Comprehensive genome data analysis establishes a triple whammy of carbapenemases, ICEs and multiple clinically relevant bacteria

Abstract

Carbapenemases inactivate most β-lactam antibiotics, including carbapenems, and have frequently been reported among Enterobacteriaceae, Acinetobacter spp. and Pseudomonas spp. Traditionally, the horizontal gene transfer of carbapenemase-encoding genes (CEGs) has been linked to plasmids. However, given that integrative and conjugative elements (ICEs) are possibly the most abundant conjugative elements among prokaryotes, we conducted an in silico analysis to ascertain the likely role of ICEs in the spread of CEGs among all bacterial genomes (n=182 663). We detected 17 520 CEGs, of which 66 were located within putative ICEs among several bacterial species (including clinically relevant bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli). Most CEGs detected within ICEs belong to the IMP, NDM and SPM metallo-beta-lactamase families, and the serine beta-lactamase KPC and GES families. Different mechanisms were likely responsible for acquisition of these genes. The majority of CEG-bearing ICEs belong to the MPF_G, MPF_T and MPF_F classes and often encode resistance to other antibiotics (e.g. aminoglycosides and fluoroquinolones). This study provides a snapshot of the different CEGs associated with ICEs among available bacterial genomes and sheds light on the underappreciated contribution of ICEs to the spread of carbapenem resistance globally.

Keywords: antibiotic resistance; carbapenemases; clinically relevant bacteria; integrative and conjugative elements.

Fig. 1.

Overview of the workflow followed in this study. All assemblies available in NCBI RefSeq were downloaded and blasted against an in-house database of carbapenemases using diamond blastx (step 1). NCBI annotated proteins from CGE-bearing genomes were then extracted (step 2) and used for the identification of relaxase and serine or tyrosine recombinase (step 3). Search of directed repeats and delimitation of putative ICEs was also performed. CONJscan was used to identify the MPF family of each element. We then looked for AR genes, restriction–modification systems, CRISPR arrays and their associated (Cas) proteins, as well as secondary metabolites within extracted ICEs. We also characterized the functional annotations of their proteomes and the MLST of the genomes carrying a CEG-bearing ICE. Abbreviations: AR, antibiotic resistance; CEG, carbapenemase-encoding gene; HMM, hidden Markov model; ICE, integrative and conjugative element; MPF, mating-pair formation; RM, restriction–modification.

Fig. 2.

Sankey diagram showing the contribution of different MPF families to the spread of CEGs among several bacterial genomes. The left, centre and right axes represent the association between the identified carbapenemases, MPF type and the bacterial genus, respectively, while the width of each connection is proportional to the number of positive hits. Abbreviations: Ax, Achromobacter xylosoxidans; Af, Alcaligenes faecalis; Bt, Bordetella trematum ; Ec, Escherichia coli; Kp, Klebsiella pneumoniae; Mm, Morganella morganii ; Pas, Pseudomonas asiatica ; Pm, Pseudomonas monteilii ; Psp, Pseudomonas sp.; Sm, Stenotrophomonas maltophilia .

Fig. 3.

Schematic representation of the different insertion site/integrase/CEG/MPF/relaxase profiles identified in this study. The figure is not to scale and the relative position of the different modules (integration, conjugation, accessory CEGs) is for illustrative purposes to show the various relationships observed. Abbreviations: CDS, Coding Sequence; CEGs, carbapenemase-encoding genes; DNA_BRE_C, DNA breaking–rejoining enzymes, C-terminal catalytic domain; INT_P4_C, P4 integrase, C-terminal catalytic domain; INT_Rci_Hp1_C, shufflon-specific DNA recombinase Rci and bacteriophage Hp1_like integrase, C-terminal catalytic domain family; MPF, mating-pair formation; tRNA, transfer RNA gene.

Fig. 4.

Grouped bar chart representing the incidence of each eggNOG function broken down by category (represented by different colours) in the CEG-bearing ICE sequences.