Plasmid copy number dynamics under antibiotic pressure
Project 1 – The effect of cryptic plasmids on the evolution of antibiotic resistance, and the effect of sub-MIC antibiotic treatment on their Plasmid Copy Number.
This project investigates the role of cryptic plasmids - small, extrachromosomal DNA elements traditionally considered functionless - in the evolution of antibiotic resistance in Escherichia coli. Recent findings suggest that these plasmids can acquire resistance genes through transposition from larger plasmids and amplify their plasmid copy number (TPCN), leading to increased resistance that is reversible in the absence of antibiotic pressure. To explore this, three blaTEM-1–carrying cryptic plasmids (ranging from 7 to 12kb) from an in-house bloodstream infection (BSI) clinical isolate collection will be transformed into E. coli isolates from the same collection. Minimum inhibitory concentrations (MICs) for the clinically relevant antibiotic combination, piperacillin-tazobactam (TZP) will be assessed using E-tests, followed by population analysis profile (PAP) testing to determine heteroresistance. Selection experiments at varying TZP concentrations above the MIC (2x, 4x, 8x and 16x) will be conducted, with subsequent analysis of evolved mutants via whole genome sequencing, fitness cost assays, and droplet digital PCR (ddPCR) to quantify plasmid copy number. Reversion experiments without antibiotic pressure will examine reversibility to the original phenotype, and bioinformatic analysis will determine plasmid features such as replicon, origin of replication as well as their prevalence in the clinics. Overall, the project aims to reveal how cryptic plasmids contribute to antibiotic resistance, how common they are, and their potential impact on treatment outcomes.
E-test of one of the project bacterial strains
Project II – Plasmid copy number variation in enterobacteria under sub-MIC antibiotic concentrations.
This project explores how sub-MIC levels of antibiotics influence PCN variation in enterobacteria, a poorly understood yet clinically relevant phenomenon. Using Yersinia pseudotuberculosis carrying the virulence plasmid pYV and clinical Escherichia coli strains with large, non-virulent plasmids, we aim to determine whether sub-MIC antibiotic exposure alters PCN and whether these effects are species- or plasmid-specific. MICs will be determined via EUCAST guidelines or E-tests, and initial screening for PCN changes under different antibiotic classes will be performed using the ddPCR, comparing treated and untreated bacteria at 26°C and 37°C for Yersinia. Conditions known to suppress Type III Secretion System (T3SS) induction (37°C + Ca²⁺) will be used to differentiate natural from stress-induced PCN changes. Promising antibiotics from the initial screen will be used for deeper analysis, including fitness cost assays and proteomic profiling to identify molecular pathways involved in PCN regulation. The same methodology will be applied to a relevant clinical E. coli strain carrying a large non-virulent plasmid. Ultimately, this project will uncover whether sub-lethal antibiotic levels universally impact PCN across different enterobacteria and contribute to adaptive resistance mechanisms.
PhD Student Angelika Ntokaki presenting a poster at a conference
Related published research
- Andersson, Dan I., Hervé Nicoloff, and Karin Hjort. 2019. “Mechanisms and Clinical Relevance of Bacterial Heteroresistance.” Nature Reviews Microbiology. Nature Publishing Group. https://doi.org/10.1038/s41579-019-0218-1.
- Nicoloff, Hervé, Karin Hjort, Dan I. Andersson, and Helen Wang. 2024. “Three Concurrent Mechanisms Generate Gene Copy Number Variation and Transient Antibiotic Heteroresistance.” Nature Communications 2024 15:1 15 (1): 1–12. https://doi.org/10.1038/s41467-024-48233-0.
- Wang, Helen, Kemal Avican, Anna Fahlgren, Saskia F. Erttmann, Aaron M. Nuss, Petra Dersch, Maria Fallman, Tomas Edgren, and Hans Wolf-Watz. “Increased Plasmid Copy Number Is Essential for Yersinia T3SS Function and Virulence.” Science 353 (6298): 492–95. https://doi.org/10.1126/science.aaf7501
- Engling, Pit, Tifaine Héchard, Tomas Edgren, Matthew Francis, Petra Dersch, and Helen Wang. 2023. “Calcium-Responsive Plasmid Copy Number Regulation Is Dependent on Discrete YopD Domains in Yersinia Pseudotuberculosis.” Plasmid 126 (May):102683. https://doi.org/10.1016/J.PLASMID.2023.102683
- Hechard, Tifaine, and Helen Wang. 2023. “Determination of Growth Rate and Virulence Plasmid Copy Number During Yersinia Pseudotuberculosis Infection Using Droplet Digital PCR.” Methods in Molecular Biology (Clifton, N.J.) 2674:101–15. https://doi.org/10.1007/978-1-0716-3243-7_7
- Schneiders, Stephan, Tifaine Hechard, Tomas Edgren, Kemal Avican, Maria Fällman, Anna Fahlgren, and Helen Wang. 2021. “Spatiotemporal Variations in Growth Rate and Virulence Plasmid Copy Number during Yersinia Pseudotuberculosis Infection.” Infection and Immunity 89 (4). https://doi.org/10.1128/IAI.00710-20