Gene dosage–driven heteroresistance in gram-negative pathogens
Heteroresistance (HR) is a hard to detect antibiotic resistance phenotype where a subset of resistant cells is present at a high frequency within a main susceptible population. This is of concern since resistant subpopulations inadvertently can be selected for under antibiotic treatment, thus resulting in treatment failure in clinic. The most common mechanism of HR in Gram-negative pathogens is transient resistance gene tandem amplifications occurring under antibiotic pressure. Furthermore, there are two additional mechanisms that frequently confer HR in Gram-negatives which recently were described, transient plasmid copy number (PCN) increase, and transposition of resistance genes onto cryptic plasmids followed by transient PCN increase. Additionally, there is one novel mechanism, phage-plasmid copy number increase mediated HR. All four of these HR conferring mechanisms do so by increasing the copy number of resistance genes resulting in an increased gene dosage of said genes. Throughout these projects, the researchers aim to increase our understanding of two of these HR causing phenomena.
Project 1 – Molecular mechanism and genetics of transient plasmid copy number increase mediated heteroresistance and phage-plasmid copy number increase mediated heteroresistance.
In this project the researchers aim to increase the understanding of the underlying genetics and the molecular mechanism of transient PCN increase mediated HR as well as phage-PCN increase mediated HR. They plan to uncover these mechanisms by implementing targeted mutagenesis, whole genome sequencing, and phenotypic characterisations through population analysis profile tests as well as PCN determination through droplet digital PCR (ddPCR).
Diagram of antibiotic resistance by gene dosage
Project 2 – Prevalence of plasmid copy number increase and phage-plasmid copy number increase mediated heteroresistance.
In this second project they aim to determine the prevalence of PCN increase and phage-PCN increase mediated HR in the World Health Organization listed priority pathogens, focusing on Escherichia coli and Klebsiella pneumoniae. Researchers will make use of our collection of >500 whole-genome sequenced microorganisms (mostly septic E. coli and K. pneumoniae) to screen for PCN and phage-PCN mediated HR. The screen will be carried out through a combination of bioinformatical analyses and selection of resistant mutants on increasing concentrations of antibiotics followed by PCN and phage-PCN determination through ddPCR.
Project 3 - Contribution of plasmid copy number increase mediated heteroresistance on the horizontal spread of antibiotic resistance.
For numerous pathogens, resistance genes are located on plasmids that often encode the conjugation machinery necessary for horizontal spread of the plasmids. Presence of antibiotics has been shown to sometimes increase conjugation rates of plasmids, and a recent study suggests that increased PCNs could also increase rates of plasmid conjugation by increasing the gene dosage and expression of the genes encoding the conjugation machinery. Thus, in this project they investigate the interplay between transient PCN increase mediated HR and the horizontal spread of antibiotic resistance through conjugation. This will help our understanding of the contribution that PCN increase mediated HR has to the horizontal spread of antibiotic resistance.
PhD student Elin Svedholm presenting her research 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 17 (8): 479–96. https://doi.org/10.1038/S41579-019-0218-1
- Heyman, Gabriel, Sofia Jonsson, Nikos Fatsis-Kavalopolous, Karin Hjort, Hervé Nicoloff, Mia Furebring, and Dan I. Andersson. 2025. ‘Prevalence, Misclassification, and Clinical Consequences of the Heteroresistant Phenotype in Escherichia Coli Bloodstream Infections in Patients in Uppsala, Sweden: A Retrospective Cohort Study’. The Lancet Microbe 6 (4): 101010. https://doi.org/10.1016/J.LANMIC.2024.101010
- Hjort, Karin, Hervé Nicoloff, and Dan I. Andersson. 2016. ‘Unstable Tandem Gene Amplification Generates Heteroresistance (Variation in Resistance within a Population) to Colistin in Salmonella Enterica’. Molecular Microbiology 102 (2): 274–89. https://doi.org/10.1111/MMI.13459
- 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
- Nicoloff, Hervé, Karin Hjort, Bruce R Levin, and Dan I Andersson. 2019. ‘The High Prevalence of Antibiotic Heteroresistance in Pathogenic Bacteria Is Mainly Caused by Gene Amplification’. Nature Microbiology 4 (3): 504–14. https://doi.org/10.1038/s41564-018-0342-0