A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center

Abstract

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of bla_KPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

Keywords: Klebsiella pneumoniae carbapenemase (KPC); burn patients; carbapenem-resistant Enterobacteriaceae (CRE); health care-associated infection; outbreak; whole-genome sequencing.

FIG 1

Timeline and location of prolonged transmission caused by carbapenem-resistant Enterobacteriaceae. Ec, E. cloacae; Kp, K. pneumoniae; BICU, burn intensive care unit; ISCU, intermediate surgical care unit; MICU, medicine intensive care unit; NSIU, neuroscience intensive care unit; SICU, surgery intensive care unit; PICU, pediatric intensive care unit; CTSU, cardiothoracic stepdown unit; MPCU, medicine progressive care unit; floor, general ward. Each strain ID corresponds to a case summarized in Table 1.

FIG 2

Characterization of acquired resistance genes and genotypes among carbapenem-resistant Enterobacteriaceae strains. Antimicrobial susceptibility profiles: S, susceptible; I, intermediate; R, resistant. Presence (black) or absence (white) of resistance genes and plasmids for each isolate is also shown. SAM, ampicillin-sulbactam; TZP, piperacillin-tazobactam; CRO, ceftriaxone; FEP, cefepime; IMP, imipenem; EPM, ertapenem; GEN, gentamicin; AMK, amikacin; CIP, ciprofloxacin; LVX, levofloxacin; SXT, trimethoprim-sulfamethoxazole; CST, colistin.

FIG 3

(A and B) Maximum-likelihood (ML) phylogram and recombination map showing the relatedness between carbapenem-resistant Enterobacteriaceae isolates. The ML phylogeny was constructed based on genetic variation in nonrecombinant genomic regions. In the horizontal tracks, each column represents a single nucleotide in the reference genome and each row represents an individual clinical isolate. Red blocks mark recombination events occurring on an internal branch of the phylogeny, while blue blocks mark potential recombination events or extensive mutation occurring on a terminal node. The E. cloacae phylogram was rooted to the most genetically distinct isolate in our sample set (Ec155). The K. pneumoniae phylogram was similarly rooted (using Kp11). Phylogenies are drawn on a log scale, and the scale bar below each image represents the phylogenetic distance (in point mutations). (C) Variant difference network, demonstrating Tn4401b-bla_KPC-3 transmission between species and between chromosomally divergent strains. Nodes represent CRE clinical isolates of E. cloacae (blue) and K. pneumoniae (red) from the burn unit outbreak. Small nodes represent single isolates, while large nodes represent two identical isolates. The two nodes within a rectangular outline represent two samples, Kp07 and Ec09, which were isolated from a single patient. All nodes with a dashed black perimeter represent samples bearing the common Tn4401b genetic element. Isolates without a dashed perimeter either had no Tn4401 element or had a non-b subtype (i.e., a or d). Lines (edges) indicate the extent of chromosomal difference between isolates. Solid lines bridging nodes indicate membership in a clonal group (defined as consecutive isolates with ≤12 variant differences; Ec_UNC for E. cloacae and Kp_UNC for K. pneumoniae), while red dashed lines connect less-related isolates. Lines are labeled with the number of SNVs that separate the clinical isolates if there are more than 2 SNVs. Line lengths are not proportional to genetic distance. Isolates depicted above a light gray matte and a dark gray matte were collected in outbreak 1 and outbreak 2 within the BICU, respectively, while isolates outside the mattes were collected outside the unit and are putatively unrelated to the outbreak.

FIG 4

Plasmid and transposon maps revealing significant gene-sharing among carbapenem-resistant Enterobacteriaceae outbreak isolates within the burn unit. (A) The bla_KPC-3-containing contig from de novo read assemblies are depicted. In every case, bla_KPC-3 was identified nested within a Tn4401 element, the boundaries of which are shown with a salmon background. Tn4401 accessory genes are shown in blue, and bla_KPC-3 is highlighted in red. For each isolate, only the plasmid region homologous to the primary outbreak plasmid identified in most Ec and two Kp isolates are shown. (B) Structure of the Tn4401-bla_KPC-3 composite genetic element as identified in each outbreak isolate. Tn4401 boundaries are marked in salmon, and Tn2-like element boundaries are marked in gray. Accessory genes are colored blue, while the β-lactamase genes bla_TEM-1 and bla_KPC-3 are highlighted in red. The single isolate bearing bla_KPC-2 (Kp150) is marked in yellow. A generic Tn2-like element is shown on the top track, and isolate-specific tracks show insertion points of Tn4401 elements into the Tn2-like element.