Emergence and dissemination of an IncC plasmid co-harbouring bla_KPC-2, bla_NDM-1 and bla_VIM-24 carbapenemase genes across multiple Gram-negative species in Colombia

Abstract

Objectives

Carbapenem-resistant Gram-negative bacilli (CRGNB) pose a public health threat, increasingly driven by plasmid-mediated dissemination of carbapenemase genes. As part of a Colombian genomic monitoring programme, we aimed to characterize CRGNB co-harbouring multiple carbapenemase genes and determine their genetic context.

Methods

We screened 832 carbapenem-resistant isolates collected during 2011–2023 using carbapenemase-targeted qPCR. Ten isolates co-harbouring bla_KPC, bla_NDM and bla_VIM underwent antimicrobial susceptibility testing, long-read whole-genome sequencing, comparative genomics and conjugation assays.

Results

Nine Enterobacterales isolates and one Aeromonas caviae isolate from unrelated patients in geographically distant Colombian hospitals carried closely related IncC plasmids ranging from 160 to 317 kb. These plasmids co-harboured bla_KPC-2, bla_NDM-1 and bla_VIM-24 within three conserved multidrug resistance regions, including a Tn21-associated region carrying bla_NDM-1 and bla_VIM-24 and a Tn6454-associated bla_KPC-2 platform, alongside class 1 integrons, insertion sequences and multiple additional antimicrobial resistance determinants. The ten isolates were resistant to multiple β-lactam/β-lactamase inhibitor combinations, and five were cefiderocol resistant. Aztreonam/avibactam remained active against all isolates; colistin resistance was confined to intrinsically resistant Providencia rettgeri and Serratia sarumanii. Conjugation assays demonstrated plasmid transfer to Escherichia coli J53, and sequencing confirmed transfer of a 179 kb IncC plasmid with functional conjugation machinery. Comparative genomic analysis showed a highly conserved IncC plasmid backbone across multiple species, supporting broad dissemination of this resistance platform.

Conclusions

This first report of a single plasmid co-harbouring three carbapenemase genes. This finding highlights a concerning mechanism for interspecies dissemination of MDR Gram-negative pathogens and underscores the importance of long-read genomic surveillance focused on high-risk plasmids.

Introduction

Carbapenem-resistant Gram-negative bacilli (CRGNB) represent a significant global health threat due to limited treatment options available for these multidrug-resistant pathogens.¹ Carbapenemases, including serine carbapenemases (e.g. KPC, OXA-48) and metallo-β-lactamases (MBL; e.g. NDM, VIM), have disseminated globally.² Carbapenemase genes disseminate through successful bacterial lineages and mobile genetic elements (MGEs), such as plasmids and transposons. KPC-2 was first reported in 2005 in Colombia, where KPC is endemic, followed by VIM (2011) and NDM (2013).^3,4  bla_KPC-2 remains the most prevalent carbapenemase gene among K. pneumoniae isolates, particularly within high-risk clones ST307 and ST1082, while bla_KPC-3 is strongly associated with ST11.⁵ These clones commonly harbour a diversity of plasmids, such as IncC and IncF replicon types.⁵

Sporadic CRGNB co-producing dual carbapenemases have been reported for more than a decade in the USA, Europe and Latin America,^2,6–9 these combinations most commonly involving a serine carbapenemase together with a MBL, thereby broadening resistance to β-lactam due to their distinct susceptibility profiles to β-lactamase inhibitors. Of particular concern, in Colombia, the proportion of Enterobacterales co-harbouring multiple carbapenemases increased markedly, rising from 1.1% in 2018 to nearly 50% of tested isolates in 2024 co-producing KPC and NDM.^10,11 The introduction of ceftazidime/avibactam and meropenem/vaborbactam has expanded treatment options for serine β-lactamase producers, although the increasing prevalence of isolates co-harbouring a MBL threatens the effectiveness of these therapies, substantially limiting therapeutic options.⁶

The limited information available on the genetic elements mobilizing multiple carbapenemase genes hinders our understanding of their dissemination. Long-read sequencing enables reconstruction of the genomic context of resistance determinants within a strain, identifying whether they are carried on separate plasmids, as is often the case, or co-localized on the same genetic element.¹² In this study, leveraging a large national genomic monitoring collection of clinically relevant CRGNB, we identified clinical isolates co-producing multiple carbapenemases and characterized a novel conjugative IncC plasmid co-harbouring bla_KPC-2, bla_NDM-1 and bla_VIM-24 across multiple Gram-negative species recovered from Colombian hospitals.

Materials and methods

Within the Grupo de Investigación en Resistencia Antimicrobiana y Epidemiología Hospitalaria (RAEH), 832 clinical or carriage CRGNB isolates collected during 2011–2023 from 37 hospitals across 17 Colombian cities were screened for carbapenemase genes by in-house qPCR.¹³ Ten isolates co-harbouring three distinct carbapenemases, confirmed with the NG-test^® CARBA5 immunoassay (NG Biotech, France), were identified and retained for this study. MICs were determined using Sensititre EUMDRXXF panels (ThermoFisher Scientific, USA) and UMIC cefiderocol strips (cat. no. UM-CID-040, Bruker Daltonics, Germany), and by gradient diffusion for aztreonam/avibactam (cat. no. 92089, Liofilchem, Italy), both ranging between 0.03–32 mg/L. Testing and result interpretation followed EUCAST V15.0 guidelines and breakpoints, including reference strain ATCC 25922. Conjugation assays were performed as previously described¹² between K. pneumoniae UEB72 (the earliest isolate) and E. coli J53, selecting on meropenem (1 mg/L). High-molecular weight DNA (Promega, USA) was extracted from exponential-phase broth cultures as previously described¹² and used for WGS of the ten clinical isolates and transconjugant using long-read sequencing (Oxford Nanopore Technologies, UK) with the Sequencing by Ligation kit V14 and R10.4.1 flow cells. Assembly was performed using Flye (v2.9.4), followed by polishing with Medaka (v1.12.1) and annotation with Center for Genomic Epidemiology databases and tools (MLST v2.0.9, ResFinder v2.4.0, PlasmidFinder v2.2.0) and MOB-Suite (V3.1.9). The cgMLST analysis was performed using Ridom SeqShere+ (v10.5.0). Plasmid alignments and regions were visualized with ProkSee (build 2026–02–25).

Results

Ten isolates consistently carrying bla_KPC-2, bla_NDM-1 and bla_VIM-24 were recovered from adult patients in hospitals across Colombia, with the earliest isolate dating back to 2019 (Tables S1–S2, available as Supplementary data at JAC-AMR Online). These isolates included five K. pneumoniae (Kpn), one K. aerogenes (Kae), one Enterobacter hormaechei (Eho), one Providencia rettgeri (Pre) and one Serratia sarumanii (Ssa; S. marcescens complex) associated with clinical infection, as well as one Aeromonas caviae (Acv) recovered from rectal screening. Genomic analysis showed that the K. pneumoniae isolates belonged to distinct genetic lineages: ST987 (Kpn_UEB72), ST17 (Kpn_UEB62), ST1441 (Kpn_UEB50) and ST307 (Kpn_UEB85 and Kpn_UEB87, differing by 11 loci based on cgMLST) (Figure 1; Table S1).

Figure 1.

Genomic characteristics and antimicrobial resistance determinants of clinical isolates co-harbouring bla_KPC-2, bla_NDM-1 and bla_VIM-24. Clinical isolates and associated plasmids are grouped by strain. Columns indicate chromosome or plasmid length, MLST, plasmid replicon type, relaxase type, predicted mobility, capsule locus (KL), O locus and the presence of antimicrobial resistance genes. Squares indicate the gene presence, with circles identifying resistance genes carried by the IncC plasmid. The conserved IncC plasmids carrying bla_KPC-2, bla_NDM-1 and bla_VIM-24 are highlighted by shared resistance profiles and conjugative MOBH relaxases across multiple bacterial species. Additional plasmid and resistance determinants identified in individual isolates are also shown. MRK257 corresponds to the E. coli J53 transconjugant obtained from conjugation assays with Kpn_UEB72.

All isolates exhibited an MDR phenotype, with broad resistance to β-lactams, including the newer β-lactam/β-lactamase inhibitor (BL/BLI) combinations ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam, while remaining susceptible to aztreonam/avibactam (Figure 1; Table S1). Five isolates, three K. pneumoniae (UEB72, UEB50 and UEB62), P. rettgeri (UEB89) and S. sarumanii (UEB92), were also resistant to cefiderocol. All isolates remained susceptible to colistin, except the intrinsically resistant P. rettgeri and S. sarumanii. Complete antimicrobial susceptibility profiles are shown in Table S1.

WGS enabled comparative genomic analysis of the genetic context associated with triple carbapenemase production. All ten genomes harboured a highly conserved IncC plasmid backbone carrying bla_KPC-2, bla_NDM-1 and bla_VIM-24. Most IncC plasmid sizes ranged from 160 to 179 kb except for a 251 kb in Kae_UEB75 and a 317 kb multireplicon plasmid in Kpn_UEB50 (Figure 1). Using the 178 kb plasmid Kpn_UEB72, recovered from the earliest isolate, as a reference, we observed a highly conserved structure across all isolates (Figure 2). Three multidrug resistance regions (MDRr), containing clustered AMR genes and MGEs, were identified. MDRr1 contained a Tn21-associated region with four modules: (i) a class 1 integron (dfrA17, aac(6’)-Ib-cr, bla_OXA-1, catB3, arr-3), (ii) bla_NDM-1 flanked by IS91 and IS4321, (iii) another class 1 integron (bla_VIM-24, aac(6’)-II, catB3, ant(2'’)-Ia, aph(2'’)-VIa and sul1) and (iv) IS1353 and the mercury resistance operon merEDCPTR. MDRr2 contained a Tn6454-associated bla_KPC-2 platform with Δbla_TEM, tnpR and tnpA genes. MDRr3 harboured arsenic, macrolide (msr(E), mph(E) and ermD) and sulphonamide (sul2) resistance determinants flanked by IS6 and IS91 (Figure 2).

Figure 2.

Comparative analysis of IncC plasmids co-harbouring bla_KPC-2, bla_NDM-1 and bla_VIM-24 in multiple CRGNB species. Circular comparative alignment of IncC plasmids identified in ten isolates representing different Gram-negative bacterial species, using pUEB72 (178 kb) as the reference plasmid. Sequence comparisons and annotations were visualized using ProkSee. Concentric rings represent individual plasmids, with sequence similarity to pUEB72 indicated by shading and gaps representing non-homologous regions. The previously reported Colombian plasmid p6234, which carries bla_NDM-1 as its sole carbapenemase gene, is included for comparison.¹⁴ The outer annotation track displays predicted coding sequences and key genetic features, including antimicrobial resistance genes, insertion sequences and transposition-associated genes, conjugation-associated genes (tra), integrases and plasmid backbone genes. Three conserved multidrug resistance regions (MDRr1–3) are indicated, including a Tn21-associated region carrying bla_NDM-1 and bla_VIM-24, and a Tn6454-associated bla_KPC-2 platform, and MDRr3, carrying macrolide and sulphonamide resistance determinants.

In the conjugation assay, K. pneumoniae UEB72 successfully transferred the plasmid to E. coli J53 (conjugation frequency: 4.4 × 10⁻³), and WGS performed on the transconjugant confirmed the presence of a 179 kb IncC plasmid co-harbouring the three carbapenemase genes along with transfer-associated features including oriT, the MOBH relaxase, type IV coupling proteins, a type IV secretion system and the higBA toxin-antitoxin system for plasmid stability (Figures 1 and 2).

Discussion

We have identified an emerging high-risk plasmid in Colombia by screening a large national CRGNB genomic monitoring collection. Our analysis revealed a novel IncC plasmid co-harbouring bla_KPC-2, bla_NDM-1 and bla_VIM-24 genes in six different species, isolated from geographically distant Colombian hospitals. Its detection in healthcare settings across the country without any clear spatiotemporal link (except for the two closely related ST307 isolates) suggests broad and successful dissemination. Our results indicate that this plasmid is broad-host-range and self-conjugative. It harboured distinct multidrug resistance regions, including a Tn21-associated region carrying both bla_NDM-1 and bla_VIM-24, and a non-Tn4401 bla_KPC-2 platform (Tn6454). To our knowledge, the co-localization of these three carbapenemase genes on a single plasmid has not been previously reported.

The combination of bla_NDM-1, bla_KPC-2 and bla_VIM-24 on a single plasmid is consistent with the molecular epidemiology in Colombia, where (i) KPC is endemic, (ii) MBLs are increasingly reported and (iii) IncC plasmids are widely associated with antimicrobial resistance in GNB.¹⁴ Globally, dual carbapenemase-producing isolates have been reported with increasing frequency. Between 2012 and 2019, the U.S. CDC recorded over 150 cases of Enterobacterales harbouring multiple carbapenemase genes, often linked to healthcare exposure abroad.¹⁵ However, these reports did not specify whether the genes were located on the same plasmid. In several countries, notably those with endemic carbapenemase circulation, multiple carbapenemase genes have been identified in the same strain, although typically located on separate genetic elements. Such cases have been reported in K. pneumoniae isolated from Greece, Colombia and elsewhere, often involving KPC, VIM and NDM on distinct plasmids or unresolved genetic platforms.^14,16,17

In our study, three K. pneumoniae, E. hormaechei and P. rettgeri isolates also carried additional copies of bla_KPC (including bla_KPC-3), while A. caviae harboured an extra copy of bla_VIM-24, located on separate plasmids or chromosome (Figure 1; Table S1). The co-localization of three carbapenemase genes on a single plasmid, as demonstrated in this study, represents yet another step in Gram-negative resistance evolution, significantly increasing the potential for plasmid-mediated gene transfer across bacterial species. Combinations of serine carbapenemases and MBLs pose a significant clinical challenge. While ceftazidime/avibactam and meropenem/vaborbactam are effective against KPC-producing Enterobacterales, their activity is compromised when MBLs are co-produced. Aztreonam/avibactam remained active against all ten isolates with low MICs; however, its limited availability in many low- and middle-income countries represents a significant issue. Colistin remained the only active option that is widely available but has limited clinical efficacy. Although five isolates were resistant to cefiderocol, this phenotype was not transferred by conjugation from K. pneumoniae UEB72 to E. coli J53, indicating that, at least in this isolate, it is not strictly plasmid encoded.

This study highlights the value of long-read sequencing for genomic surveillance of highly resistant CRGNB, enabling accurate reconstruction of complete plasmids and detection of co-located resistance genes, MGEs and plasmid backbones across diverse hosts. Such capabilities are critical for tracking and monitoring high-risk plasmids, such as the one described here. Further studies are warranted to elucidate the origin, transmission dynamics and persistence of this element. Continued genomic surveillance, infection control measures and antimicrobial stewardship programmes remain essential to curb the dissemination of these pathogens.

Funding

This study was funded by Swiss Confederation State Secretariat for Education, Research and Innovation through the Swiss Leading House Latin America Research Partnership Grant RPG2102 (D. O. A., M. V. V.), the Fondation Ernst et Lucie Schmidheiny (D. O. A., R. S.), D. O. A. received funding from the Geneva University Hospitals, Research and Development Program scheme (PRD 3-2022-II) and M. V. V. from Universidad El Bosque Research Grant PCI-2022-11023.

Transparency declarations

M. V. V. has received consulting fees and/or research grants from Merck Sharp and Dohme, Pfizer, WEST and bioMérieux. D. O. A has received presentation fees from Pfizer. All other authors declare no financial or non-financial competing interests.

Supplementary data

Tables S1 to S2 are available as Supplementary data at JAC-AMR Online.

Data availability

Data are available under BioProject No. PRJNA1292751.