Identification of a Novel blaNDM Variant, blaNDM-33, in an Escherichia coli Isolate from Hospital Wastewater in China

ABSTRACT Since the discovery of NDM-1 and the worldwide reporting of different variants have raised alarms concerning global health, the problem of carbapenem-resistant Enterobacterales (CRE) has become increasingly serious. Therefore, research on the hydrolytic activity and molecular structure of NDM variants is beneficial to the development of antibacterial drugs. NDM has been evolving into variants that possess different hydrolysis activities toward β-lactam antibiotics. Here, we characterized a novel blaNDM variant, named blaNDM-33, identified from a multidrug-resistant Escherichia coli strain from hospital sewage. NDM-33 differed from NDM-5 with a single-amino-acid substitution (A72T). blaNDM-5 was located in the Tn125-related blaNDM-33 region from an IncX3-type plasmid, pHD6415-NDM, that can be transferred horizontally. The genetic construct of blaNDM-33 showed higher MICs of carbapenems than a blaNDM-5 construct. Enzyme kinetics showed that NDM-33 had higher enzymatic activity for meropenem and cefazolin than NDM-5. The emergence of this novel NDM variant could pose a threat to public health because of its transferability and enhanced carbapenem activity. IMPORTANCE Our study described a novel NDM-33 variant from an E. coli strain isolated from hospital sewage, where it was associated with human disease and antibiotic exposure. Importantly, hospital sewage was increasingly considered to be related to CRE hosts. Pathogens were transmitted from reservoirs through direct and indirect contact, ingestion, and inhalation of contaminated water or aerosols. In addition, under the selective pressure of antibiotics, NDM variants will become the main strain in the hospital water system and evolve into high virulence and high resistance. The monitoring of NDM mutants is of great significance for preventing and controlling the evolution of superbugs.

IMPORTANCE Our study described a novel NDM-33 variant from an E. coli strain isolated from hospital sewage, where it was associated with human disease and antibiotic exposure. Importantly, hospital sewage was increasingly considered to be related to CRE hosts. Pathogens were transmitted from reservoirs through direct and indirect contact, ingestion, and inhalation of contaminated water or aerosols. In addition, under the selective pressure of antibiotics, NDM variants will become the main strain in the hospital water system and evolve into high virulence and high resistance. The monitoring of NDM mutants is of great significance for preventing and controlling the evolution of superbugs.
KEYWORDS CRE, E. coli, bla NDM-33 , hospital sewage T he prevalence of carbapenem-resistant Enterobacterales (CRE) has increased since the early 2000s, representing a tremendous public health threat (1). NDM-1 was first detected in a Klebsiella pneumoniae isolate from a Swedish patient with urinary tract infection who traveled to New Delhi in 2008 (2). Since then, 32 NDM variants have been described or the sequences have been deposited in the GenBank database (https://www .ncbi.nlm.nih.gov/). Among them, the NDM-5 variant was first found in a multidrug-resistant Escherichia coli ST648 isolate recovered from the perineum and throat of a patient in the United Kingdom, and it showed enhanced hydrolytic activity compared with NDM-1 (3). In this study, we described a novel bla NDM-33 variant, identified from a multidrug-resistant E. coli strain, HD6415, from a hospital sewage sample.
The E. coli strain HD6415 was isolated from the sewage of the second affiliated hospital of Soochow University (Suzhou, China) in July 2019. We used LB plates containing 0.5 mg/liter meropenem for primary screening of sewage separated from each department in the hospital to study the distribution of CRE and further follow-up research. Bacterial antimicrobial susceptibility testing was performed using the broth microdilution method, and the results were interpreted according to the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines (4). The EUCAST (http://www.eucast .org/) breakpoints were used for colistin and tigecycline. The testing was performed in triplicates in two different days, and Escherichia coli ATCC 25922 was used as the quality control (QC) strain.
The bla NDM-33 variant showed a single-nucleotide difference (G214A) compared with bla NDM-5 , resulting in an amino acid substitution at codon 72 (A72T). The A72T amino acid substitution in NDM-33 was in the first active-site ring between the b2 and b3 chains, which shifted away from the Zn center to accommodate substrates with different molecular structures (7). Previous studies showed that base substitutions in this region did not affect the overall folding of the protein, but different thermal stabilities have been observed in NDM variants. NDM variants with double amino acid substitutions, e.g., NDM-8 (D130G, M154L), NDM-5 (V88L, M154L), and NDM-7 (D130N, M154L), appeared to be more stable to thermal denaturation than the singly substituted NDM-6 (A233V), NDM-3 (D95N), and NDM-4 (M154L) (8).
To investigate the transferability of pHD6415-NDM, a conjugation experiment was carried out using the rifampin-resistant E. coli strain EC600 as the recipient. The transconjugant was selected on an LB agar plate containing 4 mg/liter meropenem and 200 mg/liter rifampin, and then the presence of bla NDM-33 in the transconjugant was confirmed by PCR and Sanger sequencing. Results showed bla NDM-33 can be successfully transferred via conjugation, with an efficiency of ;1.2 Â 10 22 (transconjugant/recipient). Susceptibility testing showed that the transconjugant had meropenem, imipenem, and ertapenem MICs of 64, 256, and 64 mg/liter, respectively, in consistent with the carbapenem resistance phenotype observed in the parental strains (Table 1).
To further evaluate whether the A72T substitution in NDM-33 confers different levels of resistance to b-lactam antibiotics, we cloned the full-length bla NDM-33 and bla NDM-5 along with their identical natural promoters into the pET28a vector, followed by transformation into E. coli DH5a cells. Meanwhile, the pET28a plasmid was transformed as a control (12). Susceptibility testing results showed that the carbapenem (ertapenem, imipenem, and meropenem) MICs in the bla NDM-33 construct (DH5a/pET28a-bla NDM-33 ) were 2-fold higher than those in the bla NDM-5 construct (DH5a/pET28a-bla NDM-5 ) ( Table 1).
Taken together, our study described a novel NDM-33 variant from an E. coli strain isolated from hospital sewage. Hospital wastewater serves as an important reservoir for the emergence and transmission of antimicrobial resistance genes and variants, although it represents an environmental sample in which a high density of antibiotic residues and antibiotic-resistant bacteria are present. To some extent, the phenotypic differences between NDM-5 and NDM-33 are not remarkable. However, the continuous evolution of NDM enzymes could foster the emergence of novel variants that possess different hydrolysis activities toward b-lactam antibiotics. Our study enriched our understanding of enzymatic function and evidenced the ongoing evolution of NDM enzymes. A close surveillance of NDM-producing bacteria, both environmentally and clinically, should be enacted to monitor and control the spread of NDM variants.
Data availability. The draft whole-genome sequence of strain HD6415 was submitted to GenBank under accession number JAGTHW000000000, and the complete sequence of plasmid pHD6415-NDM was submitted to GenBank under accession number MZ004933.