Plasmid Dynamics in KPC-Positive Klebsiella pneumoniae during Long-Term Patient Colonization

Abstract

Carbapenem-resistant Klebsiella pneumoniae strains are formidable hospital pathogens that pose a serious threat to patients around the globe due to a rising incidence in health care facilities, high mortality rates associated with infection, and potential to spread antibiotic resistance to other bacterial species, such as Escherichia coli Over 6 months in 2011, 17 patients at the National Institutes of Health (NIH) Clinical Center became colonized with a highly virulent, transmissible carbapenem-resistant strain of K. pneumoniae Our real-time genomic sequencing tracked patient-to-patient routes of transmission and informed epidemiologists' actions to monitor and control this outbreak. Two of these patients remained colonized with carbapenemase-producing organisms for at least 2 to 4 years, providing the opportunity to undertake a focused genomic study of long-term colonization with antibiotic-resistant bacteria. Whole-genome sequencing studies shed light on the underlying complex microbial colonization, including mixed or evolving bacterial populations and gain or loss of plasmids. Isolates from NIH patient 15 showed complex plasmid rearrangements, leaving the chromosome and the blaKPC-carrying plasmid intact but rearranging the two other plasmids of this outbreak strain. NIH patient 16 has shown continuous colonization with blaKPC-positive organisms across multiple time points spanning 2011 to 2015. Genomic studies defined a complex pattern of succession and plasmid transmission across two different K. pneumoniae sequence types and an E. coli isolate. These findings demonstrate the utility of genomic methods for understanding strain succession, genome plasticity, and long-term carriage of antibiotic-resistant organisms.

Importance: In 2011, the NIH Clinical Center had a nosocomial outbreak involving 19 patients who became colonized or infected with blaKPC-positive Klebsiella pneumoniae Patients who have intestinal colonization with blaKPC-positive K. pneumoniae are at risk for developing infections that are difficult or nearly impossible to treat with existing antibiotic options. Two of those patients remained colonized with blaKPC-positive Klebsiella pneumoniae for over a year, leading to the initiation of a detailed genomic analysis exploring mixed colonization, plasmid recombination, and plasmid diversification. Whole-genome sequence analysis identified a variety of changes, both subtle and large, in the blaKPC-positive organisms. Long-term colonization of patients with blaKPC-positive Klebsiella pneumoniae creates new opportunities for horizontal gene transfer of plasmids encoding antibiotic resistance genes and poses complications for the delivery of health care.

FIG 1

Patient carriage of carbapenemase-producing organisms, initiating with the 2011 K. pneumoniae outbreak strain. (Top panel) Patient 15 timeline of culture results. (Bottom panel) Patient 16 timeline of culture results. Cultures negative for carbapenemase-producing organisms (CPOs) are shown as red diamonds. Sequenced isolates from CPO-positive cultures are shown as filled circles. Other CPO-positive cultures are shown as plus signs. CPO positives are colored by organism as follows: unidentified, green; K. pneumoniae ST258, blue; K. pneumoniae ST37, cyan; E. coli ST127, magenta. For both the top panel and the bottom panel, CPO-negative cultures may have been positive for carbapenem-sensitive organisms. The numbers below the x axis represent the numbers of days since first collection of a bla_KPC⁺ culture, with the years indicated above the x axis.

FIG 2

Isolates from patient 15 exhibited plasmid recombination between 2011 and 2013. (A) PCR results for marker genes. Isolates from patient 15 are numbered in order of isolation as follows: 1, day 0; 2, day 437; 3, day 642. Marker genes are indicated for each set of wells. (B) Rearrangements of the pKPN-498 and pAAC154-a50 plasmids that characterized the 2011 outbreak are indicated by ribbons connecting to the 2013 plasmids pKPN-fff and pKPN-821. Ribbons are colored for visualization purposes and do not have meaning. Gene annotations are indicated on the 2011 plasmids as follows: tra genes, light green; antibiotic resistance, yellow; iron acquisition, orange; metal efflux, purple; mobile elements, brown. Other genes are indicated in dark green. Select genes described in the text are labeled. The recombination/duplication event is highlighted on the inner ring, with the recombination region shown in red and the duplicated region shown in blue.

FIG 3

Seven plasmid backbones are associated with three microbial populations isolated from patient 16. The genus, species, and sequence type are noted above each strain illustration along with the strain name and sequencing platform. Plasmid names are listed on the right, with the 2011 outbreak strain plasmids indicated in bold. The pKpQIL recombinant regions are noted in yellow and green. DIV, deletion/insertion variants. The timeline at the bottom is labeled with years indicated below the timeline and numbers of days since first bla_KPC⁺ culture indicated above the timeline.

FIG 4

pKpQIL plasmid rearrangements from 2011 to 2014. A stacked alignment of pKpQIL and recombinant variants is shown. Genes are colored by product annotation as follows: iron-related functions are indicated in orange, copper-related functions in blue, transposes/integrases/recombinases in brown, restriction/modification genes in red (R, restriction; S, specificity; M, methylase), and conjugal transfer genes in olive green. Recombinant regions are marked by yellow and green bars. nt, nucleotides.