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

Carbapenem-hydrolyzing 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 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 2014-2019 in comparison with genome sequences retrieved from 29 countries.

By combining short-read and long-read sequencing, 47 and 132 complete bla_OXA-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 bla_OXA-48 or bla_OXA-232 were mostly resistant, while E. coli bla_OXA-48, bla_OXA-181 and chromosomal bla_OXA-48 or bla_OXA-244 isolates were mostly sensitive for meropenem. Analysis of chromosomally localized bla_OXA-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 bla_OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, although a highly diverse bla_OXA-48-like plasmid subgroup was present. Chromosomally encoded bla_OXA-48-like alleles are from distinct genetic backgrounds and occurs at variable positions containing the DR, thereby indicating multiple independent transpositions.

Importance OXA-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.