Diversification of bla OXA-48-harbouring plasmids among carbapenemase-producing Enterobacterales, 11 years after a large outbreak in a general hospital in the Netherlands

<b>Introduction.</b> Genes encoding OXA-48-like carbapenem-hydrolyzing enzymes are often located on plasmids and are abundant among carbapenemase-producing <i>Enterobacterales</i> (CPE) worldwide. After a large <i>bla</i> _OXA-48 plasmid-mediated outbreak in 2011, routine screening of patients at risk of CPE carriage on admission and every 7 days during hospitalization was implemented in a large hospital in the Netherlands. The objective of this study was to investigate the dynamics of the hospitals' 2011 outbreak-associated <i>bla</i> _OXA-48 plasmid among CPE collected from 2011 to 2021.<b>Methods.</b> A selection of 86 <i>bla</i> _OXA-48-carrying CPE isolates was made from 374 isolates collected over an 11-year study period. Species included <i>Escherichia coli</i> (Eco), <i>Klebsiella pneumoniae</i> (Kpn), <i>Enterobacter cloacae complex</i> (Ecl), <i>Citrobacter freundii</i> (Cfr), <i>Citrobacter koseri</i> (Cko) and <i>Morganella morgani</i> (Mmo). Short-read sequencing was combined with long-read sequencing for all isolates to reconstruct <i>bla</i> _OXA-48-like plasmids and chromosomes of CPE. MASH, MOBsuite, ResFinder, PlasmidFinder and SNP analyses were performed to study diversity. pOXA-48 plasmids were compared to plasmid sequences that were sequenced for the Dutch CPE surveillance in the same time period.<b>Results.</b> In total for the 86 CPE, 2 failed genomic assemblies and 78 <i>bla</i> _OXA-48-encoding plasmids were reconstructed, and six <i>bla</i> _OXA-48 genes were located chromosomally. The 2011 outbreak-associated <i>bla</i> _OXA-48 plasmid of 63.6 kb with IncL replicon was found in Cfr, Ecl, Eco, Kpn and Mmo and primarily between 2011 and 2014 and indicated as LR025105 as MASH nearest neighbour. From 2014 onwards, 11 other types of <i>bla</i> _OXA-48-carrying plasmids with different antibiotic-resistant genes and replicons were discovered, representing the earlier defined distinct pOXA-48 plasmid groups found in the Netherlands. Furthermore, on a national level, the LR025105 plasmid was found after 2015 in many different bacterial backgrounds, highlighting the promiscuous nature of this pOXA-48 plasmid.<b>Conclusion.</b> After a large <i>bla</i> _OXA-48 outbreak in a large hospital in the Netherlands, the composition of the <i>bla</i> _OXA-48 plasmid population in this hospital diversified over time and is in line with national surveillance data. Plasmid sequencing provided valuable insight into the transmission dynamics of <i>bla</i> _OXA-48-encoding plasmids and showed no indication of the persistence of the 2011 <i>bla</i> _OXA-48 plasmid in the hospital environment.