bla OXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

Abstract

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

Flanker: a tool for comparative genomics of gene flanking regions

“…bla KPC has largely been associated with variants of a ~10 kb transposon, Tn 4401 [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 (, Table S2). This window length was chosen to capture the entire Tn 4401 sequence upstream of bla KPC .…”

“…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).…”

“…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 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.…”

Genomic Epidemiology of Colistin-resistant Enterobacterales from Dutch Patients: A Prospective Matched Case-control Study

“…NGS, TGS and wgMLST were performed as described before. 67 In short, genetic relatedness was assessed by wgMLST and genetic clusters were defined as collections of isolates with a maximum of 25 alleles differences for E coli and 20 for K. pneumoniae. AMR genes were identified via ResFinder software and only AMR genes with ≥97% sequence identity with the reference sequences were included.…”