Genetic and enzymatic characterization of two novel blaNDM-36, -37 variants in Escherichia coli strains

The widespread of different NDM variants in clinical Enterobacterales isolates poses a serious public health concern, which requires continuous monitoring. In this study, three E. coli strains carrying two novel blaNDM variants of blaNDM-36, -37 were identified from a patient with refractory urinary tract infection (UTI) in China. We conducted antimicrobial susceptibility testing (AST), enzyme kinetics analysis, conjugation experiment, whole-genome sequencing (WGS), and bioinformatics analysis to characterize the blaNDM-36, -37 enzymes and their carrying strains. The blaNDM-36, -37 harboring E. coli isolates belonged to ST227, O9:H10 serotype and exhibited intermediate or resistance to all β-lactams tested except aztreonam and aztreonam/avibactam. The genes of blaNDM-36, -37 were located on a conjugative IncHI2-type plasmid. NDM-37 differed from NDM-5 by a single amino acid substitution (His261Tyr). NDM-36 differed from NDM-37 by an additional missense mutation (Ala233Val). NDM-36 had increased hydrolytic activity toward ampicillin and cefotaxime relative to NDM-37 and NDM-5, while NDM-37 and NDM-36 had lower catalytic activity toward imipenem but higher activity against meropenem in comparison to NDM-5. This is the first report of co-occurrence of two novel blaNDM variants in E. coli isolated from the same patient. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of NDM enzymes. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-023-04576-y.


Introduction
The plasmid-encoded New Delhi metallo-β-lactamase (NDM) is one of the most common carbapenemases worldwide [1]. Its emergence heralds a new era of antibiotic resistance due to the ability to hydrolyze almost all known β-lactam antibiotics and the rapid worldwide dissemination [2]. In 2009, the first NDM variant (NDM-1) was reported in Klebsiella pneumoniae isolated from a Swedish patient of Indian origin who had a urinary tract infection [3]. Following the first report, 41 different NDM variants have been identified in numerous species of Enterobacteriaceae and common nonfermentative Gram-negative bacilli [4] (NDM-1 Wanshan Ma, Bo Zhu, and Wen Wang contributed equally to this work. Author order was determined by contributions to the study. to NDM-41; NDM-32 is assigned but without any information in NCBI).
The continuous evolution of NDM enzymes under the selection pressure could foster the emergence of new variants that possess different catalytic activities toward β-lactam agents [5]. For example, the NDM-5 variant showed enhanced hydrolytic activity compared with NDM-1 [6], and circular dichroism spectroscopy data revealed significant changes in the secondary structure of NDM variants [7]. Thus, a close surveillance of NDM-producing pathogens should be considered for continuous monitoring of the spread of NDM variants. Here, we describe the first detection of two novel NDM enzymes, designated NDM-36 and NDM-37, recovered from a patient with refractory urinary tract infection in China during 2020.

Bacterial strains
Three carbapenem-resistant E. coli strains (bla NDM positive) were isolated from the urine samples of a 62-year-old female patient with unilateral indwelling ureteral stents. The patient underwent cystectomy and chemotherapy for recurrence of ovarian and fallopian cancer three months ago. The first E. coli strain (JNQH497-NDM-37) was recovered in an outpatient clinic. Based on the antibiotic susceptibility testing results, empirical levofloxacin treatment (500 mg qd) was then started for urinary tract infection. After 3 weeks, the second strain (JNQH498-NDM-36) was isolated from the urine during hospital admission. The patient was then given meropenem (1000 mg intravenously [i.v.] q8h). The third E. coli strain (JNQH462-NDM-36) was identified from the urine 5 weeks after admission. Her conditions were improved after the removal of ureteral stents via cystoscopy, continuous meropenem treatment as well as implementation of nutritional support. The patient was discharged home on hospital day 16. Ethics committee approval of this study was obtained from the institutional review board of the First Affiliated Hospital of Xiamen University, and informed consent from the patient was also obtained.

Antimicrobial susceptibility testing (AST)
MICs for all the tested strains were determined by broth microdilution method using a bacterial inoculum of 5 × 10 5 CFU/ml according to CLSI performance standards. For ceftazidime-avibactam (CAZ-AVI) and aztreonamavibactam (ATM-AVI) MICs evaluation, AVI was tested at a fixed concentration of 4 mg/L, while CAZ and ATM were added at different concentrations ranged from 0.0312 to 64 mg/L and 0.0156 to 32 mg/L, respectively.

Cloning of bla NDM variants
The promoter and full length of the bla NDM genes were amplified with primers NDM-F-EcoRI (5′-CCG GAA TTC TTG AAA  CTG TCG CAC CTCAT-3′) and NDM-R-XbaI (5′-CTA GTC  TAG AAC GCC TCT GTC ACA TCGAA-3′) using PrimSTAR Max DNA Polymerase (Takara, China). After restriction enzyme digestion, the PCR products were ligated to the vector PET28a to generate PET28a-NDM-5, PET28a-NDM-36, PET28a-NDM-37 respectively. The correct constructs were confirmed by Sanger sequencing, followed by transformation into E. coli DH5α. Antimicrobial susceptibilities of these constructs were determined as described above. The empty pET28a plasmid was used as a control.

Conjugation experiment
Conjugation experiments were performed using E. coli J53AziR as recipients as previously described [10]. Briefly, overnight cultures of the donor strain (JNQH497, 498) and the recipient strains were mixed (1:1) and applied to 0.45-μm filter paper respectively, which were then placed on an LB agar plate, followed by overnight culture at 37 ℃. Transconjugants were selected on Mueller-Hinton agar containing sodium azide (100 mg/L)/meropenem (2 mg/L) for transconjugates. The selected transconjugants were confirmed by PCR targeting the bla NDM gene. Conjugation frequency was calculated by dividing the number of transconjugants by the number of recipient cells.

Pulsed-field gel electrophoresis (PFGE)
To further explore the relatedness of JNQH497, 498, 462 strains, we used PFGE to analyze the genetic relatedness. PFGE of Xbal-digested genomic DNA samples were performed with a CHEF MAPPER XA apparatus (Bio-Rad, USA), as previously described [11].

Whole-genome sequencing (WGS)
The strains were subject to next generation sequencing using the Illumina HiSeq system (Illumina, San Diego, CA, USA). Genomic DNA was isolated using a WizardR Genomic DNA Purification Kit (Promega, Madison, WI, USA). Sequencing reads were de novo assembled using Spades 3.12.0 [12]. To resolve the complete plasmid sequence carrying bla NDM in JNQH497 and JNQH498, the Oxford Nanopore (MinION system) sequencing was conducted and assembled with Illumina sequences to achieve a high-quality genome assembly. The hybrid assembly was performed using Unicycler v0·5.0 [13]. The whole-genome sequences were annotated by Prokka [14] automatically followed by manual curation.
The PFGE showed JNQH497, 498, 462 had highly similar band patterns which indicated they were closely related (Fig. 5).

Transferability of bla NDM harboring plasmids and conjugation module analysis
Conjugation assays showed the bla NDM-36 and bla NDM-37 harboring IncHI2-type plasmids were successfully transferred into E. coli J53 from JNQH498 and JNQH497 strains. E. coli J53 transconjugants acquired resistance to levofloxacin and most β-lactam antibiotics except aztreonam/avibactam (Table 1), which indicated the resistant markers to fluroquinolones were co-transferred with bla NDM genes. The conjugation frequency was 10 −3 per recipient cell for JNQH497, whereas it was only 10 −8 for JNQH498. Conjugation module analysis revealed the conjugation genes are in two separate regions: transfer region 1 carries the origin of transfer site (oriT), type IV coupling protein gene (T4CP), and genes encoding the relaxase and some type IV secretion components. Region 2 encodes most type IV secretion proteins (Fig. 2). Blastn analysis revealed all the conjugation modules were also found in JNQH498, JNQH462 strains.

Expression of the NDM proteins and enzyme activity analysis
Susceptibility testing of pET28a constructs showed that expression of the bla NDM-36 and bla NDM-37 genes in E. coli DH5α conferred resistance to most of the tested β-lactams except aztreonam and ATM/AVI. Kinetic data showed that NDM-36 had higher affinity to cefotaxime than that of NDM-37, with the Km value reduced by 82.62 μM, whereas NDM-36 displayed slightly lower affinity than those of NDM-5, -37 for imipenem and meropenem. The kcat/Km ratio for ampicillin and cefotaxime of NDM-36 was higher than those of NDM-37, -5, but imipenem kcat/Km ratio was slighter higher than those of NDM-37, -5. In comparison to NDM-5, although NDM-36, -37 had lower kcat/Km ratio for imipenem, they had higher kcat/Km ratio for meropenem. These results suggested NDM-36 had higher hydrolytic activity toward ampicillin and cefotaxime relative to NDM-37, -5, and that NDM-37, -36 had lower catalytic activity against imipenem but higher activity against meropenem relative to NDM-5 (Table 2).

Phylogenetic analysis of NDM protein sequences
Phylogenetic analysis of the protein sequence of NDM variants is represented in Fig. 6. Evolutionary analysis of the amino acid sequences showed the amino acids were substituted at 30 polymorphic sites except for NDM-18, which has five amino acids tandem repeat (QRFGD) at positions 44 to 48 relative to NDM-1 [25]. The NDM variants had most hotspot mutation at amino acid positions 88, 154, and 130 (Fig. 6).

Discussion
The widespread of NDM variants among E. coli strains and other Enterobacteriaceae isolates represents a large threat to the public health globally [26]. To date, a total of 41 NDM variants have been named and described, of which 40 sequences have been deposited in the GenBank database. In this study, two novel bla NDM variants were carried by E. coli strains isolated from the same patient. Sequence analysis showed that NDM-37 differed from NDM-5 by a single amino acid substitution (His261Tyr) due to one missense point mutations at positions 781 (C → T). One additional missense point mutation at position 698 (C → T) in bla NDM-37 resulting in NDM-36. As such, this study provides a good explanation to increase our understanding that bla NDM variants are undergoing continuous evolution and thus need to be closely monitored. WGS revealed the new variants of bla NDM-36, -37 were located on a conjugational IncHI2-type plasmid. IncHI2 plasmids are larger than most of the other conjugative plasmids and have been found to be associated with various  [27][28][29]. Complete transfer operons were identified in the plasmids, which is consistent with the finding that the bla NDM-36 and bla NDM-37 harboring IncHI2-type plasmids can be transferred by conjugation. In addition, the identification of highly similar IncHI2 plasmids in different E. coli STs suggested this plasmid had horizontal inter-species transfer between different E. coli clones, probably due to various antibiotic selection pressures as the plasmid contained multiple resistance genes.
It is noteworthy to mention, as shown in Table 1, most of the antibiotic susceptibility profiles of NDM-carrying clinical isolates were consistent with those of the corresponding E. coli DH5α transformants and J53 transconjugants, except for imipenem and meropenem. Compared with JNQH497, a premature stop codon was introduced into the coding region of ompD (c.238G > T p.Glu80*) in JNQH498 and JNQH462 strains. OmpD had been reported to be the main mechanism that mediated reduced susceptibility to imipenem in Enterobacter spp [30]. As such, it is speculated the premature stop codon likely accounts for the inconsistency of imipenem and meropenem susceptibility between pET28a-NDM-36 and pET28a-NDM-37 as compared to the corresponding source isolates. In addition, considering these isolates having MICs for meropenem ≤ 8 mg/L, the patient was given high-dose extended-infusion meropenem for urinary tract infection [31]. However, due to the excellent in vitro activity of ATM/AVI and the carriage of ESBL encoding genes, utility of aztreonam in combination with ceftazidime-avibactam might be one promising treatment strategy [32].
In summary, this study identified two novel NDM-type β-lactamases, NDM-36 and NDM-37, from E. coli strains isolated from a patient with refractory urinary tract infection. To the best of our knowledge, this is the first report describing two novel NDM variants detected from the same patient. This work extended our understanding of enzymatic function and demonstrated the ongoing evolution of NDM enzymes. Emergence of new NDM variants could be driven by de novo resistance evolution. A close surveillance of NDM-producing pathogens should be enacted for continued monitoring of the spread of NDM variants.

Data availability
The draft genome sequences of JNQH497, JNQH498, JNQH462 were deposited into NCBI Genome database under BioProject PRJNA702614. The complete genome sequences of JNQH497 and JNQH498 have been submitted to GenBank under the accession numbers of CP091925 ~ CP091927 and CP104384 ~ CP104385 respectively.
Code availability Not applicable.

Declarations
Ethical approval Ethics committee approval of this study was granted by the institutional review board of the First Affiliated Hospital of Xiamen University, and informed consent from the patient was obtained.

Consent to participate
Authors had sought consent from the individual to publish the data in a journal article.

Consent for publication
All authors have contributed to the creation of this manuscript for important intellectual content and read and approved the final manuscript to be published.

Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.