISPRi screens
Alicia Calvo-Villamañán1, Jorge Sastre-Dominguez2, Álvaro Barrera-Martín2
1Department of Microbial Biotechnology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), Madrid, Spain. aliciacalvovillamanan@gmail.com.
Abstract:
Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, blaOXA-48, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.
Insights
Antimicrobial resistance (AMR) plasmids cause fitness costs in bacteria. Plasmid-wide CRISPR interference screens reveal the blaOXA-48 gene drives these costs and identify new stability genes in carbapenem-resistant Enterobacterales.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Conjugative plasmids drive the spread of antimicrobial resistance (AMR) genes in bacteria.
- AMR plasmids confer antibiotic resistance but often incur fitness costs on host bacteria.
- Understanding plasmid-associated fitness effects is crucial for combating AMR evolution.
Purpose of the Study:
- To investigate the molecular basis of fitness costs associated with AMR plasmids.
- To utilize plasmid-wide CRISPR interference (CRISPRi) screens for dissecting these fitness effects.
- To identify specific genes responsible for fitness costs and stability of the pOXA-48 carbapenem resistance plasmid.
Main Methods:
- Designed and performed plasmid-wide CRISPRi screens targeting the pOXA-48 plasmid.
- Screened the pOXA-48 plasmid in 13 different multidrug-resistant clinical Enterobacterales strains.
- Analyzed gene-level fitness effects and plasmid stability mechanisms.
Main Results:
- Identified conserved gene-level fitness effects of the pOXA-48 plasmid across clinical strains.
- Demonstrated that the blaOXA-48 gene is the primary driver of pOXA-48 associated fitness costs.
- Highlighted the role of postsegregational killing systems and uncovered novel genes (pri, korC, DNDJGHEP_13/14, H-NS) involved in plasmid stability.
Conclusions:
- Plasmid-wide CRISPRi screens are effective for studying plasmid-mediated AMR.
- The blaOXA-48 gene significantly contributes to the fitness burden of carbapenem-resistant Enterobacterales.
- New insights into plasmid stability and evolution in clinical bacteria were gained.
