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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance
Published on: May 2, 2018
Development of pBACpAK entrapment vector derivatives to detect intracellular transfer of mobile genetic elements
Richard N Goodman1, Supathep Tansirichaiya2, Adam P Roberts1
1Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.
Abstract:
Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3')-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.
Insights
New entrapment vectors were developed to track mobile genetic elements (MGEs) in bacteria resistant to chloramphenicol. These tools enable the study of MGE movement within bacterial cells, advancing our understanding of antimicrobial resistance dissemination.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Antimicrobial resistance (AMR) spreads through bacterial populations via horizontal gene transfer (HGT).
- Mobile genetic elements (MGEs) are primary drivers of HGT and AMR dissemination.
- Existing entrapment vectors, like pBACpAK, are limited by antibiotic resistance markers, restricting their use in already resistant strains.
Purpose of the Study:
- To develop novel entrapment vectors for studying MGE intracellular transfer in chloramphenicol-resistant bacteria.
- To overcome the limitations of the pBACpAK vector in specific bacterial isolates.
- To validate new vector derivatives for tracking MGE movement within bacterial cells.
Main Methods:
- Modification of the pBACpAK vector by replacing the chloramphenicol resistance gene (catA1) with alternative resistance genes: mcr-1 (pBACpAK-COL) and aph(3')-Ia (pBACpAK-KAN).
- Verification of the engineered plasmid constructs.
- Transformation of Escherichia coli DH5α transconjugants harboring the chloramphenicol resistance plasmid pD25466 with the new vectors.
- Detection of intracellular MGE movement associated with the pD25466 plasmid.
Main Results:
- Successfully developed and validated pBACpAK-COL and pBACpAK-KAN vectors.
- Demonstrated the utility of the new vectors in studying intracellular MGE transfer in chloramphenicol-resistant E. coli.
- Confirmed the ability of the modified vectors to function in bacterial strains with pre-existing resistance.
Conclusions:
- The expanded suite of pBACpAK vectors provides versatile tools for investigating MGE intracellular transfer.
- These new vectors facilitate the study of MGE movement between and within replicons in diverse bacterial isolates, including those with varied antimicrobial resistance profiles.
- This research enhances the capacity to study the mechanisms underlying AMR spread in clinically relevant bacterial populations.
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