OXA-181 transmission confounded by a stable IncX3 plasmid

OXA-48-like carbapenemases have been historically rare, however steady increases both locally and globally have warranted further investigation into their spread. Here we present the largest genomic analysis of blaOXA-181-producing bacteria in Australia to date, focusing on a single jurisdiction over seven years (2017 - 2024). The initial investigation was prompted by an outbreak in 2017, where enhanced genomic surveillance in a single hospital identified 85 outbreak isolates related to an imported Escherichia coli ST38, carrying blaOXA-181 on an IncX3/colKP3 plasmid (previously reported as pOXA181). After four months of intensive infection control, the initial outbreak strain was eliminated. To confirm the outbreak plasmid was also contained, we collected all blaOXA-181-positive isolates from the same jurisdiction over subsequent years and sequenced with both Illumina and Oxford Nanopore Technologies to investigate clonal and mobile genetic element mediated spread. While continued surveillance post-2017 did not identify the same E. coli strain following the outbreak, pOXA181 plasmids were identified in >70% of surveillance isolates, with minimal genetic changes, which initially suggested local plasmid-mediated spread. Additional comparison to a global collection of pOXA181 plasmids found that epidemiologically unrelated pOXA181 plasmids were near identical, with no rearrangements and low, or no, single nucleotide polymorphisms. This suggests the mutation rate of pOXA-181 is incompatible with recent genomic transmission inference. This study highlights the current genomic epidemiology and drivers of blaOXA-181 and further demonstrates the necessity for detailed understanding of plasmid evolutionary rates to inform genomic surveillance. Data summaryAll assemblies and raw sequence data has been uploaded under BioProject PRJNA545001 (see accessions in Supplementary Dataset S1). Impact StatementHere we present one of the largest genomic studies of blaOXA-181-producing bacteria globally, combining seven years of local genomic surveillance with a global collection of blaOXA-181-carrying plasmids. We found that the IncX3/colKP3 plasmid carrying blaOXA-181 is remarkably stable, with epidemiologically unrelated plasmids showing little or no genetic variation. Consequently, plasmids that appear to be recently transmitted based on genomic similarity alone may instead represent independent introductions of a highly conserved plasmid. This finding highlights an important limitation of plasmid-based genomic epidemiology and demonstrates that plasmid sequence similarity cannot necessarily be interpreted as evidence of recent transmission. Our study provides a cautionary example for the increasing use of long-read sequencing and plasmid surveillance in antimicrobial resistance investigations, emphasising the need to consider plasmid evolutionary rates and epidemiological context when interpreting genomic evidence of transmission.