Tracing the evolutionary genomics of bla_NDM-harboring Salmonella typhimurium in China

Abstract

Background

Carbapenem-resistant Salmonella Typhimurium (S. Typhimurium) is occasionally reported, primarily driven by the dissemination of the bla_NDM gene. Although this poses an emerging threat, its phylogenetic structure and epidemiological patterns remain insufficiently studied.

Methods

We characterized a clinical carbapenem-resistant S. Typhimurium isolate harboring bla_NDM−5 from a tertiary hospital in China using antimicrobial susceptibility testing, plasmid conjugation, and stability assays. Whole-genome sequencing was integrated with data from GenBank to perform a series of bioinformatic analyses.

Results

The bla_NDM−5 gene was located on a conjugative IncFII-type plasmid that demonstrated long-term stability in the recipient strain. Screening of GenBank identified four structurally related bla_NDM−5-carrying plasmids, all from bacterial hosts isolated in eastern Chinese cities. Comparative analysis revealed that bla_NDM−5 was embedded within an identical truncated Tn125 (ΔTn125) unit across all plasmids, suggesting a common genetic vehicle for its dissemination. Further structural analysis indicated the presence of ISCR1 adjacent to ΔTn125, with the entire resistance region bounded by IS26 elements, implying that ISCR1 may facilitate the capture and mobilization of bla_NDM−5, while IS26 could mediate co-transfer of multiple resistance modules. In addition, we systematically analyzed all available bla_NDM-positive S. Typhimurium genomes from GenBank (n = 28). Notably, all strains originated from China, and ST34 was the dominant clone (24/28, 85.7%) among bla_NDM-positive S. Typhimurium. Phylogenetic analysis classified the strains into three major clusters (I–III), showing strong concordance with Multi-Locus Sequence Types (MLST). Plasmid replicon screening identified IncHI2 and IncFII as the most prevalent plasmid types. Resistance gene profiling further confirmed bla_NDM−5 as the predominant carbapenemase subtype, followed by bla_NDM−1.

Conclusion

This study identifies a conjugative and stable IncFII-type plasmid as an important vector for bla_NDM−5 transmission in S. Typhimurium. The plasmid carries the carbapenemase gene within a conserved ΔTn125 unit, and this plasmid type may have formed a regional epidemic in eastern China. ISCR1 likely plays a key role in capturing ΔTn125 and initiating bla_NDM−5 transfer, while IS26 facilitates co-mobilization of multidrug resistance modules. Epidemiological analysis further indicates that China is the primary region affected by bla_NDM-positive S. Typhimurium, with the ST34 clone and IncHI2/IncFII plasmids acting as potential major drivers of its dissemination. These findings provide a critical foundation for clinical and public health efforts to control the spread of carbapenem-resistant S. Typhimurium.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-026-13011-3.

Introduction

The genus Salmonella consists of Gram-negative, facultatively anaerobic bacteria belonging to the Enterobacteriaceae family and is typically equipped with fimbriae and motility [1]. This genus is divided into two principal species, Salmonella enterica and Salmonella bongori, which collectively account for over 2,600 serotypes identified so far [2]. A key representative is Salmonella Typhimurium (S. Typhimurium), which primarily causes gastroenteritis, making it a predominant agent of foodborne disease worldwide [3]. However, while infections are generally self-limiting in healthy adults, they may escalate into severe, life-threatening conditions in vulnerable populations such as infants, the elderly, and immunocompromised persons [4].

Currently, the management of severe Salmonella infections remains dependent on antibiotic therapy. However, the clinical utility of many conventional antibiotics (e.g., cefazolin, gentamicin, clindamycin, and erythromycin) is limited due to resistance [5–7]. Consequently, third-generation cephalosporins and fluoroquinolones are commonly employed in clinical practice [8, 9]. The widespread overuse of antibiotics in both clinical and agricultural settings has led to a rising prevalence of multidrug-resistant Salmonella strains, posing significant challenges to infection control [10, 11]. Although still relatively uncommon, the recent emergence of carbapenem-resistant Salmonella has attracted considerable concern [12]. Addressing the therapeutic difficulties posed by such strains, while elucidating their resistance mechanisms and molecular characteristics and implementing appropriate measures to curb the spread of resistance, is of critical public health importance.

The primary mechanism of carbapenem resistance in Salmonella is the acquisition of carbapenemase genes, which encode enzymes capable of hydrolyzing carbapenem antibiotics [13]. Based on molecular structure and substrate specificity, carbapenemases are categorized into three classes: A (e.g., GES, KPC, SME), B (e.g., NDM, IMP, VIM), and D (e.g., OXA) [14]. To date, various carbapenemase genes, including bla_NDM, bla_KPC, bla_IMP, bla_VIM, and bla_OXA-48, have been detected in carbapenem-resistant Salmonella, with associated strains distributed across multiple global regions [12, 13]. The bla_NDM gene was first reported in 2008 in Klebsiella pneumoniae, and, due to its ability to hydrolyze almost all β-lactam antibiotics except aztreonam, the NDM enzyme it encodes has garnered significant attention [15]. Currently, 84 bla_NDM variants have been identified globally (http://www.bldb.eu/BLDB.php?prot=B1#NDM). Bacterial strains carrying this gene often cause various infections and are associated with high mortality rates [16, 17]. The first case of bla_NDM-carrying S. Typhimurium was reported in 2017 in Shanghai, China, isolated from a fecal specimen of a 71-year-old male patient in an intestinal outpatient clinic [18]. Since then, sporadic cases have been reported in strains from clinical patients and animal sources [19–26]. However, there is currently a lack of systematic reports on the epidemiological distribution, genomic evolutionary characteristics, plasmid backgrounds, and repertoire of other resistance genes in bla_NDM-harboring S. Typhimurium.

Here, we isolated and characterized a clinical S. Typhimurium strain carrying bla_NDM−5, with in-depth analysis of its genomic features and the bla_NDM-harboring plasmid. The genetic context of bla_NDM was thoroughly dissected to elucidate its mobilization mechanism. By integrating data from the GenBank database, we further investigated the dissemination patterns and genomic evolution of bla_NDM-positive S. Typhimurium.

Materials and methods

Sample collection, species identification and phenotypic assays

Strain K1858 was isolated in 2024 from a pediatric diarrhea patient at a tertiary hospital in Zhejiang, China. The isolate was initially identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Genomic confirmation of the species was achieved through Average Nucleotide Identity (ANI) analysis [27]. Antimicrobial susceptibility testing was performed with the bioMérieux VITEK 2 system, following the Clinical and Laboratory Standards Institute (CLSI 2024) breakpoints [28]. Carbapenemase production was assessed via the NG-Test CARBA 5 immunochromatographic assay.

Sequencing and sequence assembly

Genomic DNA was extracted using the Gentra Puregene Yeast/Bacteria Kit (Qiagen, Valencia, CA, USA). Sequencing libraries were constructed with the TruePrep DNA Library Prep Kit V2 and the SQK-LSK109 Ligation Sequencing Kit, followed by sequencing on both the Illumina HiSeq X Ten platform (Illumina, San Diego, CA, USA) for short reads and the GridION X5 platform (Oxford Nanopore Technologies, Oxford, UK) for long reads. The raw data from both platforms were quality-trimmed using Canu (v1.8) to obtain high-quality clean reads. A hybrid de novo assembly was subsequently performed by integrating the Illumina short-read and Nanopore long-read data with Unicycler (v0.4.5).

Genomic phylogeny and Average Nucleotide Identity (ANI) analysis

A total of 32,510 publicly available S. Typhimurium genome assemblies (contig-level or higher) were obtained from GenBank (accession date: August 7, 2025). Initial screening using ABRicate (v1.2.0) identified genomes harboring bla_NDM gene variants. These subsequently underwent rigorous quality assessment: assembly continuity was evaluated with Quast (v5.0.2) applying an N50 ≥ 20 kb threshold; genome quality was verified using CheckM (v1.2.2) with thresholds of ≥ 95% completeness and ≤ 5% contamination. Species identification was confirmed through ANI analysis using FastANI (v1.33) against reference strain LT2 (GCA_000006945.2), applying a ≥ 95% species demarcation threshold. This curation process yielded 27 high-quality bla_NDM-positive S. Typhimurium genomes for subsequent analysis.

Phylogenetic analysis was performed by aligning the whole-genome sequences of all 27 genomes against the complete chromosome of the reference strain LT2 (GenBank accession: GCA_000006945.2) using MUMmer v3.2. Core single-nucleotide polymorphisms (SNPs) were identified from the alignments, followed by masking of repetitive genomic regions with RepeatMasker. A maximum-likelihood phylogenetic tree was constructed based on the recombinant-filtered core SNP alignment using RAxML under the GTR model with 1000 bootstrap replicates. The final tree was visualized and annotated using the Interactive Tree of Life (iTOL) platform (https://itol.embl.de).

Screening of Antibiotic Resistance Genes (ARGs), Multi-Locus Sequence Types (MLST), and plasmid replicons

ARGs were identified using abricate (v1.2.0) with the ResFinder database (accessed 2024-03-22), applying thresholds of ≥ 90% for both identity and coverage. The ARG profiles were converted into a binary presence/absence matrix. Visualization was performed by generating a heatmap using the pheatmap package (v1.0.12) in R (v3.27.1; https://www.r-project.org). MLST of the bla_NDM-harboring S. Typhimurium isolates was conducted in silico via the PubMLST (https://pubmlst.org/) platform. To enable plasmid typing of bacterial strains, a custom plasmid sequence database was constructed by integrating data from the PlasmidFinder database (https://cge.food.dtu.dk/services/PlasmidFinder/) with plasmid sequences obtained through laboratory sequencing.

Genomic annotation and comparative analysis

The genome sequence was initially annotated for general biological functions using the RAST 2.0 online platform. Subsequent detailed annotation was performed using specialized databases, including the CARD [29], ResFinder [30], ISFinder [31] and DANMEL [32], for the identification of ARGs, mobile genetic elements, and other specific features. The comparative genomic structures of plasmids, particularly the mobile elements and resistance regions, were visualized using Inkscape (https://inkscape.org/en/).

Conjugal transfer

Conjugation assays were performed using rifampin-resistant Escherichia coli EC600 as the recipient and the K1858 isolate as the donor. Individual colonies of both strains were inoculated into 5 mL of LB broth and cultured at 37℃ with shaking at 200 rpm for 12–18 h. Subsequently, 500 µL of each culture was mixed into 4 mL of fresh LB medium and incubated with shaking at 37℃ for 4 h, followed by static incubation for another 4 h. A 100 µL aliquot of the mating mixture was spread onto Mueller-Hinton agar plates supplemented with 200 µg/mL rifampin (for counterselection of the EC600 recipient) and 1 µg/mL meropenem (for selection of bla_NDM-carrying transconjugants). After 18 h of incubation, putative transconjugant colonies growing on the selection plates were subjected to MALDI-TOF MS identification, PCR verification, and AST.

Nucleotide sequence accession number

The complete genome sequence of strain K1858, comprising its chromosome cK1858 and three plasmids (pK1858-NDM, pK1858-NR1, and pK1858-NR2), has been deposited in the GenBank database under the accession numbers CP185808, CP185809, CP185810, and CP185811, respectively.

Results

Species identification and antimicrobial susceptibility test

Strain K1858 was initially identified as Salmonella sp. by MALDI-TOF MS. To confirm its taxonomic assignment, ANI was performed, revealing 99.88% identity with the S. Typhimurium reference strain LT2 (GenBank accession no. GCA_000006945.2), thereby confirming its identity as S. Typhimurium at the genomic level. AST demonstrated that K1858 was resistant to carbapenems (imipenem and meropenem), cephalosporins (ceftazidime, ceftriaxone, and cefuroxime), and tetracyclines (minocycline and doxycycline). In contrast, the isolate remained susceptible to fluoroquinolones (levofloxacin and ciprofloxacin), trimethoprim-sulfamethoxazole, and aztreonam (Table 1). In addition, production of NDM-type carbapenemase was confirmed phenotypically using the NG-Test CARBA 5 immunochromatographic assay (Figure S1).

Table 1.

Antimicrobial drug susceptibility profiles

Antibiotics	MIC (mg/L)/antimicrobial susceptibility
	K1858	K1858-NDM-EC600	EC600
Ceftazidime	≥ 64/R	≥ 64/R	0.5/S
Piperacilin/Tazobactam	≥ 128/R	≥ 128/R	≤ 4/S
Cefuroxime	≥ 64/R	≥ 64/R	≤ 0.25/S
Imipenem	≥ 16/R	8/R	≤ 0.25/S
Meropenem	≥ 16/R	≥ 16/R	≤ 0.25/S
Ceftriaxone	≥ 64/R	≥ 64/R	≤ 0.25/S
Cefoperazone/Sulbactam	≥ 64/R	≥ 64/R	≤ 8/S
Ciprofloxacin	≤ 0.25/S	≤ 0.25/S	≤ 0.25/S
Levofloxacin	0.5/S	0.5/S	0.5/S
Tigecycline	≤ 0.5/S	≤ 0.5/S	≤ 0. 5/S
Doxycycline	≥ 16/R	1/S	1/S
Minocycline	≥ 16/R	≤ 1/S	≤ 1/S
Aztreonam	≤ 1/S	≤ 1/S	≤ 1/S
Trimethoprim/Sulfamethoxazole	≤ 20/S	≤ 20/S	≤ 20/S

S Sensitive, R Resistant

Overview of the genomic information of S. Typhimurium K1858

Whole-genome sequencing revealed that strain K1858 possesses a chromosome of 4,967,444 bp with a GC content of 52.2%, encoding 4,932 predicted open reading frames (ORFs) (Table 2). Two resistance genes, aac(6’)-Iaa and tet(B), were identified on the chromosome. In silico MLST via the PubMLST database assigned the isolate to sequence type ST34. Furthermore, K1858 harbored three circular plasmids, designated pK1858-NDM, pK1858-NR1, and pK1858-NR2 (Table 2). The plasmid pK1858-NDM was 112,188 bp in length with a GC content of 49.5%, carried 122 ORFs, and harbored the resistance genes bla_NDM-5, sul1, and ble_MBL. This plasmid was assigned to replicon type IncFII. The remaining two plasmids, pK1858-NR1 and pK1858-NR2, did not carry any known resistance genes (Table 2).

Table 2.

Whole genome information of Salmonella typhimurium K1858

Sequence	Mean G + C content (%)	Length (bp)	Total number of ORFs	MLST	Inc type	Accession number	Resistance genes
cK1858	52.2%	4,967,444	4,932	ST34	-	CP185808	aac(6’)-Ia, tet(B)
pK1858-NDM	49.5%	112,188	122	-	IncFII	CP185809	blaNDM−5, sul1, bleMBL
pK1858-NR1	47.7%	95,578	118	-	IncFIB	CP185810	-
pK1858-NR2	55.4%	4,263	3	-	Unknown	CP185811	-

-, not available

Genetic characterization of pK1858-NDM and comparative analysis with related plasmids

To further resolve the genetic structure of the bla_NDM-5-harboring plasmid pK1858-NDM, we performed an in-depth genomic analysis. The plasmid architecture can be divided into a backbone region and accessory module. The backbone region (93.2 kb) encompasses functional units responsible for replication, maintenance, and conjugation: the replication region contains the origin of replication and its regulatory genes; the maintenance region includes genes such as stbA/stbB; and the conjugation region encodes a complete set of type IV secretion system proteins (e.g., traA/B/C/D). The accessory module consists of a 17.6 kb bla_NDM-5-bearing resistance region (Fig. 1).

Fig. 1.

Circular diagram of plasmid pK1858-NDM. Genes are denoted by arrows and colored based on gene function classification.The innermost circle presents GC-skew [(G-C)/(G + C)], with a window size of 500 bp and a step size of 20 bp. Next-to-innermost circle presents GC content

To investigate the structural and evolutionary relationships between pK1858-NDM and related plasmids within the same incompatibility group, we retrieved four plasmids pNDM5_LS003, pPM4-5-ndm-5, pHZZ201-114 K, and pKP-NDM-5 from GenBank based on homology to the replication initiation gene repA1 and the carbapenemase gene bla_NDM (Table S1). Among these, pNDM5_LS003, pPM4-5-ndm-5, and pHZZ201-114 K were derived from Escherichia coli, while pKP-NDM-5 originated from Klebsiella pneumoniae. The plasmid described in this study, pK1858-NDM, was isolated from S. Typhimurium. Notably, all host strains were isolated from cities in Eastern China (Table S1).

Structural comparison of the five plasmids revealed that, with the exception of pNDM5_LS003, the remaining four (pK1858-NDM, pPM4-5-ndm-5, pHZZ201-114 K, and pKP-NDM-5) share a highly conserved backbone (Fig. 2). pNDM5_LS003 carries an additional 11.6 kb maintenance segment but lacks a 5.7 kb conjugation region present in the others. Furthermore, all plasmids except pHZZ201-114 K carry two foreign insertion regions. Among them, pK1858-NDM, pPM4-5-ndm-5, and pKP-NDM-5 share the bla_NDM region, inserted downstream of the ΔydfA gene, leading to its truncation. In contrast, pNDM5_LS003 carries the mph(A) gene inserted within traD. Additionally, pHZZ201-114 K contains a Group IIB Intron E.c.I8 inserted into the maintenance gene orf348, resulting in its disruption (Fig. 2).

Fig. 2.

Comparison of plasmids pK1858-NDM, pNDM5_LS003, pPM4-5-ndm-5, pHZZ201-114 K, and pKP-NDM-5. Genes are denoted by arrows. Genes and other features are colored based on their functional classification. Shaded regions denote regions of homology (nucleotide identity ≥ 95%)

Genetic comparison of the bla_NDM−5 regions from pK1858-NDM and related plasmids

Comparative analysis revealed that the bla_NDM region in all five plasmids contains an identical core mobile element, ΔTn125 (ΔISAba125-bla_NDM-5-ble_MBL-trpF-dsbD-ΔcutA), whose mobility is mediated by ISCR1 (Fig. 3). However, structural variations were observed in the regions flanking this core element: the resistance modules in pK1858-NDM and pHZZ201-114 K are flanked by IS26 and consist of three components (ISCR1-ΔTn125, the 3’-end of a class 1 integron, and ISCR28-erm); those in pKP-NDM-5 and pPM4-5-ndm-5 are also IS26-flanked but lack the ISCR28-erm module; whereas pNDM5_LS003 carries two truncated units (ISCR2-floR and ISCR1-ΔTn125). These findings suggest that despite the diversity in flanking structures, the dissemination of the bla_NDM-5 gene among these plasmids likely depends on a conserved, ISCR1-driven ΔTn125 core unit (Fig. 3).

Fig. 3.

Organization of bla_NDM−5 region from pK1858-NDM, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥ 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmids. Accession numbers of Tn125 used as reference is JN872328

Conjugation and plasmid stability assays

The plasmid pK1858-NDM was successfully transferred from the wild-type strain K1858 to the recipient Escherichia coli EC600, yielding the transconjugant K1858-NDM-EC600. Due to the acquisition of the bla_NDM-5 gene, this transconjugant exhibited high-level resistance to both imipenem and meropenem (Table 1). To assess plasmid stability in the transconjugant, serial passaging was performed for 15 days without antibiotic selection pressure, with PCR detection of the key plasmid genes repA1 and bla_NDM conducted every 3 days. Results demonstrated that both repA1 and bla_NDM remained consistently detectable throughout the passaging period, confirming the stable maintenance of pK1858-NDM in the recipient strain (Figure S2).

Epidemiology and phylogenetics of bla_NDM-harboring S. Typhimurium

A total of 32,510 S. Typhimurium genome assemblies were retrieved from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/datasets/genome/, accessed August 7, 2025). After comprehensive quality control and screening for bla_NDM variants, 27 high-quality genomes were obtained. Combined with the newly sequenced strain from this study, a final set of 28 genomes was included in the analysis. Geographically, all strains originated from various provinces in China, with Zhejiang province accounting for the highest proportion (13/28, 46.4%), followed by Guangdong, Jiangsu, and others. Temporally, these bla_NDM-positive isolates were recovered over a decade (2013–2024), with 23/28 (82.1%) collected after 2020. Due to the limited number of cases, which reflects the genuine rarity of bla_NDM in S. Typhimurium, a detailed temporal trend analysis was not feasible. The year-by-year distribution is as follows: 2013 (n = 1), 2015 (n = 2), 2016 (n = 1), 2017 (n = 1), 2020 (n = 4), 2021 (n = 6), 2022 (n = 6), 2023 (n = 3), and 2024 (n = 4).(Table S2). MLST analysis revealed that ST34 was the predominant sequence type (24/28, 85.7%), followed by ST36 (3 strains) and ST19 (1 strain).

To elucidate the genetic background of bla_NDM-harboring S. Typhimurium, a maximum-likelihood phylogenetic tree was constructed based on 1,289 recombinant-filtered core SNPs extracted from the chromosomal sequences of the 28 strains, using S. Typhimurium LT2 (GCA_000006945.2) as the reference and Salmonella Heidelberg SL476 (NC_011083.1) as the outgroup (Fig. 4). Phylogenetic analysis revealed three distinct evolutionary clusters (Cluster I–III). A strong correlation was observed between phylogenetic clustering and MLST profiles: all dominant ST34 strains fell within Cluster III, while ST36 and ST19 strains were exclusively assigned to Cluster I and Cluster II, respectively. The three ST36 strains in Cluster I were all isolated from the same hospital in Hubei Province, suggesting potential nosocomial clonal transmission. In contrast, ST34 strains in Cluster III were widely distributed across multiple provinces, including Zhejiang, Shanghai, and Jiangsu, indicating that this sequence type has become the predominant and widely disseminated lineage among bla_NDM-positive S. Typhimurium in China.

Fig. 4.

Phylogenetic tree of 28 S. Typhimurium isolates carrying the bla_NDM gene. The reference strain used was S. Typhimurium LT2 (GCA_000006945.2), Salmonella Heidelberg SL476 (NC_011083.1) as the outgroup. The red font indicates the strains sequenced in our study

Distribution of plasmids and antimicrobial resistance genes in bla_NDM-positive S. Typhimurium

Plasmid replicon screening identified five distinct incompatibility groups: IncHI2, IncQ1, IncFII, IncX3, and IncFIB. Among these, IncHI2 was the most prevalent (20/28, 71.4%), followed by IncFII (14/28, 50.0%) and IncQ1 (11/28, 39.3%), while IncX3 and IncFIB were less frequently detected (Fig. 5).

Fig. 5.

Distribution of geographical location, year, ST types, ARGs, and plasmid types among the 28 S. Typhimurium isolates carrying the bla_NDM gene. The phylogenetic tree is shown on the left, following the distribution of geographical location, year, and ST types, the antibiotic resistance gene matrix in the middle, and the plasmid type matrix on the right, with all aligned in the same order. Color coding at the top distinguishes the categories of antibiotic resistance genes and plasmid labels. Colored cells in the matrices indicate the presence or absence of the corresponding antibiotic resistance gene clusters or plasmid types

ARGs across the 28 S. Typhimurium genomes revealed a diverse repertoire of acquired resistance determinants. A total of 45 distinct ARG types were identified, conferring resistance to 12 antimicrobial classes: β-lactams, aminoglycosides, quinolones, phenicols, rifamycins, sulfonamides, tetracyclines, macrolides, trimethoprim, fosfomycin, lincosamides, and bleomycin. The highest diversity was observed among β-lactam (9 genes) and aminoglycoside (14 genes) resistance genes. In terms of prevalence, the aminoglycoside acetyltransferase gene aac(6′)-Iaa was detected in all strains. Although this gene is commonly found in S. Typhimurium, it is often considered a non-functional or cryptic gene that does not contribute to phenotypic aminoglycoside resistance [33, 34]. Its universal presence in our strains is consistent with its role as an intrinsic chromosomal marker rather than an acquired resistance determinant. The sulfonamide resistance gene sul1 and tetracycline resistance gene tet(B) were also widely distributed. Among the bla_NDM variants, three subtypes bla_NDM-1, bla_NDM-5, and bla_NDM-13 were identified, with bla_NDM-5 being the most common (17/28, 60.7%), followed by bla_NDM-1 (10/28, 35.7%) and bla_NDM-13 (1/28, 3.6%). The three ST36 strains in Cluster I exhibited highly concordant ARG and plasmid replicon profiles, further supporting their clonal relatedness (Fig. 5).

Discussion

This study provides a comprehensive genomic and phenotypic characterization of a bla_NDM-5-harboring S. Typhimurium strain K1858. By integrating data from GenBank, we further investigated the epidemiology of bla_NDM-positive S. Typhimurium across China, revealing a potential clonal dissemination trend for ST36, while ST34 strains exhibited greater diversity in their resistance profiles and NDM variants.

Strain K1858 exhibited resistance to multiple critical antimicrobial classes, including carbapenems and cephalosporins, while remaining susceptible to quinolones and aztreonam, confirming that this phenotype is consistent with its identified resistance gene profile. Carbapenem resistance was mediated by the bla_NDM-5 gene located on the plasmid pK1858-NDM. Structural analysis revealed that pK1858-NDM carries a complete conjugation system, and experimental conjugation confirmed its successful transfer to Escherichia coli EC600, rendering the transconjugant resistant to carbapenems. This demonstrates the plasmid’s capacity for horizontal gene transfer, highlighting its role as a key vector for bla_NDM-5 dissemination across bacterial species. Moreover, plasmid stability assays indicated that pK1858-NDM could be maintained in the recipient strain even in the absence of antimicrobial selective pressure, suggesting a potential risk for its persistent circulation in the environment or within hosts, thereby facilitating long-term resistance dissemination.

Comparative analysis of pK1858-NDM with other IncFII bla_NDM-carrying plasmids pNDM5_LS003, pPM4-5-ndm-5, pHZZ201-114 K, and pKP-NDM-5 revealed that all are derived from Enterobacterales isolates originating from cities in Eastern China. This suggests an ongoing regional spread of this plasmid type among clinically important bacteria in this geographic area. ISCR1 can mobilize adjacent genes and gene cassettes as a single unit via a rolling-circle replication mechanism [35]. A structurally conserved ΔTn125 core mobile element (ΔISAba125-bla_NDM-5-ble_MBL-trpF-dsbD-ΔcutA), mobilized by ISCR1, was identified in all five plasmids. In four of the plasmids, the resistance region was flanked by IS26 elements, indicating that further resistance gene accumulation may have occurred through IS26-mediated recombination events. The frequent co-occurrence of ISCR1 and IS26 in the genetic environment of bla_NDM, as also reported in previous studies [36–38], suggests that ISCR1 may have been involved in the initial capture of the gene, while IS26 likely plays a major role in its subsequent mobilization and dissemination.

Taken together, these structural features point to two complementary mechanisms driving the dissemination of bla_NDM-5 in S. Typhimurium. On one hand, conjugative plasmids such as the IncFII-type pK1858-NDM serve as direct vehicles for inter-bacterial horizontal gene transfer, as demonstrated by our experimental conjugation assays. On the other hand, the conserved ISCR1-ΔTn125-IS26 architecture identified across multiple structurally related plasmids highlights a critical role for transposition-mediated mobility. In this model, ISCR1 initiates rolling-circle transposition to capture and mobilize the ΔTn125 unit containing bla_NDM-5, while flanking IS26 elements facilitate the integration of this resistance region into diverse plasmid backbones and promote the co-accumulation of additional resistance genes.

Despite the global scope of the initial data retrieval, all high-quality bla_NDM-positive S. Typhimurium genomes obtained were exclusively of Chinese origin. This observation underscores either a potential epidemiological concentration of these resistant clones in China or a substantial ascertainment bias in publicly available datasets. Phylogenetic analysis of 28 bla_NDM-positive S. Typhimurium genomes, delineated three distinct evolutionary clusters that strongly correlated with MLST types. ST34 strains not only constituted the vast majority (24/28) but were also exclusively grouped within Cluster III and were widely distributed across multiple provinces in Eastern China. This pattern suggests that S. Typhimurium ST34 serves as a primary recipient lineage for bla_NDM-carrying plasmids in China. The observation that diverse bla_NDM variants and distinct plasmid backbones are present within the ST34 genetic background indicates that multiple independent acquisition events of bla_NDM-harboring plasmids have occurred within this successful clone, rather than the dissemination of a single plasmid-carrying strain. The wide geographic distribution of ST34 across multiple provinces in China likely reflects both the background prevalence of this successful lineage and its ongoing capacity to capture resistance determinants. This finding aligns with global reports of ST34 emerging as a successful multidrug-resistant clone in various countries [39–41].

More notably, three ST36 strains from the same hospital in Cluster I shared nearly identical resistance gene and plasmid profiles, indicating potential nosocomial clonal transmission and emphasizing the critical need for robust infection control measures in healthcare settings.

Plasmids serve as critical vectors for the dissemination of ARGs. IncHI2-type plasmids, a major group within the IncHI incompatibility complex, are characterized by their large size, broad host range, and high stability, enabling them to carry and spread multiple resistance determinants [42]. These features underscore their pivotal role in propagating antimicrobial resistance (AMR) among Enterobacteriaceae [42, 43]. Previous studies have reported the widespread circulation of bla_NDM-5-carrying IncHI2 plasmids in China [24]. In our study, the high prevalence of the IncHI2 replicon (20/28, 71.4%) among the bla_NDM-positive S. Typhimurium genomes, which collectively harbored 45 distinct resistance genes, suggests that IncHI2 plasmids may play an important role in facilitating the spread of resistance genes in this pathogen. Additionally, IncFII-type plasmids are commonly reported in Salmonella [44], and their notable prevalence (50.0%) in our dataset, including in our strain K1858, indicates they could also serve as potential vehicles for resistance dissemination.While IncX3 plasmids have been identified as major vectors for bla_NDM in various Enterobacterales [45, 46], they were less prevalent in our S. Typhimurium collection. Although definitive assignment of specific resistance genes to individual plasmid replicons is limited for the majority of our genomes that were sequenced using short-read platforms, the overall replicon distributions observed, supported by available assemblies from the remaining genomes, suggest that for bla_NDM-carrying S. Typhimurium in China, IncHI2 and IncFII-type plasmids are likely the vectors responsible for the dissemination of carbapenem resistance. Further studies incorporating long-read sequencing would help confirm these associations at the individual plasmid level.

Conclusion

This study demonstrates that the bla_NDM-5 gene can be carried by a conjugative and highly stable IncFII-type plasmid in S. Typhimurium. Plasmids harboring bla_NDM detected in multiple bacterial species have so far been reported only in eastern China, suggesting the emergence of regional dissemination in this area. The bla_NDM-5-carrying plasmid exhibits a conserved backbone and accessory modules, with the carbapenemase gene embedded within a structurally uniform ΔTn125 unit. ISCR1 likely played a critical role in capturing and mobilizing ΔTn125, thereby facilitating the transfer of bla_NDM-5, while IS26 further promotes the co-transfer of additional resistance determinants. Furthermore, we performed a systematic analysis of all bla_NDM-positive S. Typhimurium genomes available in GenBank, revealing distinct epidemiological characteristics. China appears to be the primary region affected by these strains, with ST34 being the dominant epidemic clone, although several different bla_NDM variants are harbored in different plasmid families. IncHI2 and IncFII-type plasmids are potentially associated with the dissemination of bla_NDM in S. Typhimurium. In terms of gene subtype distribution, bla_NDM-5 is predominant, followed by bla_NDM-1. These findings provide important insights for clinical and public health authorities in developing targeted surveillance and control strategies against carbapenem-resistant Salmonella infections.

Abbreviations

ANI

Average Nucleotide Identity

CLSI

Clinical and Laboratory Standards Institute

SNPs

Single-nucleotide polymorphisms

ARGs

Antibiotic Resistance Genes

MLST

Multi-Locus Sequence Types

ORFs

Open reading frames

S. Typhimurium

Salmonella Typhimurium

Authors’ contributions

Conceptualization, XH.L., JB.Y., and YL.Z.; methodology, JB.Y., YL.Z. , DK.H., HM.C., YT.J., and JY.H.; data analysis, XH.L., YL.Z., JB.Y., JY.H and QR.M.; resources, DK.H., JB.Y. and HM.C.; writing-original draft, JB.Y. and YL.Z.; writing-review and editing, XH.L. All authors read and approved the final manuscript.

Funding

This work was supported by the Medical and Health Science and Technology Project of Zhejiang Province (2025KY461 and 2024KY543), and the Natural Science Foundation of Zhejiang Province (LTGY23H190003).

Data availability

The datasets generated and analyzed during the current study are available in the public NCBI GenBank database, under the following accession numbers: CP185808, CP185809, CP185810, and CP185811.

Declarations

Ethics approval and consent to participate

The use of human specimens and all related experimental protocols was reviewed and approved by the Ethics Committee of Taizhou Municipal Hospital, Zhejiang, China, in accordance with the medical research regulations of the Ministry of Health, China. Research and all related procedures involving biohazardous materials were approved by the Biosafety Committee of Taizhou Municipal Hospital. This research was conducted in China.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.