bla_OXA-48-like genome architecture among carbapenemase-producingEscherichia coliandKlebsiella pneumoniaein the Netherlands

Abstract

Carbapenem-hydrolyzing enzymes belonging to the OXA-48-like group are encoded by blaOXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective was to investigate the characteristics, gene content, and diversity of the blaOXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014-2019 in comparison with genome sequences retrieved from 29 countries. By combining short-read and long-read sequencing, 47 and 132 complete blaOXA-48-like plasmids were reconstructed for E. coli and K. pneumoniae, respectively. Distinct plasmid groups designated as pOXA-48, pOXA-181, and pOXA-232 were identified in the Netherlands and varied in size, % G+C, presence of antibiotic resistance genes, replicons and gene content. The pOXA-48 plasmids were variable, while pOXA-181 and pOXA-232 plasmids were conserved. A group of non-related pOXA-48 plasmids contained different resistance genes, non-IncL type replicons or carried no replicons. K. pneumoniae isolates carrying blaOXA-48 or blaOXA-232 were mostly resistant, while E. coli blaOXA-48, blaOXA-181 and chromosomal blaOXA-48 or blaOXA-244 isolates were mostly sensitive for meropenem. Analysis of chromosomally localized blaOXA-48-like alleles revealed that these were flanked by a direct repeat (DR) upstream of IS1R, which were found at multiple locations in the chromosome of distinct genetic backgrounds. In conclusion, the overall blaOXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, although a highly diverse blaOXA-48-like plasmid subgroup was present. Chromosomally encoded blaOXA-48-like alleles are from distinct genetic backgrounds and occurs at variable positions containing the DR, thereby indicating multiple independent transpositions.ImportanceOXA-48-type of carbapenem hydrolyzing enzymes encoded by blaOXA-48-like genes from transmissible plasmids or chromosomes of Escherichia coli and Klebsiella pneumoniae have spread world-wide and are of concern. Dissecting the blaOXA-48-like genome architecture at the molecular level by combining short-read and long-read sequencing will lead to understanding trends in the plasmid reservoir of E. coli and K. pneumoniae in the Netherlands and may enhance future international pathogen surveillance.

Flanker: a tool for comparative genomics of gene flanking regions

“…To validate Flanker, demonstrate its application and provide a comparison with existing tools, we used two recent datasets of complete plasmids (derived from hybrid long-/ short-read assemblies) containing carbapenemase genes of clinical importance [23,28]. The first dataset comprised 51 complete bla OXA-48 -harbouring plasmids; 42/51 came from carbapenem-resistant Escherichia coli and Klebsiella pneumoniae isolates from patients in the Netherlands [28] and 9/51 from EuSCAPE (a European surveillance programme investigating carbapenem resistance in Enterobacterales) [23]. The second dataset comprised 50 bla KPC-2 or bla KPC-3 -(K.…”

“…pneumoniae carbapenemase)-harbouring plasmids in carbapenem-resistant K. pneumoniae isolated from the Netherlands [28] (8/50) and as part of the EuSCAPE study (42/50) [23]. The EuSCAPE dataset [23,29] additionally contains a large collection of short-read sequencing data for Klebsiella spp.…”

“…Using complete plasmids from the Netherlands [28]/EuSCAPE [23] hybrid assembly datasets, Flanker identified 17 distinct FPs in the 2200 bp upstream sequence of bla OXA-48 of which 7 occurred in L/M(pOXA-48)-type plasmids (Fig. 2, Table S3).…”

“…bla KPC has largely been associated with variants of a ~10 kb transposon, Tn4401 [43,44]. From the combined EuSCAPE [23] and Dutch CPE collection [28] of 50 hybrid assembled KPC-containing plasmids, Flanker identified eight distinct FPs over a 7200 bp window upstream of bla KPC-2/3 (Fig. 3, Table S2).…”

Flanker: a tool for comparative genomics of gene flanking regions

“…3). 2 Annotation (using Galileo AMR; see methods) of these revealed that whereas FP16 contains Tn1999, 3 FP6 contains Tn1999.2, which has been previously described as creating a strong promoter which 4 [26] of 50 hybrid assembled KPC-containing 1 plasmids, Flanker identified 8 distinct FPs over a 7200bp window upstream of bla KPC-2/3 (Fig. 4; Table 2 S2).…”