Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5

Purpose The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. Methods The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. Results Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. Conclusion Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most β-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-024-04765-3.

Here, we report sequence characteristics of carbapenem and ceftazidime-avibactam-resistant monophasic S. Typhimurium isolate, recovered from a Chinese hospital with the bla CTX-M variant (bla CTX-M-261 ) and bla NDM-5 gene located on two specific transmissible plasmids, and their mobile genetic elements (MGEs).MGEs are closely associated with the formation and spread of antibiotic resistance genes (ARGs), including insertion sequences (IS), transposons, integrons, plasmids, and genomic islands [12].Therefore, we investigated the whole genome sequence of S. 1,4, [5], 12: i:-ST3606, to determine the related antibiotic resistance genes and their genetic environments, especially the mobile genetic elements associated with the ARGs.In addition, we characterized the hydrolytic activity of the novel CTX-M enzyme (CTX-M-261), which differed from CTX-M-55 by an Val251Met substitution, which increased hydrolytic activities toward ceftazidime and cefepime at the expense of hydrolytic activity to cefotaxime.

Bacterial isolation
In September 12, 2021, at the Clinical Microbiology Laboratory of Zhuhai hospital affiliated with Jinan University (Zhuhai, Guangdong, China), one Salmonella enterica serovar 1,4, [5], 12: i:-strain was obtained from a stool specimen of a boy (2 years old).The outpatient was suffering from a diarrhea of 6 days (2-8 times daily) and a fever of 38.5-38.9℃.Before admission, antibiotic treatment (intravenous infusion of ceftazidime for 4 days and then ceftriaxone for 2 days (700 mg intravenously q12h)) was initiated, but the fever persisted.A carbapenem-resistant Salmonella isolate was discovered from his stool sample (designated ST3606).According to antibiotics susceptibility test result obtained by Vitek® 2 Automated Susceptibility System, the patient was then given trimethoprim-sulfamethoxazole (300 mg orally q12h).His conditions were improved after continuous antibiotic treatment and then he was discharged home after 15 days in hospital.Ethics committee approval of this study was obtained from the institutional review board of Zhuhai hospital affiliated with Jinan University, and informed consent from the patient was also obtained (code:【2022 】No.51).

Confirmation and antibiotics susceptibility testing
The species of this strain was identified using Vitek® MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry, bioMérieux, Marcy-l'Étoile, France) and confirmed by single-molecule real-time (SMRT) sequencing.Serotyping of this isolate revealed 1, 4, [5], 12: i:-(Diagnostic Serum Kit, Tianrun Bio-Pharmaceutical Co. Ltd, Ningbo) confirmed by multiplex PCR [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, cefotaximeclavulanic 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.

Cloning of bla CTX-M variants
The full lengths of the bla CTX-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 CaC1 2 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.

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., Beijing, 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 bla CTX-M gene and sequencing.

Genetic contexts associated with bla CTX-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 bla CTX-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 bla CTX-M-261 , a novel bla CTX-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.bla CTX-M-261 , bracketed by IS1 elements and IS4 elements, could encode extended-spectrum beta-lactamase (ESBL) conferring resistance to the extended-spectrum cephalosporins.The bla CTX-M gene of pST3606-2 was organized as "IS1-IS26-bla CTX-M-261 -WbuC-bla TEM-1 -IS26-IS4" (Fig. 3), which among plasmids in the NCBI nucleotide database, IncFII plasmid was positive for bla CTX-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 bla CTX-M-261 and bla TEM-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).

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 k cat and K m of  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 K m value of CTX-M-261 catalyzing ceftazidime could be determined, but its k cat /K m value was also too low (0.06 μM −1 s −1 ).However, regarding the MICs of ceftazidime for E. coli TOP10 carrying bla CTX-M , CTX-M-261 mediated the highest MIC (Table 1), which seem to be in agreement with the K m value.Sometimes, in β-lactamaseoverproducing 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.
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 bla CTX-M-261 variant, that belong to bla CTX-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 bla CTX-M variants are undergoing continuous evolution and thus need to be closely monitored.
WGS revealed the new variant bla CTX-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 bla CTX-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 bla CTX-M-261 but bla NDM-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 bla CTX-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 bla CTX-M-1/-55 (0.125 mg/L), which speculated bla CTX-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 bla NDM-5 and a novel bla CTX-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-bla NDM-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 bla CTX-M-261 and bla NDM-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 bla NDM-5 , and the IS26 element upstream and downstream of bla CTX-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 bla NDM-5 -carrying S.1,4, [5], 12: i:-strains and evolution of bla CTX-M in clinic.

Table 1
MICs for the clinical isolate ST3606, the corresponding E. coli C600 transconjugant carrying bla CTX-M-261 , and CTX-M-producing E.

Table 3
Kinetic parameters of CTX-M-261, CTX-M-1, and CTX-M-55 a Data are the averages of the results obtained from three independent experiments.CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; IPM, imipenem; MEM, meropenem b ND, not determined due to a low initial rate of hydrolysis The protein expression levels of CTX-M-1/55/261 in E. coli BL21 and E. coli TOP10 were compared 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