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Author affiliation: Author affiliations: Université Paris-Saclay, Fontenay-aux-Roses and Le Kremlin-Bicêtre, France (I. Rezzoug, D. Girlich, C. Emeraud, L. Dortet); Assistance Publique-Hôpitaux de Paris, Le Kremlin-Bicêtre (I. Rezzoug, J. Poiraud, L. Dortet); Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-Producing Enterobacteriaceae, Le Kremlin-Bicêtre (I. Rezzoug, C. Emeraud, L. Dortet); Microbes, Intestin, Inflammation et Susceptibilité de l’Hôte, Clermont-Ferrand, France (A. Birer, R. Bonnett); Centre National de Référence de la Résistance aux antibiotiques, service de Bactériologie, CHU Gabriel-Montpied, Clermont-Ferrand (A. Birer, R. Bonnett); National Reference Laboratory for Monitoring of Antimicrobial Resistance in Gram-Negative Bacteria, CHU Dinant-Godinne, UCL Namur, Yvoir, Belgium (P. Bogaerts)
Carbapenems are among the last-resort antimicrobial agents available to treat infections caused by multidrug-resistant gram-negative bacteria. Extensive use of carbapenems has led to emergence of carbapenem-hydrolyzing enzymes, known as carbapenemases, that hamper last-resort antimicrobial drug therapies (1). Carbapenemase-encoding genes are mostly carried on plasmids, which enable rapid dissemination of those genes among gram-negative bacteria.
In France, OXA-48–like enzymes are the most prevalent carbapenemases in Enterobacterales (2). However, in recent years, the prevalence of metallo-β-lactamases, particularly New Delhi metallo-β-lactamase (NDM), increased exponentially (3), and 86 NDM variants had been identified by April 2025 (http://www.bldb.eu). Diverse plasmid types, such as IncF, IncA/C, IncL/M, IncH, IncN, and IncX3, carry blaNDM genes (3,4). We characterized NDM-producing Enterobacterales circulating in France during 2021–2023 to decipher the prevalence of NDM variants among Enterobacterales species.
Figure 1
Figure 1. Variants and species involved in emergence and polyclonal dissemination of blaNDM-7–carrying InX3 plasmid in Enterobacter cloacaecomplex, France, 2021–2023. A) Distribution of NDM variants among CPE….
Among 11,825 carbapenemase-producing Enterobacterales (CPE) isolates received at the French National Reference Center for Antimicrobial Resistance (F-NRC) during January 1, 2021–December 31, 2023, we recovered and genetically characterized 3,367 NDM-producing isolates (Appendix 1). We found that the number of NDM producers increased from 22.5% (592/2,582) of all CPE isolates in 2021 to 27.1% (1,135/4,187) in 2022 and 32.4% (1,640/5,056) in 2023 (Figure 1, panel A; Appendix 2). Among isolates, NDM-1, NDM-5, NDM-7, and NDM-14 represented 98% of all NDM enzymes (Figure 1, panel B).
We identified NDM-type carbapenemases in 10 different genera (Appendix 1), but the distribution of NDM variants varied drastically within Enterobacterales species (Figure 1, panel B). We identified NDM-1 mainly in Klebsiella pneumoniae (43%) and NDM-5 predominantly in Escherichia coli (67%), and we found that NDM-14 was nearly exclusively associated with K. pneumoniae (98%). Unexpectedly, Enterobacter cloacae complex (ECC) accounted for 41% (107/261) of the NDM-7 producers.
Figure 2
Figure 2. Distribution of STs among 107 Enterobacter cloacae complex isolates used in a study of emergence and polyclonal dissemination of blaNDM-7–carrying InX3 plasmid, France, 2021–2023. ST,…
Among the 107 NDM-7–producing ECC isolates, we identified 32 different sequence types (STs), the most prevalent of which were ST873 (22.4%), ST135 (10.2%), ST145 (10.2%), ST683 (9.3%), ST252 (6.5%), and ST32 (5.6%) (Figure 2). Those STs corresponded to 8 different Enterobacter species, including 46.7% of E. hormaechei subspecies hoffmanii (mostly ST135, ST145, and ST683), 22.4% E. quasihormaechei (all ST873), 7.4% E. hormaechei subsp. steigerwaltii, 6.5% (7/107) E. xianfangensis, 6.5% E. asburiae, 5.6% E. kobei (all ST32), 2.8% E. cloacae, 0.9% E. hormaechei subsp. oharae, and 0.9% E. hormaechei subsp. hormaechei. Phylogenetic analysis confirmed the polyclonal dissemination of NDM-7–producing ECC, including in the 4 major STs (ST873, ST145, ST135, and ST683) comprising several isolates (Appendix 1 Figure 1).
Among the 107 NDM-7–producing ECC isolates, short-read sequencing enabled us to identify 12 different plasmid replicases (Appendix 2). Long-read sequencing performed on 30 representative NDM-7–producing ECC showed that blaNDM-7 was carried on an IncX3-type plasmid in all isolates (Appendix 1). Of note, 93.3% (28/30) of the IncX3 plasmids were 46,161-bp long, and 6.7% (2/30) were 49,830-bp long (Appendix 1 Figure 2). The size difference corresponded to the Tn5403 transposon. Those 2 plasmids shared 99.9% nucleotide identity with the 46,161-bp blaNDM-5–carrying plasmid pEC21Z078-46K (GenBank accession no. CP10126), previously described in an E. coli isolate from China (5).
To compare the blaNDM-7–carrying plasmids with those harboring blaNDM-5 in Enterobacter spp., we performed long-read sequencing on 19 additional NDM-5–ECC isolates (Appendix 2). We found that 96% of those isolates also harbored an IncX3 plasmid. Among them, 84.2% (16/19) harbored an IncX3 plasmid almost identical to one of the blaNDM-7–carrying plasmids (except the 2 single-point mutations between blaNDM-5 and blaNDM-7); 12 harbored the 46,161-bp plasmid and 4 harbored the 49,830-bp plasmid. Among the other 3 NDM-5–producing ECC isolates, we localized blaNDM-5 on different IncFII-type plasmids: a 177,571-bp and a 96,517-bp plasmid similar to pABC143C-NDM (GenBank accession no. KY130431.1), and a 187,303-bp plasmid similar to p60214CZ (GenBank accession no. CP085746.1).
To investigate the origin of the blaNDM-7 IncX3 plasmid, we further explored 79 full-length sequences of NDM-1–producing ECC for IncX3 plasmids. Among the 11 different replicases identified, IncX3 was in only 14% of NDM-1–producing ECC. Long-read whole-genome sequencing of those 11 IncX3-positive NDM-1–producing ECC identified the blaNDM-1 gene on IncX3 plasmids of 39,582-bp (n = 7) and 44,682-bp (n = 2) length (Appendix 1 Figure 2). Both of those plasmids were close to blaNDM-5– and blaNDM-7–carrying IncX3 plasmids but showed notable differences in the genetic region surrounding the blaNDM gene (Appendix 1 Figure 2). Despite detection of an InX3 plasmid, the other 2 strains harbored blaNDM-1, either directly localized on the chromosome or on a 51,088-bp IncFII plasmid that had no similarity to plasmids available in GenBank.
The close genetic context of blaNDM genes localized on IncX3 plasmids showed that both blaNDM-7 and blaNDM-5 were flanked upstream by ΔTn2–ΔIS3000–ISAba125–IS5 and downstream by genes encoding bleMBL, PAI, ccdA, IS26, UmuD protein, ISKox3, a resolvase encoding gene, and Tn5403 (Appendix 1 Figure 2). Several elements were truncated or absent in the blaNDM-1 genetic environment compared with blaNDM-5 and blaNDM-7, including truncation of ISAba125 and deletions of IS5, ISKox3, and Tn5403.
Finally, to decipher whether the IncX3 plasmids were more prevalent in ECC, we looked for IncX3 replicase in 20,028 multidrug-resistant Enterobacterales genomes in the F-NRC database collected since 2022. Of those, 11.9% (n = 2,393) of isolates carried the IncX3-encoding replicase gene, 27.5% (n = 658) of which were Klebsiella spp., 27.3% (n = 653) were Citrobacter spp., 26.8% (n = 642) were E. coli, and 17.7% (n = 423) were ECC (Appendix 1 Figure 3).
During 2021–2023, F-NRC received increasing numbers of carbapenem-resistant Enterobacterales isolates: 2,582 in 2021, 4,187 in 2022, and 5,056 in 2023. In addition, the number of NDM-producing isolates more than tripled during that timeframe (Figure 1).
Although >86 NDM variants have been reported globally in the Beta-Lactamase DataBase (http://bldb.eu), 4 main variants are dominant in Europe: NDM-1, NDM-5, NDM-7, and NDM-14. In France, our results demonstrated that NDM-1 remains predominantly associated to K. pneumoniae, among which ST147 has been described as highly prevalent (48%) (6,7). The blaNDM-1 gene is carried on various plasmid types, including IncX3 (8), IncFIB, IncHI1B, and IncL, that harbor both resistance and virulence factors (9). In France, NDM-1 producers have been progressively replaced by NDM-5 producers since 2023. NDM-5 and NDM-7 differ from NDM-1 by substitutions responsible for an enhanced carbapenemase activity (7). In Europe, dissemination of NDM-5 is mostly caused by E. coli (10), which represented 70% of NDM-5 producers in our study. As previously described (11), our results confirmed that blaNDM-5 gene dissemination is mainly mediated by IncX3 plasmids. Our results confirmed that NDM-14–producing K. pneumoniae ST147 became established after it emerged in France in 2022 (12).
Our results highlight the emergence of NDM-7 in ECC, as noted in 41% of the isolates in our study. However, we did not identify any exclusive or particularly dominant clone involved in the dissemination of NDM-7 ECC, except ST873, which was slightly overrepresented. Of note, ECC isolates have been reported to trigger dissemination of Verona-integron–encoded metallo-β-lactamases in France, of which ST893 is the most prevalent ST (13). Our results also raise concerns about E. quasihormaechei ST873 as a high-risk clone for acquiring multidrug resistance, particularly carbapenemase resistance.
In summary, we demonstrated that the IncX3 blaNDM-7–carrying plasmids most probably derived of IncX3 blaNDM-5–carrying plasmids that were derived from IncX3 blaNDM-1–carrying plasmids and contain several additional features, including IS5, ISKox3, and Tn5403. Despite the high transfer frequency (14) and low fitness cost (15) of IncX3 plasmids, their implication in the dissemination of blaNDM remain unequal between Enterobacterales species. The strong association between IncX3 blaNDM-7–carrying plasmids and ECC underscores the need for enhanced surveillance to monitor spread of antimicrobial resistance.
