Introduction: Carbapenems are a class of broad-spectrum beta-lactam antibiotics that are used to treat severe infections, often as a last resort treatment when other antibiotics have failed. Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug resistant bacteria and an urgent threat in the United States and many other countries. Monitoring the frequency and spread of CRE is, therefore, of utmost importance. CRE infections are most frequently contracted in hospitals and long-term care facilities. Many CRE organisms are uropathogenic, and the urinary tract is a common site of infection due to catheter use. Many CRE isolates carry carbapenemase encoding genes on plasmids, which can be horizontally transferred between bacteria via conjugation. Our hypothesis is that carbapenem resistance is proliferating through horizontal gene transfer via plasmid conjugation.
Methods: In this study we characterize a collection of 40 strains isolated from patients at Duke University Hospital in 2017-2018 that were identified as CRE strains through antimicrobial susceptibility tests. Genomic DNA was extracted from each isolate and sequenced using Illumina (short-read) and PacBio (long-read) methods. Unicycler assembler was used to construct de novo whole genome sequences of the CRE isolates using both short- and long-read data. Genomes were analyzed for presence of antibiotic resistance genes and plasmid replicon types via ResFinder and PlasmidFinder search tools using curated databases. Plasmids carrying carbapenemase genes were inspected for conjugation gene modules using oriTfinder.
Results: Results showed that the majority of samples contain multiple resistance markers located on both chromosomal and plasmid DNA elements. Carbapenemase-encoding genes were detected in 29 (72.5%) of samples, with some sample genomes containing two copies of the same carbapenemase gene. Sixteen plasmids are predicted to be self-transmissible, and four additional plasmids are predicted to be mobilizable.
Conclusions: This evidence suggests that transfer of carbapenem resistance is taking place via conjugation. Additionally, eleven isolates possess alternate modes of carbapenem resistance from carbapenemase production, indicating a limitation of current WGS methods for carbapenem resistance detection.
Source of Funding: This study was in part sponsored by K12 Duke KURe (DK100024) NIDDK and UAH Startup funds to TAS.
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