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PCR-based typing of IncC plasmids.
Christopher J Harmer1, Ruth M Hall1
1School of Molecular Bioscience, The University of Sydney, 2006, Australia.
Plasmid
|September 4, 2016
Summary
A new PCR method quickly classifies IncC plasmids into type 1 or type 2 lineages. This tool aids in tracking antibiotic resistance genes and understanding plasmid evolution.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- IncC (A/C2) plasmids are key vectors for spreading antibiotic resistance genes, including extended-spectrum β-lactamases and carbapenamases, in Gram-negative bacteria.
- Identifying and tracking these plasmids is crucial for epidemiological and clinical surveillance.
- Previous analyses revealed two distinct evolutionary lineages of IncC plasmids: type 1 and type 2.
Purpose of the Study:
- To develop a rapid Polymerase Chain Reaction (PCR) method for classifying IncC plasmids into type 1 or type 2 lineages.
- To create PCR assays for detecting specific genetic elements like i1 and i2 insertions (type 2 specific), and the ARI-A and ARI-B antibiotic resistance islands.
Main Methods:
- Designed and validated a PCR scheme using specific primer pairs to differentiate between IncC plasmid lineages.
- Developed additional PCRs to detect lineage-specific insertions (i1, i2) and antibiotic resistance islands (ARI-A, ARI-B).
- Tested the PCR strategy on known IncC plasmids (pRMH760, pDGO100, pSRC119-A/C) and a collection of non-IncC plasmids in various bacterial hosts.
Main Results:
- Successfully developed a simple PCR method to assign IncC plasmids to type 1 or type 2 lineages.
- Validated the PCR scheme's accuracy using reference plasmids and diverse bacterial isolates.
- Identified an IncC plasmid in a susceptible E. coli as type 1, lacking resistance islands and a backbone segment.
- Analysis of the historical pIP40a IncC plasmid confirmed its type 1 classification and revealed a complete GIsul2 element, unlike more recent isolates.
Conclusions:
- The developed PCR method provides a rapid and reliable tool for IncC plasmid lineage determination.
- This technique facilitates epidemiological studies by enabling efficient tracking of IncC plasmid types and associated resistance determinants.
- The findings shed light on the evolutionary dynamics of IncC plasmids, including the presence and loss of specific genetic elements like GIsul2 over time.
