Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae

Antibiotic-resistant microbes are spreading at an alarming rate in health care facilities throughout the world. Conlan et al. use a new DNA sequencing method to take a close look at one way in which antibiotic resistance spreads. With single-molecule sequencing, the authors completely characterized individual plasmids, the circular bits of DNA that carry the genes for antibiotic resistance in bacteria. They focused on resistance to the carbapenems, a class of antibiotics that is often used for infections that do not respond to more conventional antimicrobial agents. By using this approach in their microbial surveillance program at the NIH Clinical Center, the authors found evidence that plasmids carrying carbapenemase genes moved from one microbial species to another within the hospital environment. They also used the technique to test hypotheses about patient-to-patient transmission and to characterize a previously undescribed carbapenemase-encoding plasmid carried by diverse bacterial species that could cause dangerous clinical infections.

Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common health care–associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.