Two IncHI1 megaplasmids in Klebsiella species reveal transposable-element-mediated bla IMP-1 mobilisation

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Abstract Introduction Carbapenemase-producing Enterobacterales (CPE) represent a major threat to hospitalised patients worldwide. The dissemination of carbapenemase genes, such as bla IMP , is frequently mediated by mobile genetic elements including plasmids. During a previously described multispecies, healthcare-associated outbreak of bla IMP -positive CPE in North West London, two unusual isolates, IMP47 ( Klebsiella grimontii ) and IMP76 ( K. pneumoniae ), recovered in 2019 from routinely collected rectal swabs of inpatients, were predicted to harbour bla IMP-1 -carrying IncHI1 megaplasmids. Aims This study aimed to determine complete genomic sequences of IMP47 and IMP76, resolve the genetic context of bla IMP-1 , and assess the conjugative mobility of bla IMP-1 -carrying megaplasmids. Methods Genomic sequences of both isolates were recovered through hybrid assembly of Oxford Nanopore and Illumina sequencing reads. Complete plasmid sequences were characterised to determine replicons, conjugation machinery, and genes encoding resistance to antimicrobials or other stress factors. Integrons and transposable elements (TEs) within flanking regions of bla IMP-1 were resolved through genome annotation and search against public databases. Liquid-mating experiments were performed to assess the mobility of bla IMP-1 -carrying plasmids. Results Completed genome assemblies were generated from both isolates, confirming two bla IMP-1 -carrying megaplasmids, pIMP47 (391 kbp) and pIMP76_1 (519 kbp), of the replicon type IncHI1A(pNDM-CIT)/IncHI1B(pNDM-CIT). The bla IMP-1 locus was carried by nearly identical class 1 integrons in both plasmids and a closely related IncHI1 megaplasmid pEB3_IMP1 (361 kbp) previously identified in South West England. Comparative analysis revealed conserved genetic structures linking bla IMP-1 to mercury-resistance genes and TEs Tn c025 , Tn As3 , IS 2c , and IS 5075 , suggesting a history of recombination and potential for TE-mediated mobilisation. Conjugation experiments confirmed transfer of pIMP76_1 into a recipient K. pneumoniae strain, resulting in acquisition of ertapenem resistance, whereas transfer of pIMP47 was not observed under the tested conditions. Conclusion Two IMP-producing IncHI1 megaplasmids in gut-colonising Klebsiella species revealed TE-mediated bla IMP-1 mobilisation. The co-localisation of bla IMP-1 and metal-resistance genes in both plasmids highlights the potential for co-selection of bla IMP-1 in environments enriched with metal ions. Our findings underscore the importance of longitudinal genomic surveillance of carbapenemase-encoding megaplasmids in healthcare settings. Impact statement This study characterises two bla IMP-1 -carrying IncHI1 megaplasmids in Klebsiella isolates recovered in 2019 from rectal swabs of inpatients in London. Comparative genomic analysis revealed that bla IMP-1 was embedded within a conserved class 1 integron linked to insertion sequences and transposon-borne mercury-resistance genes, suggesting that transposable-element-mediated recombination has contributed to the acquisition of bla IMP-1 in IncHI1 plasmids and may drive further mobilisation of this carbapenem-resistance determinant. Experimental evidence confirmed conjugative transfer of one megaplasmid and associated ertapenem resistance encoded by this plasmid. Our findings identify IncHI1 megaplasmids as potential emerging vectors of bla IMP-1 and emphasise the importance of genomic surveillance for monitoring the spread of carbapenemase-encoding megaplasmids in healthcare settings. Data summary Raw Illumina and nanopore whole-genome sequencing reads of isolates IMP47 and IMP76 have been deposited in the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena ) under BioSample accessions SAMEA6990777 and SAMEA6990795, respectively. Complete genome assemblies of these isolates are accessible in the ENA under Assembly accessions GCA_903936125.2 (IMP47) and GCA_903936275.2 (IMP76). All data accessions are available in supplementary Table S1.

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