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

Abstract

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 healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. 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. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment.

Clinical Performance of a Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Method for Detection of Certain bla KPC -Containing Plasmids

“…The ϳ11,109-Da peak was detected by two independent operators in 25 of the 26 isolates that were positive for p019 by PCR (96% overall sensitivity; 95% confidence interval [CI], 80% to 100%) ( Table 2). All nine replicates were detected in c bla KPC was carried on an IncN plasmid lacking the p019 gene, consistent with the PCR results in this study (12). d A subset of these carbapenem-resistant organisms were published previously as supplemental data (8): C. freundii complex (n ϭ 1), E. aerogenes (n ϭ 1), E. cloacae complex (n ϭ 4), E. coli (n ϭ 10), K. pneumoniae (n ϭ 8), and Serratia spp.…”

“…This simple methodology is applicable to other clinical microbiology laboratories because it can provide real-time detection of certain carbapenem-resistant bacteria that contain a bla KPC -containing plasmid encoding an associated p019 protein on that same plasmid with significant benefits for infection control efforts, using mass spectra acquired as part of routine organism identification without placing additional constraints on laboratory resources. This methodology has proven to work well in our institution because of the known presence of pKpQIL-containing organisms in our hospital environment (8,12,22). This methodology has now been implemented in the routine clinical workflow of our laboratory, with all spectra scanned by the automated script for peaks within a window of 11,109 Ϯ 15 Da using the Flex Analysis program (Bruker Daltonics).…”

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae , an urgent threat to public health

“…There also is a high risk that current hypervirulent clones may acquire AMR, and MDR ST23 already has been reported in Korea, Vietnam, China, Madagascar, and Brazil (12,(61)(62)(63). Now that the associations have been recognized, new efforts can be more focused on the task of reconstructing the mobile elements that carry the critical virulence genes, ideally using long-read sequencing approaches that have proven successful for unraveling MDR plasmids (64). However, given the plasticity of the K. pneumoniae pangenome and the broad distribution of key virulence and AMR genes, we need to place all K. pneumoniae within a wider population framework to target known epidemic clones and increase the likelihood of identifying new and emerging clones.…”

Practical Value of Food Pathogen Traceability through Building a Whole-Genome Sequencing Network and Database

“…Hospitals represent the first responders to epidemic and zoonotic infectious agents, as they emerge in the population. To this end, we are closely watching pilot clinical work flows that permit hospitals to feed WGS data through to the NCBI pipeline for direct upload and curation (18)(19)(20)(21). Laboratories like these are successfully using WGS data to identify common resistance genes and for the purposes of evaluating transmissibility and are part of clinical laboratory networks working to integrate WGS data and a controlled level of associated metadata with a public interface and opensource curation.…”