Introduction

Since Salmonella enterica serovar 1,4,[5], 12: i:-(S. 1,4,[5], 12: i:-) was reported in Spain in 1997 [1], the detection rate has increased and in recent years has surpassed Salmonella enterica serovar typhimurium (STM) as the most common serotype globally [2]. S. 1,4,[5], 12: i:—has high genetic similarity with STM, and it is speculated that 1,4,[5], 12: i:-may be a monophasic variant of phase I of STM. S. 1,4,[5], 12: i:-was deficient in fljB gene, which is a target site of multiplex quantitative PCR for distinguishing S. 1,4,[5], 12: i:-and STM [3]. Carbapenem has been considered as the last drug against most of multi-drug–resistant Enterobacteriaceae, including Salmonella spp., and remains a major public health problem [4]. So far, carbapenemase found in Salmonella strains includes KPC-2 [5, 6], NDM-1 [4], NDM-5 [7,3], a monophasic variant of S. enterica serovar Typhimurium, which was recognized as an emerging cause of infection worldwide. Antimicrobial susceptibility was performed using the broth microdilution method, which employed the following antimicrobial agents: piperacillin, cefotaxime, cefotaxime-clavulanic acid, ceftazidime, ceftazidime-clavulanic acid, ceftazidime-avibactam, ceftriaxone, cefepime, imipenem, and meropenem, and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI M100–S32) (CLSI, 2022). Disk diffusion method was performed for cefiderocol susceptibility. Piperacillin, ceftriaxone, imipenem, ceftazidime, clavulanic acid, and avibactam were purchased from Sigma-Aldrich, St. Louis, MO, USA. Cefepime, cefotaxime, and meropenem were purchased from MedChemExpress, New Jersey, USA. Disks of cefiderocol (30 μg) were purchased from Liofilchem, Roseto degli Abruzzi, Italy. For ceftazidime-avibactam MIC evaluation, avibactam was tested at a fixed concentration of 4 mg/L, while ceftazidime was added at different concentrations that ranged from 0.0625 to 128 mg/L. MICs were determined in triplicate on three separate days. Escherichia coli TOP10 (pHSG396) and E. coli C600 were used as quality control strains.

Whole-genome sequencing and annotation

The genomic DNA of Salmonella enterica serovar 1,4,[5], 12: i:-ST3606 was extracted using NucleoBond® HMW DNA kit (MACHEREY–NAGEL, Germany) and SMRT sequencing was performed using Illumina Novaseq 6000 (Illumina, San Diego, USA) and PacBio sequencer (Suzhou Genewiz Biotechnology Co. Ltd., Suzhou, China). Raw data generated using short-read technology were optimized by the software cutadapt (v1.9.1) to generate clean data by removing adapters as well as low-quality sequences. PacBio reads were assembled using HGAP 4.0/Falcon 0.3 (Celera Assembler 8.2) [13]. Assembly polishing was performed with Pilon (version 1.22) using Illumina reads. Annotation of the ST3606 genome was completed using the National Center for Biotechnology Information (NCBI) prokaryotic annotation pipeline (PGAP). ResFinder 3.2 [14], PlasmidFinder [15], OriTfinder database [18] were utilized to detect resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606, respectively. Sequence comparisons, map generation, and plasmid circular representation diagram were performed using BLAST (version BLAST + 2.11.0), Easyfig (version 2.2.5), and BLAST Ring Image Generator, respectively.

Cloning of blaCTX-M variants

The full lengths of the blaCTX-M-1/-55/-261 genes were synthesized and ligated to the vector pET28a and pHSG396 by BGI Genomics Co., Ltd, to generate CTX-M-1/-55/-261-pET28a and CTX-M-1/-55/-261-pHSG396 respectively. The correct constructs were confirmed by Sanger sequencing and transformed into E. coli TOP10 treated with 100 mM CaC12 and subjected to heat-shock at 42° for 1 min. Antimicrobial susceptibilities of these constructs were determined as described above. The empty pHSG396 plasmid was used as a control.

CTX-M-261 β-lactamase production and steady-state kinetic parameters

The recombinant CTX-M-1/-55/-261-pET28a plasmids were transformed into E. coli BL21 Rosetta-gami™ DE3 and grown in LB medium containing 50 mg/L kanamycin at 37 ℃ until an optical density of 0.4–0.6 (OD600) was reached. Next, 0.2 mM IPTG (isopropyl-β-d-thiogalactoside) was added and the temperature was lowered to 20 ℃ and allowed to incubate for 22 h. Cells were then harvested, resuspended in ice-cold buffer A (10 mM imidazole, 10 mM sodium phosphate, pH 7.4, and 300 mM NaCl), and then lysed by sonication in an ice-bath. The cell pellet was removed by centrifugation steps at 12,000 g for 30 h at 4 ℃, the supernatant was filtered, and the resulting soluble fraction applied to HisTrap™ HP column (GE Healthcare) prebalanced by buffer A. After washing with buffer B (60 mM imidazole, 10 mM sodium phosphate pH 7.4, and 300 mM NaCl), the protein was eluted from the resin with buffer C (500 mM imidazole, 10 mM sodium phosphate, pH 7.4, and 300 mM NaCl). Finally, the eluted protein was loaded into a dialysis bag and was dialyzed with buffer D (10 mM sodium phosphate, pH 7.4, and 10 mM NaCl) overnight for desalination and removing imidazole. The purity of the protein was estimated to be higher than 95% by SDS-PAGE. The concentrations were determined by Pierce™ BCA Protein Assay Kit.

Kinetic parameters of cefotaxime, ceftazidime, ceftriaxone, cefepime, imipenem, and meropenem were determined using purified CTX-M-1/-55/-261 β-lactamases in 50 mM sodium phosphate buffer (pH 7.0) at 30 ℃. BSA at 20 mg/L was added to the dilute solution of CTX-M to prevent denaturation. The real-time absorbances of meropenem (298 nm), imipenem (297 nm), ceftazidime (257 nm), cefepime (254 nm), cefotaxime (257 nm), and ceftriaxone (240 nm) were determined under initial-rate conditions with a SHIMADZU UV2550 spectrophotometer (Kyoto, Japan) for 5 min. The initial velocities versus substrate concentrations were measured at least thrice. The molar extinction coefficients for tested substrates were obtained from a previous study: nitrocefin (Δε482 nm = 15,000 M−1 cm−1), ceftriaxone (Δε240 nm =  − 10,351 M−1 cm−1), cefotaxime (Δε257 nm =  − 7,500 M−1 cm−1), cefepime (Δε254 nm =  − 10,000 M−1 cm−1), ceftazidime (Δε257 nm =  − 9000 M−1 cm−1), imipenem (Δε297 nm =  − 9000 M−1 cm−1), and meropenem (Δε298 nm =  − 6500 M−1 cm−1) [19, 20]. Notably, the estimated molecular weight of the tag-free CTX-M enzyme, as determined using the ExPASy-Compute pI/MW tool, approximated 28 kDa, and the isoelectric point, was determined at 7.8. The kcat and Km values were calculated with the GraphPad Prism 8.4 (San Diego, USA) using the Michaelis–Menten equation as follows [19, 21]:

$$\text{E}+\text{S}\begin{array}{c}k_{+1}\\\Leftrightarrow\\k_{-1}\end{array}\mathrm{ES}\xrightarrow{k_{+2}}{\mathrm E\mathrm S'}\xrightarrow{k_{+3}}\mathrm{ES}''\rightarrow\rightarrow\rightarrow\xrightarrow{k_{+n}}\text{E}+\text{P}$$

Cefotaxime and ceftriaxone behave as the best substrates of CTX-M, and the steady-state kinetic parameters, Km and kcat, were determined by Hanes–Woolf linearization or by non-linear least-square fit of the Michaelis–Menten equation [19, 21]. The reaction is described by the model: S0/v = (Km + S0)/V, where V is the maximum rate, observed when S0 >  > Km (substrate saturation). For ceftazidime and cefepime that is rather stable to the action of the CTX-M, with imipenem acting as transient inactivator and meropenem acting as inactivator, the Km value of ceftazidime and cefepime and the Kcat value of four drugs were determined from experiments involving competition between the poor substrate and 100 μM of nitrocefin as a reporter substrate at 482 nm [21, 22].

Western blotting

The expression levels of CTX-M-1, CTX-M-55, and CTX-M-261 in E. coli BL21 and E. coli TOP10 were determined by His-antibody. Briefly, E. coli carrying CTX-M-1/-55/-261-pET28a was self-induced without IPTG, and then recovered and resuspended in B-PER buffer (Thermo Scientific, MA, USA). The 30-μg total protein was subjected to SDS-PAGE, transferred onto a PVDF membrane, and probed with an His-antibody to determine the protein levels of the three CTX-M enzymes in the same E. coli strains.

Conjugation experiment

The agar mating method was used to transfer β-lactam resistance to the rifampin-resistant E. coli C600 recipient. ST3606 and E. coli C600 with a McFarland standard value of 2.0 were mixed 1:1, and then the mixture dropped to the membrane placed on the solid LB medium without antibiotics, which was incubated at 37 ℃ for 18–24 h. After swirling the filtration membrane with 2 mL of liquid LB medium without antibiotics, 20 µL suspension was seeded on LB plate containing 100 mg/L ampicillin (Genview Co., Bei**g, China) and 750 mg/L rifampin (Sangon Biotech Co., Shanghai, China) and cultured for 24 h. The selected transconjugant colonies were identified by PCR targeting the blaCTX-M gene and sequencing.

Nucleotide sequence accession numbers

The nucleotide sequences of genome, pST3606-1, pST3606-2, pST3606-3, pST3606-4, and pST3606-5 plasmids were deposited in the GenBank under the accession numbers CP094332, CP094333, CP094334, CP094335, CP094336, and CP094337, respectively. The nucleotide sequences of blaCTX-M-261 were deposited in the GenBank under the accession number OQ942222.

Results

Antibiotic resistance profiles of Salmonella enterica serovar 1,4,[5], 12: i:-ST3606 and conjugant

We isolated the strain and identified it as a Salmonella enterica serovar by MALDI-TOF MS and Diagnostic Serum Kit. We further confirmed the serotype of the strain as 1,4,[5], 12: i:-by polymerase chain reaction for amplification of fliB and fliB-fliA as described previously [3]. The result showed that only one band at 1000 bp (fliB-fliA) but none of band at 1389 bp (fliB) was found, which verified this strain was Salmonella enterica serovar 1,4,[5], 12: i: -, a monophasic variant of S. enterica serovar Typhimurium (Fig. 1). Based on the results of the antibiotic susceptibility test, ST3606 exhibited resistance to most of the commonly used antibiotics, including piperacillin (> 2048 μg/mL), cefotaxime (2048 μg/mL), cefotaxime-clavulanic acid (> 128 μg/mL), ceftazidime (> 128 μg/mL), ceftazidime-clavulanic acid (> 128 μg/mL), ceftazidime-avibactam (> 128 μg/mL), ceftriaxone (> 128 μg/mL), cefepime (> 128 μg/mL), imipenem (16 μg/mL), and meropenem (32 μg/mL) (Table 1), except for levofloxacin (≤ 0.125 μg/mL), amikacin (≤ 2 μg/mL), and trimethoprim-sulfamethoxazole (≤ 20 μg/mL) (Table S1).

Fig. 1
figure 1

Differentiation of diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium (S. 1,4,[5], 12: i: -). A 1389-bp product from Salmonella Typhimurium that possesses a phase 2 flagellar antigen and no product from S. 1,4,[5], 12: i:—that lacks a phase 2 flagellar antigen

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Table 1 MICs for the clinical isolate ST3606, the corresponding E. coli C600 transconjugant carrying blaCTX-M-261, and CTX-M-producing E. coli TOP10 clones
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Overview of the Salmonella enterica serovar 1,4,[5], 12: iST3606

Whole-genome sequencing and MLST 2.0 tool analysis showed Salmonella enterica serovar 1,4,[5], 12: i:- ST3606 belonged to sequence type 34 (ST34). Salmonella enterica serovar 1,4,[5], 12: i:-ST3606 contained one chromosome and five plasmids (pST3606-1 to pST3606-5). The chromosome was 4,959,696 bp in size and contained 4769 genes with a guanine–cytosine content of 52.16%. pST3606-1 to pST3606-5 plasmids were 109,070 bp, 70,455 bp, 3592 bp, 4059 bp, and 3001 bp in size, respectively. ARGs were located on the chromosome, pST3606-1 and pST3606-2, but not on pST3606-3/-4/-5. Only one ARG coding for aminoglycoside resistance (aac(6’)-laa) was identified on the chromosome. Five ARGs, including blaNDM-5 coding for β-lactam resistance, sul1 coding for sulfonamide resistance, qacE coding for antiseptic resistance, aadA2 coding for aminoglycoside resistance, and dfrA12 coding for trimethoprim resistance, and three ARGs, including blaCTX-M-261 and blaTEM-1B coding for β-lactam resistance and aac(3)-lld coding for fluoroquinolone/aminoglycoside resistance, were identified on pST3606-1 and pST3606-2 plasmids, respectively (Table 2). blaNDM-5 has been reported in Salmonella enterica serovar 1,4,[5], 12: i:-for the first time in this article, although it was previously reported in Salmonella enterica serovar Typhimurium. Interestingly, we also found a novel blaCTX-M gene on another plasmid. According to their genetic locations, the ARGs were divided into three drug-resistance regions, for which the genetic structures are described in the following sections. Through BLAST analysis and the GenBank database, we detected identical or similar sequences in other bacteria.

Table 2 Genomic characteristics of Salmonella enterica serovar 1,4,[5], 12: i:—isolate ST3606
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Genetic contexts associated with blaNDM-5 and class I integron

Salmonella enterica serovar 1,4,[5], 12: i:-ST3606 contained blaNDM-5, which encoded for New Delhi metallo-β lactamase that hydrolyzes almost all β-lactam antibiotics including carbapenem. The blaNDM-5 gene was located on the coordinate 79,483-80,295 of pST3606-1 with a GC content of 50.6%, which was identified as a typical IncI1-I(α) plasmid containing regions involved in plasmid stability, replication, and conjugative transfer (Fig. 2). The blaNDM-5 was embedded in an IS26, IS15, and Aba125-mediated transposition unit, with IS26, IS15, and ISAba125 located upstream of blaNDM-5, forming the genetic structure “IS26-IS15-ISAba125-blaNDM-5” (77,884-80,295), which has also been found in diverse isolates in different genetic contexts, including the E. coli plasmid pC-F-164_A-OXA140 (91% query coverage and 99.98% identity, GenBank accession no. CP048368), E. coli plasmid pE-T654-NDM-5 (100% query coverage and 99.98% identity, GenBank accession no. CP090291), the Klebsiella pneumoniae plasmid p_dm760b_NDM-5 (96% query coverage and 99.98% identity, GenBank accession no. CP095648), and Citrobacter sedlakii plasmid p_dmcr749c_NDM-5 (100% query coverage and 99.96% identity, GenBank accession no. CP095669) (Fig. 2). The sul1, qacE, aadA2, and dfrA12 genes, which mediated sulfonamide, disinfectant, aminoglycoside, and trimethoprim resistance, were located upstream of integrase type I (Intl1) and on the coordinate 84,608-85,447; 85,507-85,788; 85,952-86,743; and 87,151-87,648, respectively. The class I integron of Salmonella enterica serovar 1,4,[5], 12: i:-ST3606 was 3547 bp in size (coordinate: 84,608–88,155) and contained the trimethoprim resistance gene dfrA12, aminoglycoside resistance gene aadA2, disinfectant resistance gene qacE, trimethoprim resistance gene sul1, and integrase (intI1). We found that ISVsa3 was located upstream of class I integron followed by the IS26 and TnAs1 transposons, forming a typical TnAs1 transposition unit (Fig. 2).

Fig. 2
figure 2

A 10.9-kb IncI1-α sequence of the genetic context of the NDM-5-harboring plasmid pST3606-1 is shown. Linear genetic structure comparison of plasmid pST3606-1 (Salmonella Typhimurium (S. 1,4,[5], 12: i: -), GenBank accession number CP094333), pC-F-164_A-OXA140 (Escherichia coli, GenBank accession number CP048368), pE-T654-NDM-5 (Escherichia coli, GenBank accession number CP090291), p_dm760b_NDM-5 (Klebsiella pneumoniae, GenBank accession number CP095648), and p_dmcr749c_NDM-5 (Citrobacter sedlakii, GenBank accession number CP095669). The arrows indicate open reading frames. The red arrows represent antibiotic resistance gene. The blue arrows represent the transposon and IS elements. The yellow arrows represent Integrase gene. Light green shading denotes homology regions. The depth of shadowing is indicative of the BLASTn matching degree. IS, insertion sequences. Color images are available online. Comparisons performed using a nucleotide basic local alignment sequence tool search and visualized using in silico molecular cloning genomics edition software

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Genetic contexts associated with blaCTX-M-261

The IncFII-type plasmid pST3606-2 carried by ST3606 shares a similar backbone with the plasmid pST90-1 (84% query coverage and 100% identity, GenBank accession no. CP050735) which was identified in a strain of S. enterica but carrying blaCTX-M-27 isolated from a patient in the USA (Fig. 3). The main difference between pST3606-2 and pST90-1 was that pST3606-2 contained a 4941-bp complex transposon structure carrying blaCTX-M-261, a novel blaCTX-M gene carrying a single-nucleotide mutation 751G > A leading to amino acid substitution of Val for Met at position 251 (Val251Met) on the coordinate 70,155-70,455, 1-575. blaCTX-M-261, bracketed by IS1 elements and IS4 elements, could encode extended-spectrum beta-lactamase (ESBL) conferring resistance to the extended-spectrum cephalosporins. The blaCTX-M gene of pST3606-2 was organized as “IS1-IS26-blaCTX-M-261-WbuC-blaTEM-1-IS26-IS4” (Fig. 3), which among plasmids in the NCBI nucleotide database, IncFII plasmid was positive for blaCTX-M. The conjugation experiment demonstrated that pST3606-2 was successfully transferred from the donor strain (ST3606) to the recipient (Escherichia coli C600) and conferred beta-lactam but not carbapenem resistance to the recipient strain due to the pST3606-2 containing blaCTX-M-261 and blaTEM-1 genes (Table 1 and 2). The conjugation frequency of pST3606-2 was 10–3 per recipient cell for ST3606. We further analyzed the conjugative transfer region of the conjugative plasmid pST3606-2 using software oriTfinder, and the results showed that it contained the intact conjugative transfer region, including origin of transfer site (oriT) on the coordinate 49,414-49,499, relaxase gene on the coordinate 16,618-21,888, gene encoding type IV coupling protein (T4CP) on the coordinate 21,888-24,113, and gene cluster for Tra_F-like IV secretion system (T4SS) on the coordinate 15,852-50,067 (Fig. 3).

Fig. 3
figure 3

Circular representation of pST3606-2. The innermost circle represents GC content; the second inner circle represents GC skew (green, GC + ; purple, GC-). These mcr-8-carrying plasmids pST3606-2 (Salmonella Typhimurium (S. 1,4,[5], 12: i: -), GenBank accession number CP094334), p51015_CTX_M_15 (Klebsiella pneumoniae, GenBank accession number CP050379), pST90-1 (Salmonella enterica, GenBank accession number CP050735), pEC31-1 (Escherichia coli, GenBank accession number CP056037), and plasmid unnamed 2 (Shigella flexneri, GenBank accession number CP058841) were represented by pink, light blue, purple, dark blue, and yellow circles, respectively. The outermost red circle depicts gene distribution of the resistance gene, transposon, IS elements, and integrative and conjugative element. blaCTX-M-261, CTX-M-261 extended spectrum β-lactamase gene; aac(3)-lld gene, aminoglycoside resistance gene; ATPase, adenosine triphosphate synthase; hypothetical protein

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Steady-state kinetics of CTX-M-261

The purities of CTX-M proteins were more than 95% as estimated by SDS-PAGE. CTX-M-55, differing from CTX-M-261 variant by only one single amino acid substitution, has the highest homology and was employed as a contrast enzyme. The steady-state kinetic parameters kcat and Km of CTX-M-1, CTX-M-55, and CTX-M-261 among a set of substrates (Table 3) indicated that all cephalosporins tested but not carbapenem could be hydrolysed by CTX-M. Cefotaxime was the best substrate for CTX-M-261, with a catalytic efficiency of 7.9 μM−1 s−1. But, for CTX-M-1 and CTX-M-55, the highest catalytic efficiency happened to ceftriaxone (9.98 μM−1 s−1 and 16.67 μM−1 s−1, respectively), which was consistent with a previous report [20]. Compared to CTX-M-55, CTX-M-261 exhibited significantly decreased affinity and diminished turnover for ceftriaxone (0.7 μM−1 s−1 vs. 16.67 μM−1 s−1), which was also confirmed by their MICs of this drug (64 μg/mL vs. > 128 μg/mL) (Table 1). Intriguingly, the Km value of CTX-M-261 catalyzing ceftazidime could be determined, but its kcat/Km value was also too low (0.06 μM−1 s−1). However, regarding the MICs of ceftazidime for E. coli TOP10 carrying blaCTX-M, CTX-M-261 mediated the highest MIC (Table 1), which seem to be in agreement with the Km value. Sometimes, in β-lactamase-overproducing strains, very poor activities against some substrates can nonetheless lead to amazingly increased MIC values for these drugs. So, we examined the protein expression of three CTX-Ms. CTX-M-261 was significantly increased compared with CTX-M-1/-55 in the same bacterial background environment, which was not consistent with the result of He D et al. (Fig. 4). The hydrolytic activities of the three CTX-M were undetectable against imipenem and meropenem as inactivators.

Table 3 Kinetic parameters of CTX-M-261, CTX-M-1, and CTX-M-55
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Fig. 4
figure 4

The protein expression levels of CTX-M-1/55/261 in E. coli BL21 and E. coli TOP10 were compared

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Discussion

Salmonella enterica serovar Typhimurium, especially monophasic S. enterica serovar Typhimurium (S. 1,4,[5], 12: i: -) is one of the most prevalent Nontyphoidal Salmonella (NTS) bacterial causes of gastrointestinal infections worldwide in the last two decades [23]. There have been lots of reports describing the multidrug resistance of Salmonella Typhimurium, including β-lactams, aminoglycosides, and colistin [24]. But there have been no reports about S. 1,4,[5], 12: i:-resistant to both carbapenem and ceftazidime-avibactam. We report for the first time a S. 1,4,[5], 12:i-strain carrying blaNDM and blaCTX-M genes and resistant to carbapenem and ceftazidime-avibactam. Although the blaNDM and blaCTX-M genes were identified in S. Typhimurium from pork [7, 9] and a clinical patient [8] in China, this is the first case of the occurrence of blaNDM-5-positive and blaCTX-M-261-positive, coding a novel CTX-M enzyme variant that differed from CTX-M-55 by a single amino acid substitution (Val251Met) due to one missense point mutation at position 751 (G → A), in S. 1,4,[5], 12: i: -. Notably, the genetic context of blaNDM in this isolate ST3606 (IS26-IS15-ISAba125-blaNDM-5-ISVsa3-sul1-qacE-aadA2-dfrA12-IntI1) was different from that of pNDM5-SSH006 in S. Typhimurium SSH006 (ISSWi1-IS3000-ΔISAba125-IS5-blaNDM-5-ble-trpF-tat-IS26-ΔumuD). Moreover, on the downstream of blaNDM-5 on pST3606-2, we found a IntI1 Integrase gene considered as a mobilizable platform that promotes ARG transfer and transformation in the environment and reflect the impact of human activities [25]. Surprisingly, the E. coli C600 transconjugant (pST3606-2) was not sensitive to cefiderocol with inhibition zones of 14 mm at 30 μg/disk according to the antimicrobial disk susceptibility tests (Zone Diameter Breakpoints: S, ≥ 16 mm; I, 9–15 mm; R, ≤ 8 mm; Figure S1). Cefiderocol, a novel siderophore-substituted cephalosporin with antibacterial activity against a broad spectrum of multidrug-resistant Gram-negative bacteria even including those that produce either KPC enzymes or NDM family, was approved by the FDA on November 2019 [26]. Hence, the analyzed genome content draws attention to the problem of multidrug-resistant S. 1,4,[5], 12: i:-isolated in clinic and a potential threat to human health.

The assembled genome sequences showed that ST3606 possesses a 4.96-Mb chromosome and two plasmids carrying ARGs: a 109 070-bp IncI1-Iα plasmid NDM-5 (designated pST3606-1) encoding acquired resistance genes, such as blaNDM-5, sul1, qacE, aadA2, and dfrA12, and a 70,455-bp IncFII plasmid (designated pST3606-2) encoding genes that confer resistance to β-lactams (blaCTX-M-261 and blaTEM-1B), aminoglycosides (aac(3)-lld) (Figs. 1 and 2). The major plasmid types carrying blaNDM-5 from reference NCBI database included IncX3 (29.68%), IncFII (15.41%), IncFIB (12.79%), and IncC (9.59%) [27]. The blaNDM gene previously reported to be carried by Salmonella appears on the IncX3 (blaNDM-5), IncA/C (blaNDM-1), IncFII (blaNDM-5), IncFIB (blaNDM-5), and IncI1 (blaNDM-13) plasmids, and chromosome (blaNDM-9 and blaNDM-1) [28]; particularly, the IncX3 and IncA/C plasmids are the most prevalent. However, IncI1-Iα plasmid carrying blaNDM-5 has not appeared in Salmonella, only a Salmonella Rissen ST469 harboring IncI1 plasmid carrying blaNDM-13 [29].

The wide spread of CTX-M variants among Salmonella isolates represents a large threat to the public health globally [30]. To date, more than 260 CTX-M variants have been named and deposited in the GenBank database. In this study, one novel blaCTX-M-261 variant, that belong to blaCTX-M-1like group according to Ambler classification method [31], was carried by S. 1,4,[5], 12: i:-isolated from the patient. Compared to CTX-M-55, amino acid substitution (Val251Met) conferred CTX-M-261 enzyme higher affinity (166.7 μM vs 736 μM) with ceftazidime but not higher hydrolytic activity (0.06 μM−1 s−1 vs 0.04 μM−1 s−1) in enzyme kinetics experiment. However, MICs in the E. coli TOP10 clones producing CTX-M-261 were higher due to the higher expression. In addition, CTX-M-261 may be an evolution leading to development of cefiderocol susceptibility decrease [32]. We speculate the presence of cross-resistance of CTX-M-261 between cefatzidime and cefiderocol. As such, this study increases our understanding that blaCTX-M variants are undergoing continuous evolution and thus need to be closely monitored.

WGS revealed the new variant blaCTX-M-261 was located on a conjugational IncFII-type plasmid. IncFII plasmids have been found to be associated with various resistance genes including ESBLs, and carbapenemase encoding genes in Salmonella [33, 34]. Complete conjugative transfer region was identified in the plasmid, which is consistent with the finding that the blaCTX-M-261 harboring IncFII-type plasmid can be transferred by conjugation [35]. It is noteworthy to mention, as shown in Table 1, that most of the antibiotic susceptibility profiles of ST3606 were consistent with E. coli C600 transconjugant, except for imipenem, meropenem, and ceftazidime-avibactam, which indicated that not blaCTX-M-261 but blaNDM-5 plays a dominant role in yielding to resistance of carbapenem and ceftazidime-avibactam. However, we found MIC for ceftazidime-avibactam of E. coli TOP10 transformant carrying blaCTX-M-261 (0.5 mg/L) is consistent with E. coli C600 transconjugant (0.5 mg/L) and higher than those of E. coli TOP10 transformant carrying blaCTX-M-1/-55 (0.125 mg/L), which speculated blaCTX-M-261 may be an evolution leading to development of ceftazidime susceptibility decrease [36].

To the best of our knowledge, this is the first report describing blaNDM-5 and a novel blaCTX-M variant in S. 1,4,[5], 12: i:-isolate with susceptibility decrease of ceftazidime. On the one hand, this work extended our understanding of enzymatic function and demonstrated the ongoing evolution of CTX-M enzymes. While focusing on the evolution of NDM carbapenemase, a close surveillance of CTX-M-producing pathogens should be enacted for continued monitoring of the spread of CTX-M variants [37]. On the other hand, the comparison of pST3606-1 showed that E. coli, Klebsiella pneumoniae, and Citrobacter sedlakii share a complete conserved plasmid backbone (IS26-IS15-ISAba125-blaNDM-5-ISVsa3-sul1-qacE-aadA2-dfrA12-IntI1), which shows the prevalence of the plasmid with a strong transmissibility among different species widely [38]. Especially, it has been confirmed that persistent Salmonella isolates could promote the spread of antibiotic resistance plasmids in the gut [39]. Hence, identifying the mechanism of the spread of carbapenem-resistant Salmonella in the environment has become a substantial global health concern.

In this study, we investigated the genetic characteristics of Salmonella enterica Serovar 1,4,[5], 12: i:-isolate ST3606 carrying blaCTX-M-261 and blaNDM-5, and characterized steady-state kinetics of CTX-M-261. Notably, this is the first report finding the S.1,4,[5], 12: i:- carrying both NDM and a novel CTX-M (CTX-M-261). S.1,4,[5], 12: i:-is the the predominant serovar in both humans and animals in China. CTX-M-261 may be an evolution leading to development of ceftazidime susceptibility decrease. The IS element upstream and IntI1 element downstream of blaNDM-5, and the IS26 element upstream and downstream of blaCTX-M-261 will contribute to horizontal gene transfer between different bacteria in environment. Further surveillance and increased measures should be adapted to prevent the transmission of blaNDM-5-carrying S.1,4,[5], 12: i:-strains and evolution of blaCTX-M in clinic.