Carbapenem resistance in Escherichia marmotae: characterization of IncL plasmids carrying bla_OXA-48

Dear Sir,

Carbapenemase-producing Enterobacterales (CPE) represent a significant concern to human health. Among these, the bla_OXA-48 gene, often located on the highly stable incompatibility group L (IncL) plasmids. These plasmids enable rapid horizonal gene transfer across bacterial species, posing an emerging threat.

Escherichia marmotae was first described in 2015. It was initially considered susceptible to antimicrobials. However, present-day findings indicate resistance to last-resort agents and high pathogenic potential. The genetic proximity to E. coli raises concerns about its potential in AMR dissemination. A recent study identified E. marmotae isolates harbouring extended-spectrum β-lactamase (ESBL) genes located on plasmids, suggesting the capacity to acquire and propagate AMR via horizontal gene transfer.¹ Sivertsen et al. reported bla_KPC-carrying E. marmotae, reinforcing its invasive pathogen potential,² aligning with findings by Liu et al.³

Since 2014, CPE samples have been collected and whole genomes sequenced as part of the Danish CPO surveillance programme. In 2023 and 2024, two carbapenemase-producing E. marmotae isolates were submitted to the National Reference Laboratory at Statens Serum Institut (NRL, SSI) for verification and characterization. Whole-genome sequencing using short- and long-read technologies identified resistance genes, virulence factors, plasmid content and assessed genetic relatedness. The bla_OXA-48-carrying IncL plasmids were compared for structural similarities.

The two E. marmotae isolates originated from geographically distinct patients. The first isolate, submitted in 2023 by Aalborg University Hospital, was confirmed as E. marmotae carrying bla_OXA-48. Retrospective data revealed that Patient 1 had a previous infection with bla_OXA-48-producing K. oxytoca 6 months earlier. In the E. marmotae, virulence factors chuA, iss, ompT and sitA were identified.

In 2024, a second E. marmotae isolate was submitted by Copenhagen University Hospital, Amager and Hvidovre. This isolate also carried bla_OXA-48 and a broad array of virulence genes, including the porcine colonization factor F4 (faeD/E/F/H/I) typical of enterotoxigenetic E. coli (ETEC), ExPEC associated genes (papAH/C and kpsMII) and genes characteristic of hybrid ExPEC-STEC O80:H2 strains (cia, etsC, iss, traJ, ompT, hlyF, iroN).⁴ The presence of haemolysin (hlyF) and haem uptake system (chuA) suggest additional virulence factors enhancing bacterial survival and pathogenicity. Retrospective searches revealed that Patient 2 had a previous carbapenemase-negative E. marmotae, which was neither collected nor sequenced.

E. marmotae has often been misidentified as E. coli, partly because it was not included in the Bruker^™ MALDI-TOF database until 2020. Consequently, a retrospective rMLST analysis of 907 Danish CPE E. coli isolates was performed, but no additional E. marmotae cases were identified.

Although reports of resistant E. marmotae remain limited, Sivertsen et al. found bla_KPC in one and bla_CTX-M in two out of 45 E. marmotae genomes.² By mid-2024, 259 E. marmotae genomes were available in the NCBI database, with 23 (9%) carrying ESBL (bla_CTX-M-14; n = 10, bla_CTX-M-15; n = 2, bla_CTX-M-1; n = 1, bla_CTX-M-32; n = 1), pAmpC (bla_CMY-2; n = 6) or carbapenemase genes (bla_KPC-2; n = 1, bla_KPC-3; n = 1), whereas one isolate carried both bla_CTX-M-14 and bla_CMY-2. Thus, the two Danish isolates represent, to our knowledge, the first reported cases of OXA-48 producing E. marmotae.

Genomic comparison revealed 28 757 SNP differences between the two Danish E. marmotae isolates. They did not show close genetic relatedness to any of the 257 publicly available E. marmotae genomes. The closest matches were 163 and 251 SNPs apart, respectively, suggesting two independent acquisitions. Epidemiological investigations found no links between the patients, further supporting this conclusion.

Both E. marmotae, as well as the earlier K. oxytoca from Patient 1, carried IncL plasmids associated with bla_OXA-48.^5,6 Genomes annotated with Bakta from long-read assemblies and visualized with Clinker⁷ confirmed similar plasmid backbones (Figure 1). Using Pling,⁸ structural comparison of the E. marmotae plasmid from Patient 1 (pEm_PT1) and the K. oxytoca plasmid (pKo_PT1) revealed a single rearrangement; an IS1 insertion upstream of bla_OXA-48 gene (Figure 1). Such rearrangements are common in interspecies plasmid transmissions, suggesting plasmid transfer between the K. oxytoca isolate and the E. marmotae isolate in Patient 1.

Figure 1.

Comparison of three IncL plasmids carrying bla_OXA-48, from two Danish patients. pKo_PT1 reconstructed from a Klebsiella oxytoca and pEm_PT1 from an Escherichia marmotae from Patient 1. pEm_PT2 from an E. marmotae from Patient 2.

Comparison of pEm_PT1 from Patient 1 and the plasmid from Patient 2 (pEm_PT2) identified two additional rearrangements; an inversion between IS10A elements and the insert/deletion of a KilA-N domain protein and ISL3 insertion element downstream of bla_OXA-48. Despite structural similarity of the two plasmids, no epidemiological links were found between the two patients. With IncL plasmids carrying bla_OXA-48 now near endemic status, the plasmids were probably acquired from another bacterial host.

In conclusion, E. marmotae, traditionally considered susceptible, has now been shown to acquire bla_OXA-48-carrying IncL plasmids. Here we report two independent acquisition events, adding E. marmotae to the list of potential recipients of this worldwide disseminated plasmid. The combination of hybrid ETEC-ExPEC virulence traits and resistance potential of E. marmotae, reinforces the importance of continued CPE surveillance and genomic characterization at both species and plasmid level.

Funding

Part of this work was supported by the Danish Ministry of Health.

Transparency declarations

All authors: None to declare.