Plasmid-Mediated Spread of Carbapenem Resistance in Enterobacterales: A Three-Year Genome-Based Survey

Abstract

The worldwide emergence and dissemination of carbapenem-resistant Gram-negative bacteria (CRGNB) is a challenging problem of antimicrobial resistance today. Outbreaks with CRGNB have severe consequences for both the affected healthcare settings as well as the patients with infection. Thus, bloodstream infections caused by metallo-ß-lactamase-producing Enterobacterales can often have clinical implications, resulting in high mortality rates due to delays in administering effective treatment and the limited availability of treatment options. The overall threat of CRGNB is substantial because carbapenems are used to treat infections caused by ESBL-producing Enterobacterales which also exist with high frequency within the same geographical regions. A genome-based surveillance of 589 CRGNB from 61 hospitals across the federal state Hesse in Germany was implemented using next-generation sequencing (NGS) technology to obtain a high-resolution landscape of carbapenem-resistant isolates over a three-year period (2017-2019). The study examined all reportable CRGNB isolates submitted by participating hospitals. This included isolates carrying known carbapenemases (435) together with carbapenem-resistant non-carbapenemase producers (154). Predominant carbapenemase producers included Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii and Acinetobacter baumannii. Over 80% of 375 carbapenem-resistant determinants including KPC-, NDM-, VIM- and OXA-48-like ones detected in 520 Enterobacterales were plasmid-encoded, and half of these were dominated by a few incompatibility (Inc) types, viz., IncN, IncL/M, IncFII and IncF(K). Our results revealed that plasmids play an extraordinary role in the dissemination of carbapenem resistance in the heterogeneous CRGNB population. The plasmids were also associated with several multispecies dissemination events and local outbreaks throughout the study period, indicating the substantial role of horizontal gene transfer in carbapenemase spread. Furthermore, due to vertical and horizontal plasmid transfer, this can have an impact on implant-associated infections and is therefore important for antibiotic-loaded bone cement and drug-containing devices in orthopedic surgery. Future genomic surveillance projects should increase their focus on plasmid characterization.

Keywords: Germany; Gram-negative bacteria; WGS; carbapenem resistance; implant-associated infections; plasmid; surveillance.

Figure 1

Overview of the CRGNB studied from Hesse, 2016–2019. (A) Percentage distribution of carbapenemase-producing (CP) and non-carbapenemase-producing (NCP) CRGBN; (B) Year-wise species distribution of all carbapenem-resistant bacteria, CP and NCP-CRGNB; (C) Numbers of identified carbapenemase types; (D) Carbapenemase distribution according to the species. * = OXA-48-like.

Figure 2

Phylogenetic tree of the carbapenem-resistant Escherichia coli isolates. The predicted plasmid types that bore KPC-2, OXA-48 or NDM-5 are indicated directly on the tree branches. MLST types, carbapenemase types and genes encoding other β-lactamases are shown. The different color lines indicate the predominant ST-types: ST38 (green), ST69 (blue), ST131 (magenta), and ST58 (red). The remaining ST’s are shown in black.

Figure 3

Phylogenetic tree of the carbapenem-resistant Klebsiella pneumoniae isolates and the identified carbapenemase-encoding plasmids. Clonal clades belonging to KPC-3-positive ST512, ST101 and ST307, NDM-1-carrying ST147 and OXA-232-encoding ST231 were present. KPC-2 and OXA-48 were distributed broadly in 30 and 18 different ST types, respectively. A total of 80% of KPC-2 genes were predicted to be located on an IncN (pMLST-15) plasmid and 67% of the OXA-48 genes were carried by the plasmid of the IncL/M(pOXA-48) group.

Figure 4

Phylogenetic tree and genomic characterization of the Citrobacter spp. The identified carbapenemase-encoding plasmid types are indicated on the tree. Genomes of Citrobacter freundii have a large diversity. MLST types, carbapenemase types and other beta-lactamase genes are shown. Citrobacter species other as “freundii” are indicated: C. koseri (red), C. portucalensis (green), C. braakii (magenta).

Figure 5

Phylogenetic tree of the carbapenem-resistant Enterobacter spp. based on whole genome alignment (MAUVE). MLST types and carbapenemase types as well as other beta-lactamase genes identified are indicated.

Figure 6

Phylogenetic tree of the Acinetobacter baumannii isolates. OXA-23 and OXA-72 were the most predominant carbapenemase types and associated with genome types ST2 and ST636 according to the Pasteur-MLST scheme.

Figure 7

Phylogenetic tree based on whole genome sequence alignment (MAUVE) and the genetic characteristics of all remaining species except E. coli, K. pneumoniae, Acinetobacter baumannii, Citrobacter spp. and Enterobacter spp.

Figure 8

Relative distribution of carbapenemase types within the predominant species Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Acinetobacter baumannii and Enterobacter spp. according to the study periods.

Figure 9

Characteristics of the most prevalent plasmids harboring KPC-2, KPC-3, VIM-1, NDM-5 and OXA-48 in Hesse, 2016–2019. (A) Geographical distribution; (B) Their occurrence in the bacterial species in Hesse from 2017 to 2019.