Characterization of blaNDM–5-carrying plasmids in two clinical Salmonella isolates from Jiaxing city, China

Introduction: Salmonella is an important cause of foodborne diarrheal diseases worldwide. The emergence of bla_NDM-positive carbapenem-resistant Salmonella enterica isolates in recent years poses a huge public health challenge.

Methods: In this study, two clinical S. enterica isolates carrying bla_NDM–5: a serotype 4,[5],12:i:- strain (2023JX045) and a serovar Stanley strain (2024–406) were analyzed using antimicrobial susceptibility testing and whole genome sequencing.

Results: Both isolates were multidrug resistant, with insusceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, ceftiofur, cefazolin, cefoxitin, cefotaxime, ceftazidime, cefepime, ceftazidime/avibactam, meropenem, imipenem, ertapenem, tetracycline, gentamicin, trimethoprim/sulfamethoxazole, florfenicol, chloramphenicol, ciprofloxacin, colistin, and polymixin B. The bla_NDM–5-carrying plasmids in 2023JX045 and 2024–406 were named p23045-NDM5 and p2024406-NDM5, respectively, with both belonging to the incompatibility (Inc)HI2/IncHI2A group and sequence type ST3. In 2023JX045, bla_NDM–5 was flanked by two same-oriented copies of IS26 elements (IS26-dsbC-trpF-ble_MBL-bla_NDM–5-IS5-ΔIS3000-ΔISKox3-umuC-umuD-IS26). In 2024–406, p2024406-NDM5 was found to carry two copies of bla_NDM–5, possibly resulting from duplication of IS26-dsbC-trpF-ble_MBL-bla_NDM–5-IS5-ΔISAba125-ΔIS3000-ΔISKox3-umuC-umuD-IS26 and interrupted by mobile element IS1 at the upstream region of ΔIS3000.

Discussion: This is the first report to describe the presence of two bla_NDM–5 copies on an IncHI2/IncHI2A plasmid carried by serovar Stanley, as well as the dissemination of bla_NDM–5 in Salmonella in Jiaxing City, China. IS26-flanked composite transposons appeared to play an important role in the formation of this region. The dissemination of blaNDM in Salmonella isolates and the complexity of the bla_NDM–5 region highlight the urgent need to monitor carbapenem-resistant S. enterica.

1 Introduction

Non-typhoidal Salmonella (NTS) is one of the most prevalent foodborne pathogens, consistently causing gastrointestinal infections. More than 2,600 serovars of NTS have been identified, among which S. Typhimurium is one of the most common (Lamichhane et al., 2024). Previous studies have indicated that Salmonella enterica may act as a reservoir for carbapenemase genes, contributing to the transmission of carbapenem resistance via the food chain (Day et al., 2015). Whole-genome sequencing (WGS) data of global carbapenem-resistant S. enterica (CRSE) isolates revealed S. Typhimurium (21.8%) to be the most prevalent serotype of CRSE worldwide (Wu et al., 2023). Additionally, Typhimurium (25.8%) and Senftenberg (19.4%) were the most prevalent serovars of global CRSE isolates harboring the New Delhi metallo-β-lactamase (bla_NDM) gene (Zhao et al., 2025). In recent years, the bla_NDM gene has been identified in S. Stanley isolated from clinical and environmental samples (Deng et al., 2024, Huang et al., 2013).

The worldwide spread of multidrug-resistant (MDR) Enterobacteriaceae strains, particularly carbapenem-resistant Enterobacteriaceae (CRE), has become an increasing public health threat (Nordmann et al., 2012). Mobile resistance elements carrying bla_NDM–1 have contributed to the dramatic increase in the prevalence of CRE in clinical settings (Huang et al., 2016). Twenty-nine NDM protein variants have been identified since 2009 (Mojica et al., 2022). The blaNDM-5 gene was first identified in a clinical Escherichia coli strain (EC045) from India in 2011, and was later commonly identified among strains of E. coli (Yang et al., 2014, Sassi et al., 2014), Klebsiella pneumoniae (Bathoorn et al., 2015), and Morganella morganii (Guo et al., 2019). In China, a report on bla_NDM–1 in Acinetobacter baumannii isolates appeared in early 2011 (Chen et al., 2011). Among Salmonella spp., bla_NDM–1 was first identified in a Senftenberg isolate in 2011, and was located on an incompatibility (Inc)L/M group plasmid (Savard et al., 2011, Rasheed et al., 2013). A 2012 report described the identification of a bla_NDM–1-bearing strain of Salmonella Stanley isolated from the feces of an 11-month-old girl (Huang et al., 2013). The first bla_NDM–5-positive IncFII plasmid, isolated from an S. Typhimurium sequence type (ST) 34 isolate in China, was reported in 2015 (Li et al., 2017). Compared with bla_NDM–1, bla_NDM–5 has two amino acid substitutions (Val88Leu and Met154Leu) and confers a high level of resistance to carbapenems and broad-spectrum cephalosporins (Hornsey et al., 2011).

Here, we aimed to better understand the antimicrobial resistance determinants and transmission risk of bla_NDM-positive Salmonella in Jiaxing City, China by conducting a comprehensive investigation of two bla_NDM–5-carrying carbapenem-resistant Salmonella isolates that were recovered from clinical samples. To the best of our knowledge, this is the first report of Salmonella isolates carrying bla_NDM in this part of China. Notably, it is also the first report of an IncHI2/IncHI2A plasmid co-carrying two copies of bla_NDM–5 in S. Stanley. Jiaxing is located in the Yangtze River Delta region, with well-developed water and land transportation and a continuously growing population. It is an important economic and population aggregation area in Zhejiang Province. These findings further complicate the challenges of establishing effective treatment modalities and management strategies.

2 Materials and methods

2.1 Bacterial collection and characterization

Fecal samples from patients with acute clinical diarrhea were collected to isolate Salmonella spp. Within 4 h of collection, undiluted samples were streaked onto Columbia Blood Agar plates (CHROMagar, Shanghai, China) and cultured overnight at 37°C. Suspected Salmonella spp. colonies were analyzed using matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Serotyping was conducted using the slide agglutination method to detect somatic (O) antigen and flagellar (H) antigens (phase 1 and 2) following the White–Kaufmann–Le Minor Scheme. Salmonella Serotyping by Whole Genome Sequencing was confirmed using the Sequence query tool implemented in SeqSero2/SeqSero2S.¹

2.2 Antimicrobial susceptibility testing

AST of the following antimicrobial agents was performed to determine the minimum inhibitory concentration (MIC) of each using the microdilution method: ampicillin, ampicillin/ sulbactam, amoxicillin/clavulanic acid, cefuroxime, ceftiofur, cefazolin, cefoxitin, cefotaxime, ceftazidime, cefepime, cefotaxime/ clavulanate, ceftazidime/clavulanic acid, ceftazidime/avibactam, meropenem, imipenem, ertapenem, tetracycline, gentamicin, amikacin, trimethoprim/sulfamethoxazole, florfenicol, chloramphenicol, ciprofloxacin, nalidixic acid, colistin, polymixin, and azithromycin. The resistance breakpoints of ampicillin, ceftiofur, imipenem, meropenem, ertapenem, azithromycin, tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol were determined in accordance with the principles outlined in relevant documents from the Clinical and Laboratory Standards Institute (CLSI) (M100-S32, M45-A3). Amoxicillin/clavulanic acid, ampicillin/sulbactam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, cefuroxime, ceftazidime/avibactam, gentamicin, and amikacin were determined in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Colistin, Polymixin B, and florfenicol were interpreted in accordance with the “National Food Contamination and Hazardous Factor Risk Monitoring Work Manual 2024” (China National Center for Food Safety Risk Assessment, 2024). Escherichia coli ATCC 25922, Enterococcus faecalis ATCC29212, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC29213 was used as a quality control strains for AST.

2.3 Genomic DNA extraction and WGS

Total genomic DNA was extracted from overnight (16–18 h) cultures of strains 2023JX045 and 2024-406 using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. WGS was performed on the two strains using both the long-read Nanopore MinION (Nanopore, Oxford, United Kingdom) and the short-read NextSeq 550 (Illumina, San Diego, CA, United States) platforms. The derived short reads and long reads were assembled using SPAde v.3.6 software.

2.4 Bioinformatic analysis

The ST of Salmonella isolates were determined using multilocus sequence typing software.² Antimicrobial-resistant genes and plasmid profiles were analyzed used ResFinder³ and PlasmidFinder.⁴ Annotation of mobile elements was carried out using online databases, such as ISfinder.⁵ Plasmid sequence alignment was performed using BRIG v0.95 (Alikhan et al., 2011) and Easyfig v2.2.5.⁶

To investigate the epidemic characteristics of bla_NDM-carrying plasmids in Salmonella, we obtained 31 bla_NDM-positive plasmids from the GenBank core nucleotide database (last accessed on 6th January, 2025. Plasmids from Salmonella isolates (taxid: 590) were selected). For the input sequences, multiple sequence alignment (MSA) was performed with the Multiple Alignment Using Fast Fourier Transform in auto mode. The resulting MSA was then entered into ModelTest using default parameters to estimate the best model for constructing the evolutionary tree. Subsequently, the MSA and the selected model were used as input for RAxML-NG with the arguments –all –seed 12345 –bs-trees 1000) to generate the final evolutionary tree. Visualization and annotation of the phylogenetic tree were performed using iTOL v7.⁷

2.5 Nucleotide sequence accession number

The sequences of plasmids p23045-NDM5 and p2024406-NDM5 were submitted to the GenBank database and assigned accession numbers OR497833 and PQ844496, respectively.

3 Results

3.1 Characterization of two carbapenem-resistant Salmonella strains

S. enterica strains 2023JX045 and 2024-406 were isolated from clinical diarrhea samples collected from a 20-month-old boy and 57-year-old woman, respectively. The main symptoms of case 1 were a fever of 39.3°C and watery diarrhea 10 times per day. No history of suspected food exposure was found. Case 2 did not have a fever. The digestive system symptoms were abdominal pain and watery diarrhea five times a day. It is suspected that it might be related to the consumption of bulk fruits and their products. The patients and his/her family had not traveled to any country in recent 7 days, and no family members were affected. Strain 2023JX045, identified as 4,[5],12:i:-, a ST34 monophasic variant of S. enterica serovar Typhimurium, was found to carry the following antimicrobial resistance genes: aph(4)-Ia, aph(3’)-Ia, aac(3)-IV, aadA2b, bla_NDM–5, bla_OXA–10, bla_TEM–1B, lnu(F), qnrS1, and sul3. Strain 2024-406 was identified as belonging to ST29 S. enterica serovar Stanley (4,12:d:2). The antimicrobial resistance genes identified in this isolate included the following: aph(4)-Ia, aph(6)-Id, aph(3’)-Ia, aph(3”)-Ib, aadA1, aadA1, aadA2b, aac(3)-IV, bla_TEM–1B, bla_NDM–5, bla_NDM–5, bla_OXA–10, cmlA1, cmlA1, floR, qnrS1, ARR-2, sul3, tet(A), tet(A), and dfrA14. Furthermore, 2024-406 was shown to possess a single point mutation in the quinolone resistance-determining region of parC (T57S). While a sole plasmid carrying IncHI2/IncHI2A replicons was identified in 2024-406, 2023JX045 was found to carry multiple plasmid replicons, namely Col (pHAD28), IncHI2/IncHI2A, IncI2 (Delta), IncQ1, and p0111 (Table 1).

TABLE 1

Table 1. Information about the NDM-5-harboring Salmonella strains 2023JX045 and 2024-406 identified in this study and its plasmids.

The AST analysis showed that these two isolates were MDR to β-lactams, including penicillins (ampicillin, ampicillin/sulbactam, and amoxicillin/clavulanic acid) and cephalosporins (cefuroxime, ceftiofur, cefazolin, cefoxitin, cefotaxime, ceftazidime, cefepime, and ceftazidime/avibactam), carbapenems (meropenem, imipenem, and ertapenem), tetracycline (tetracycline), aminoglycosides (gentamicin), sulfonamides (trimethoprim/sulfamethoxazole), amphenicols (florfenicol and chloramphenicol), fluoroquinolones (ciprofloxacin), and polymyxins (colistin and polymixin B), but remained susceptible to amikacin and azithromycin (Table 2).

TABLE 2

Table 2. AST of the 2023JX045 and 2024-406 isolates using a panel of 27 antimicrobial agents.

3.2 Plasmid characterization

The carbapenemase-encoding gene bla_NDM–5 was found on a 266,011-bp plasmid (p23045-NDM5) with 47.1% GC content in strain 2023JX045. Strain 2024-406 was found to carry a 277,574-bp plasmid (p2024406-NDM5) with 47.3% GC content. Both p23045-NDM5 and p2024406-NDM5 were classified as IncHI2/IncHI2A and ST3 plasmids. Exhibiting 99% coverage and 100% identity with p23045-NDM5, p2024406-NDM5 is distinguished by numerous rearrangements and inversions within accessory regions.

The resistance genes in p23045-NDM5 were found to be arranged in three regions. The first region contains △tet(A), qnrS1, aadA22, lnu(F), aph(3”)-Ib, aph(6)-Id, aph(3’)-Ia, aph(4)-Ia, aac(3)-Iva, sul3, aadA1, cmlA1, and aadA2b, with the major antimicrobial resistance genes arranged within two class 1 integrons (IntI1). The bla_NDM–5-region carries resistance genes including floR, tet(A), dfrA14, aadA1, bla_OXA–10, cmlA, ARR-2, ble_MBL, and bla_NDM–5. This plasmid also carries bla_TEM–1 within a truncated Tn2.

In p2024406-NDM5, we found the resistance genes aph(4)-Ia, aph(6)-Id, aph(3’)-Ia, aph(3”)-Ib, aadA1, aadA1, aadA2b, aac(3)-IV, bla_TEM–1B, bla_OXA–10, cmlA1, cmlA1, floR, qnrS1, ARR-2, sul3, tet(A), tet(A), dfrA14, along with two copies of bla_NDM–5 are clustered in a complicated accessory region. The bla_OXA–10-region containing tet(A), qnrS1, △ bla_TEM–1B, tet(A), floR, and a class 1 integron carrying ARR-2, cmlA1, bla_OXA–10, aadA1, and dfrA14 resistant genes.

Compared with the genetic background of the typical bla_NDM5–IncX3 plasmid pNDM_MGR194 (IS3000-△ ISAba125-IS5-bla_NDM–5-ble-trpF-dsbC-IS26), the bla_NDM–5 gene on plasmid p23045-NDM5 was found to be flanked by two same-oriented copies of IS26 elements (IS26-umuD-umuC-△ ISKox3-△ IS3000-IS5-bla_NDM–5-ble_MBL-trpF-dsbC-IS26). Note the absence of the △ ISAba125 feature in this region. We identified more complex genetic arrangements in p2024406-NDM5, formed by duplication of the IS26-umuD-umuC-△ ISKox3-△ IS3000-△ ISAba125-IS5-bla_NDM–5-ble_MBL-trpF-dsbC-IS26 unit. Additionally, mobile element IS1 was found upstream of △ IS3000, which may have led to the loss of IS26-umuD-umuC-△ ISKox3 (Figure 1). A direct repeat was not detected immediately upstream or downstream of those two IS26-flanked regions.

FIGURE 1

Figure 1. Schematic representation and comparison of the genetic environments of the bla_NDM-flanking region in p23045-NDM5 and p2024406-NDM5 recovered from this study. Arrows indicate the direction of transcription of each gene. Numbers in brackets indicate nucleotide positions within corresponding plasmid sequences.

3.3 Comparative study with other NDM-positive plasmids in S. enterica strains already identified

A total of 31 S. enterica strains with positivity for bla_NDM plasmids were obtained from the GenBank core nucleotide database (Table 3). The serovars of these strains, mostly isolated from Homo sapiens, were Senftenberg (n = 2), Bareilly (n = 1), London (n = 2), Lomita (n = 1), Enteritidis (n = 1), Mbandaka (n = 2), Rissen (n = 1), 1,4,[5],12:i:- (n = 11), Stanley (n = 2), Corvallis (n = 2), Kottbus (n = 1), 1,4,[5],12:i:2 (n = 2), Typhimurium (n = 1), and Indiana (n = 1). The most prevalent bla_NDM gene was bla_NDM–1, followed by bla_NDM–5, which was the major bla_NDM gene in 1,4,[5],12:i:- isolates. Two S. Stanley strains carried a bla_NDM gene, with one isolate harboring bla_NDM–1 and the other bla_NDM–5. Despite the presence of bla_NDM, other β-lactamase genes (e.g., bla_CMY, bla_OXA, and bla_TEM) were also detected in some of these strains.

TABLE 3

Table 3. 31 S. enterica strains with positivity for bla_NDM plasmids obtained from the GenBank core nucleotide database.

Plasmids of different Inc groups, including IncC, IncFII, IncM, IncX3, and IncHI2/IncHI2A, were also found to carry bla_NDM genes (Table 3), with IncC and IncHI2/IncHI2A being the most common. Plasmids in S. Stanley strains, namely pHS36-NDM and p2024406-NDM5 (this study), belong to IncC and IncHI2/IncHI2A, respectively. Multiple copies of bla_NDM on a single plasmid were found in pSM28_NDM_1 (three copies of bla_NDM–1), pSM30_NDM_1 (seven copies of bla_NDM–1), and pST_HI2_NDM-1 (eight copies of bla_NDM–1). Both pSM28_NDM_1 and pSM30_NDM_1 are IncC-type plasmids, hosted by S. Mbandaka. The IncHI2/IncHI2A-type plasmid pST_HI2_NDM-1 was isolated from a 1,4,[5],12:i:2 strain in 2020. Two copies of bla_NDM–5 were found in p2024406-NDM5. Overall, IncHI2/IncHI2A and IncC plasmids carry more resistant genes than IncX3, IncFII, and IncN2 plasmids.

Phylogenetic analysis showed similarity between p23045-NDM5 and pST_HI2_NDM-1 carried by 1,4,[5],12:i:2. Eight copies of bla_NDM–1, in addition to 14 other resistance genes, were found in pST_HI2_NDM-1. The highest homology was between p2024406-NDM5 and pST2742-1, which harbors bla_NDM–5 and was isolated from a 1,4,[5],12:i:- strain. IncHI2/IncHI2A- and IncX3-type plasmids were more similar than IncC- and IncFII-type NDM-positive plasmids in Salmonella isolates (Figure 2).

FIGURE 2

Figure 2. Phylogenetic tree of p23045-NDM5 and p2024406-NDM5 and 29 screened bla_NDM-carrying plasmid of Salmonella isolates from GenBank.

4 Discussion

Carbapenems are last-resort antimicrobial agents against infections caused by MDR Gram-negative bacteria. Infection with CRE has become an urgent and continuous threat to public health worldwide (Temkin et al., 2014). Resistance to carbapenems among Salmonella isolates is primarily attributed to the presence of mobile genetic elements encoding various classes of β-lactamases. These include carbapenemase, temoneira, NDM, oxacillinase, imipenemase, and Verona integron-encoded metallo-β-lactamase. The carrier isolates with a single copy gene have high minimum inhibitory concentration (MIC) values for all β-lactams (Han et al., 2020, Miao et al., 2018).

In China, the most prevalent carbapenemase gene among bla_NDM-positive isolates is bla_NDM–1, followed by bla_NDM–5 and bla_NDM–3 (Hu et al., 2017). In recent years, bla_NDM genes have also been identified in rare Salmonella serotypes, such as S. Kottbus, S. Corvallis, and S. Lomita (Nielsen et al., 2021, Villa et al., 2015, Li et al., 2020). ST34 S. Typhimurium is often characterized by MDR expressed through several resistance genes, including mcr-1, bla_CTX–M–55, and qnrS. A previous report characterized an ST34 S. Typhimurium isolate carrying bla_NDM–5 and the clonal dissemination of bla_NDM–1-positive ST34 S. Typhimurium in South China (Deng et al., 2024). Here, we have described the first identification of Salmonella isolates carrying bla_NDM in Jiaxing City. Our isolation of two unrelated clinical Salmonella isolates of different serovars, both carrying bla_NDM–5, indicates that the major NDM type in Jiaxing is NDM-5.

Horizontal transmission mediated by various Inc groups of plasmids constitute the major route for the ongoing spread of carbapenem resistance, and include IncC, IncC/IncFIB(K), IncC/IncX3, IncFIB(K), IncI1-I(Alpha), IncFII, IncFII(pCoo), IncFII(pCoo)/p0111, IncM2, IncN2, IncX3, IncHI2/IncHI2A, and IncHI2/IncHI2A/IncN. All of these were isolated from different serotypes across various countries from 2012 to 2024, highlighting the global burden of bla_NDM-positive plasmid in S. enterica. Most IncHI2 plasmids found in ST34 S. Typhimurium strains shared a similar backbone, with the capture of bla_NDM–1 through an IncHI2/ST3 plasmid (Deng et al., 2024). Although IncX3 has been deemed the primary vehicle for bla_NDM transmission worldwide in Enterobacteriaceae (Guo et al., 2019), IncHI2/ST3 plasmids have replaced IncX3 plasmids as the primary plasmid vector for bla_NDM–5 transmission on some farms (He et al., 2023).

ST3-IncHI2 plasmids exhibit high sequence conservation in backbones, but possess highly genetic plasticity in accessory regions, allowing for the acquisition of numerous antibiotic resistance genes through mobile elements (Fang et al., 2018). Many mobile elements have played crucial roles in the dissemination of bla_NDM, including IS26, ISAba125, IS5, ISCR1, Tn3, Tn125, and Tn3000 (Feng et al., 2018, Zhao et al., 2021, Li et al., 2021). A novel IS26-flanked composite transposon (Tn7540) in the chromosome of an S. Indiana isolate was found to carry bla_NDM–9 and fosA3 (Sun et al., 2023). An IS15DIV-flanked composite transposon also contributed to the dissemination of bla_NDM–5 in S. Typhimurium (Zhao et al., 2025). NDM-positive isolates consistently carry either a complete or fragmented ISAba125, providing a promoter region for bla_NDM and playing a critical role in the horizontal transmission of bla_NDM–5 and other resistance determinants (Zhao et al., 2025). Our comparative plasmid analysis showed that the deletion of ISAba125 may have been occurred late in the evolution of p23045-NDM5. Up to eight tandem copies of an ISCR1 unit (ISCR1-dsbD-trpF-ble-bla_NDM–1-ΔISAba125) were found on an HI2 plasmid in S. Typhimurium (Song et al., 2023). Although plasmid-borne bla_NDM–5 is usually found as a single copy, we previously identified two non-tandem copies of bla_NDM–5 on a 144,225-bp IncF plasmid from a carbapenem-resistant clinical isolate of E. coli (Feng et al., 2018). The coexistence of two bla_NDM–5 genes was attributed to duplication of an IS26-bracketed region containing ISCR1. In the present study, the two bla_NDM–5 regions within one IncHI2/IncHI2A plasmid carried by S. Stanley may have resulted from the duplication of a unit comprising IS26-umuD-umuC-△ ISKox3-△ IS3000-△ ISAba125-IS5-bla_NDM–5-ble_MBL-trpF-dsbC-IS26 that was subsequently interrupted by IS1 upstream of △ IS3000. No ISCR1 sequences were found in p23045-NDM5 or p2024406-NDM5.

5 Conclusion

In conclusion, we described two MDR Salmonella strains carrying bla_NDM–5 that were isolated in Jiaxing City, China, specifically 2023JX045 (4,[5],12:i:-) and 2024-406 (S. Stanley). Each strain was shown to harbor a bla_NDM–5-positive IncHI2/IncHI2A plasmid (p23045-NDM5 in 2023JX045 and p2024406-NDM5 in 2024-406), exhibiting signs of multiple evolutionary events that contributed to the diversity of the bla_NDM–5-region. IS26-flanked composite transposons appeared to play an important role in the formation of this region. The complex diversity of the bla_NDM–5 region is one explanation for the common development of MDR host strains. To the best of our knowledge, this is the first report of a bla_NDM gene carried by Salmonella, a major foodborne pathogen, in this region of China. Importantly, this is also the first report of a single IncHI2/IncHI2A plasmid carrying two copies of bla_NDM–5 in an S. Stanley host. The identification of CRSE isolates harboring bla_NDM and the expanding diversity of bla_NDM–5-positive plasmids indicate the potential for widespread dissemination.

This study has several limitations. Since only two isolates were analyzed in this study, the transmission and evolution mechanism of NDM in Salmonella has not been fully explained. The sources of infection of the two cases were also not successfully identified. Therefore, we recommend heightened vigilance and international cooperation to mitigate the public health impact of these pathogens.

Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

Author contributions

PL: Formal Analysis, Writing – original draft, Writing – review & editing. YYu: Data curation, Formal Analysis, Investigation, Visualization, Writing – review & editing. YYa: Software, Writing – review & editing. MJ: Software, Writing – review & editing. LG: Software, Writing – review & editing. XL: Conceptualization, Writing – review & editing. YS: Conceptualization, Visualization, Writing – original draft, Writing – review & editing. GZ: Methodology, Writing – review & editing. ZC: Data curation, Writing – review & editing.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. This work was supported by the National Key Research and Development Program of China (2023YFC260510401 and 2023YFC2605100), the Medical Science and Technology Project of Zhejiang Province (2024KY1697) and the Science and Technology Program of Jiaxing City (2023AY31028 and 2023AY11037).

Acknowledgments

We thank Michelle Kahmeyer-Gabbe, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The authors declare that no Generative AI was used in the creation of this manuscript.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Footnotes

1. ^http://denglab.info/SeqSero2

2. ^https://www.pubmlst.org

3. ^https://cge.food.dtu.dk/services/ResFinder/

4. ^https://cge.food.dtu.dk/services/PlasmidFinder/

5. ^https://www.is.biotoul.fr

6. ^https://mjsull.github.io/Easyfig/

7. ^https://itol.embl.de/

References

Alikhan, N., Petty, N., Ben Zakour, N., and Beatson, S. (2011). BLAST Ring Image Generator (BRIG): Simple prokaryote genome comparisons. BMC Genomics 12:402. doi: 10.1186/1471-2164-12-402

PubMed Abstract | Crossref Full Text | Google Scholar

Bathoorn, E., Rossen, J., Lokate, M., Friedrich, A., and Hammerum, A. (2015). Isolation of an NDM-5-producing ST16 Klebsiella pneumoniae from a Dutch patient without travel history abroad, August 2015. Euro Surveill. 20:30040. doi: 10.2807/1560-7917.ES.2015.20.41.30040

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, Y., Zhou, Z., Jiang, Y., and Yu, Y. (2011). Emergence of NDM-1-producing Acinetobacter baumannii in China. J. Antimicrob Chemother. 66, 1255–1259. doi: 10.1093/jac/dkr082

PubMed Abstract | Crossref Full Text | Google Scholar

China National Center for Food Safety Risk Assessment (2024). National foodborne disease surveillance manual in 2024. Beijing: China National Center for Food Safety Risk Assessment.

Google Scholar

Day, M., Meunier, D., Doumith, M., de Pinna, E., Woodford, N., and Hopkins, K. (2015). Carbapenemase-producing Salmonella enterica isolates in the UK. J. Antimicrob Chemother. 70, 2165–2167. doi: 10.1093/jac/dkv075

PubMed Abstract | Crossref Full Text | Google Scholar

Deng, L., Lv, L., Tu, J., Yue, C., Bai, Y., He, X., et al. (2024). Clonal spread of blaNDM-1-carrying Salmonella enterica serovar Typhimurium clone ST34 and wide spread of IncHI2/ST3-blaNDM-5 plasmid in China. J. Antimicrob Chemother. 79, 1900–1909. doi: 10.1093/jac/dkae178

PubMed Abstract | Crossref Full Text | Google Scholar

Fang, L., Li, X., Deng, G., Li, S., Yang, R., Wu, Z., et al. (2018). High genetic plasticity in multidrug-resistant sequence type 3-IncHI2 plasmids revealed by sequence comparison and phylogenetic analysis. Antimicrob Agents Chemother. 62:e02068-17. doi: 10.1128/AAC.02068-17.

PubMed Abstract | Crossref Full Text | Google Scholar

Feng, Y., Liu, L., McNally, A., and Zong, Z. (2018). Coexistence of two blaNDM-5 genes on an IncF plasmid as revealed by nanopore sequencing. Antimicrob Agents Chemother. 62, e110–e118. doi: 10.1128/AAC.00110-18

PubMed Abstract | Crossref Full Text | Google Scholar

Guo, X., Rao, Y., Guo, L., Xu, H., Lv, T., Yu, X., et al. (2019). Detection and genomic characterization of a Morganella morganii isolate from China that produces NDM-5. Front. Microbiol. 10:1156. doi: 10.3389/fmicb.2019.01156

PubMed Abstract | Crossref Full Text | Google Scholar

Han, R., Shi, Q., Wu, S., Yin, D., Peng, M., Dong, D., et al. (2020). Dissemination of Carbapenemases (KPC, NDM, OXA-48, IMP, and VIM) among carbapenem-resistant Enterobacteriaceae isolated from adult and children patients in China. Front. Cell Infect. Microbiol. 10:314. doi: 10.3389/fcimb.2020.00314

PubMed Abstract | Crossref Full Text | Google Scholar

He, W., Gao, M., Lv, L., Wang, J., Cai, Z., Bai, Y., et al. (2023). Persistence and molecular epidemiology of blaNDM-positive Gram-negative bacteria in three broiler farms: A longitudinal study (2015-2021). J. Hazard Mater. 446:130725. doi: 10.1016/j.jhazmat.2023.130725

PubMed Abstract | Crossref Full Text | Google Scholar

Hornsey, M., Phee, L., and Wareham, D. (2011). A novel variant, NDM-5, of the New Delhi metallo-β-lactamase in a multidrug-resistant Escherichia coli ST648 isolate recovered from a patient in the United Kingdom. Antimicrob Agents Chemother. 55, 5952–5954. doi: 10.1128/AAC.05108-11

PubMed Abstract | Crossref Full Text | Google Scholar

Hu, X., Xu, X., Wang, X., Xue, W., Zhou, H., Zhang, L., et al. (2017). Diversity of New Delhi metallo-beta-lactamase-producing bacteria in China. Int. J. Infect. Dis. 55, 92–95. doi: 10.1016/j.ijid.2017.01.011

PubMed Abstract | Crossref Full Text | Google Scholar

Huang, J., Wang, M., Ding, H., Ye, M., Hu, F., Guo, Q., et al. (2013). New Delhi metallo-β-lactamase-1 in carbapenem-resistant Salmonella strain, China. Emerg. Infect. Dis. 19, 2049–2051. doi: 10.3201/eid1912.130051

PubMed Abstract | Crossref Full Text | Google Scholar

Huang, Y., Yu, X., Xie, M., Wang, X., Liao, K., Xue, W., et al. (2016). Widespread dissemination of carbapenem-resistant Escherichia coli sequence type 167 strains harboring blaNDM-5 in clinical settings in China. Antimicrob Agents Chemother. 60, 4364–4368. doi: 10.1128/AAC.00859-16

PubMed Abstract | Crossref Full Text | Google Scholar

Lamichhane, B., Mawad, A., Saleh, M., Kelley, W., Harrington, P., Lovestad, C., et al. (2024). Salmonellosis: An overview of epidemiology, pathogenesis, and innovative approaches to mitigate the antimicrobial resistant infections. Antibiotics. 13:10. doi: 10.3390/antibiotics13010076

PubMed Abstract | Crossref Full Text | Google Scholar

Li, R., Xie, M., Liu, L., Huang, Y., Wu, X., Wang, Z., et al. (2020). Characterisation of a cointegrate plasmid harbouring blaNDM-1 in a clinical Salmonella Lomita strain. Int. J. Antimicrob Agents 55:105817. doi: 10.1016/j.ijantimicag.2019.09.021

PubMed Abstract | Crossref Full Text | Google Scholar

Li, X., He, J., Jiang, Y., Peng, M., Yu, Y., and Fu, Y. (2021). Genetic characterization and passage instability of a hybrid plasmid Co-Harboring blaIMP-4 and blaNDM-1 reveal the contribution of insertion sequences during plasmid formation and evolution. Microbiol. Spectr. 9:e0157721. doi: 10.1128/Spectrum.01577-21

PubMed Abstract | Crossref Full Text | Google Scholar

Li, X., Jiang, Y., Wu, K., Zhou, Y., Liu, R., Cao, Y., et al. (2017). Whole-genome sequencing identification of a multidrug-resistant Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong. China. Infect Genet Evol. 55, 195–198. doi: 10.1016/j.meegid.2017.09.005

PubMed Abstract | Crossref Full Text | Google Scholar

Miao, M., Wen, H., Xu, P., Niu, S., Lv, J., Xie, X., et al. (2018). Genetic Diversity of Carbapenem-Resistant Enterobacteriaceae (CRE) clinical isolates from a tertiary hospital in Eastern China. Front. Microbiol. 9:3341. doi: 10.3389/fmicb.2018.03341

PubMed Abstract | Crossref Full Text | Google Scholar

Mojica, M., Rossi, M., Vila, A., and Bonomo, R. (2022). The urgent need for metallo-β-lactamase inhibitors: An unattended global threat. Lancet Infect Dis. 22, e28–e34. doi: 10.1016/S1473-3099(20)30868-9

PubMed Abstract | Crossref Full Text | Google Scholar

Nielsen, H., Thomsen, P., Litrup, E., Torpdahl, M., Overballe-Petersen, S., Hansen, F., et al. (2021). A case of bla NDM-1-positive Salmonella Kottbus, Denmark, November 2021. Euro Surveill. 26:2100569. doi: 10.2807/1560-7917.ES.2021.26.26.2100569

PubMed Abstract | Crossref Full Text | Google Scholar

Nordmann, P., Dortet, L., and Poirel, L. (2012). Carbapenem resistance in Enterobacteriaceae: Here is the storm! Trends Mol. Med. 18, 263–272. doi: 10.1016/j.molmed.2012.03.003

PubMed Abstract | Crossref Full Text | Google Scholar

Rasheed, J., Kitchel, B., Zhu, W., Anderson, K., Clark, N., Ferraro, M., et al. (2013). New Delhi metallo-β-lactamase-producing Enterobacteriaceae United States. Emerg. Infect. Dis. 19, 870–878. doi: 10.3201/eid1906.121515

PubMed Abstract | Crossref Full Text | Google Scholar

Sassi, A., Loucif, L., Gupta, S., Dekhil, M., Chettibi, H., and Rolain, J. (2014). NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria. Antimicrob Agents Chemother. 58, 5606–5608. doi: 10.1128/AAC.02818-13

PubMed Abstract | Crossref Full Text | Google Scholar

Savard, P., Gopinath, R., Zhu, W., Kitchel, B., Rasheed, J., Tekle, T., et al. (2011). First NDM-positive Salmonella sp. strain identified in the United States. Antimicrob Agents Chemother. 55, 5957–5958. doi: 10.1128/AAC.05719-11

PubMed Abstract | Crossref Full Text | Google Scholar

Song, H., Zou, S., Huang, Y., Jian, C., Liu, W., Tian, L., et al. (2023). Salmonella Typhimurium with Eight Tandem Copies of blaNDM-1 on a HI2 Plasmid. Microorganisms 12:10. doi: 10.3390/microorganisms12010020

PubMed Abstract | Crossref Full Text | Google Scholar

Sun, Y., Han, Y., Qian, C., Zhang, Q., Yao, Z., Zeng, W., et al. (2023). A novel transposon Tn7540 carrying blaNDM-9 and fosA3 in chromosome of a pathogenic multidrug-resistant Salmonella enterica serovar Indiana isolated from human faeces. J. Glob. Antimicrob Resist. 33, 72–77. doi: 10.1016/j.jgar.2023.01.013

PubMed Abstract | Crossref Full Text | Google Scholar

Temkin, E., Adler, A., Lerner, A., and Carmeli, Y. (2014). Carbapenem-resistant Enterobacteriaceae: Biology, epidemiology, and management. Ann. N. Y. Acad. Sci. 1323, 22–42. doi: 10.1111/nyas.12537

PubMed Abstract | Crossref Full Text | Google Scholar

Villa, L., Guerra, B., Schmoger, S., Fischer, J., Helmuth, R., Zong, Z., et al. (2015). IncA/C plasmid carrying bla(NDM-1), bla(CMY-16), and fosA3 in a Salmonella enterica serovar corvallis strain isolated from a migratory wild bird in Germany. Antimicrob Agents Chemother. 59, 6597–6600. doi: 10.1128/AAC.00944-15

PubMed Abstract | Crossref Full Text | Google Scholar

Wu, Y., Jiang, T., Bao, D., Yue, M., Jia, H., Wu, J., et al. (2023). Global population structure and genomic surveillance framework of carbapenem-resistant Salmonella enterica. Drug Resist. Updat. 68:100953. doi: 10.1016/j.drup.2023.100953

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, P., Xie, Y., Feng, P., and Zong, Z. (2014). blaNDM-5 carried by an IncX3 plasmid in Escherichia coli sequence type 167. Antimicrob Agents Chemother. 58, 7548–7552. doi: 10.1128/AAC.03911-14

PubMed Abstract | Crossref Full Text | Google Scholar

Zhao, K., Jin, J., Liao, Y., Liu, A., Liu, W., and Wu, W. (2025). IS 15DIV-flanked composite transposon harboring bla NDM-5 in multidrug-resistant Salmonella typhimurium. iScience 28:111720. doi: 10.1016/j.isci.2024.111720

PubMed Abstract | Crossref Full Text | Google Scholar

Zhao, Q., Zhu, J., Cai, R., Zheng, X., Zhang, L., Chang, M., et al. (2021). IS 26 is responsible for the evolution and transmission of blaNDM-harboring plasmids in Escherichia coli of Poultry Origin in China. mSystems 6:e0064621. doi: 10.1128/mSystems.00646-21

PubMed Abstract | Crossref Full Text | Google Scholar