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

Introduction. Genes encoding OXA-48-like carbapenem-hydrolyzing enzymes are often located on plasmids and are abundant among carbapenemase-producing Enterobacterales (CPE) worldwide. After a large bla _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 bla _OXA-48 plasmid among CPE collected from 2011 to 2021.Methods. A selection of 86 bla _OXA-48-carrying CPE isolates was made from 374 isolates collected over an 11-year study period. Species included Escherichia coli (Eco), Klebsiella pneumoniae (Kpn), Enterobacter cloacae complex (Ecl), Citrobacter freundii (Cfr), Citrobacter koseri (Cko) and Morganella morgani (Mmo). Short-read sequencing was combined with long-read sequencing for all isolates to reconstruct bla _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.Results. In total for the 86 CPE, 2 failed genomic assemblies and 78 bla _OXA-48-encoding plasmids were reconstructed, and six bla _OXA-48 genes were located chromosomally. The 2011 outbreak-associated bla _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 bla _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.Conclusion. After a large bla _OXA-48 outbreak in a large hospital in the Netherlands, the composition of the bla _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 bla _OXA-48-encoding plasmids and showed no indication of the persistence of the 2011 bla _OXA-48 plasmid in the hospital environment.