Co-existence of plasmid-mediated blaNDM-1 and blaNDM-5 in Escherichia coli sequence type 167 and ST101 and their discrimination through restriction digestion.

The concurrent presence of multiple New Delhi metallo-β-lactamase (bla_NDM) variants within an isolate often goes undetected without next-generation sequencing. This study detects and characterizes dual bla_NDM variants in Escherichia coli through Sanger and whole-genome sequencing. Additionally, a rapid identification method utilizing restriction digestion was designed for detecting bla_NDM variants carrying M154L mutation. Antibiotic susceptibility, minimal inhibitory concentration for meropenem and ertapenem, PCR, and Sanger sequencing of bla_NDM along with genome sequencing using Illumina and Nanopore technology were conducted. Transmissibility and replicon types of bla_NDM-harboring plasmids were evaluated. Restriction digestion using restriction enzyme, BtsCI was developed to distinguish between bla_NDM-1 and bla_NDM variants possessing M154L mutation, such as bla_NDM-5, bla_NDM-7 etc. Two isolates belonging to phylogroups A; ST167 and B1; ST101 and resistant to meropenem and ertapenem (≥16 mg/L) were recovered from the blood of a neonate and the rectal swab of a pregnant woman, respectively. bla_NDM was detected by PCR, and Sanger sequences of bla_NDM showed two peaks at 262 (G and T) and 460 (A and C) nucleotide positions indicative of more than one bla_NDM variant. Hybrid assembly confirmed co-existence of bla_NDM-1 and bla_NDM-5 in each isolate. bla_NDM-1 was located on IncY (ST167) and IncHI1A/HI1B (ST101), while bla_NDM-5 was on IncFIA/FII (ST167) and IncC (ST101) plasmids in the two isolates. Digestion with BtsC1 could discriminate between bla_NDM-1 and bla_NDM-5. The co-existence of multiple bla_NDMs, bla_NDM-1, and bla_NDM-5 in epidemic clones of E. coli is concerning. Restriction digestion method and Sanger sequencing can facilitate quick identification of dual bla_NDM variants in a single isolate.IMPORTANCEThe global dissemination of antimicrobial resistance genes is a serious concern. One such gene, bla_NDM, has spread globally via plasmids. bla_NDM confers resistance against all β-lactam antibiotics, except monobactams. Most of the earlier literature reported the presence of single bla_NDM variant. However, this study reports the prevalence of dual bla_NDM variants (bla_NDM-1 and bla_NDM-5) located on two separate plasmids identified in two distinct Escherichia coli epidemic clones ST167 and ST101 isolated from a septicemic neonate and a pregnant mother, respectively. bla_NDM-5 differs from bla_NDM-1 due to the presence of two point mutations (i.e., V88L and M154L). This study detected dual bla_NDM variants through Sanger sequences and further validated them through hybrid-genome assembly. Detection of multiple bla_NDM variants in a single isolate remains difficult until genome sequencing or southern blotting is carried out. Hence, a simple restriction digestion method was devised to rapidly screen dual bla_NDM variants containing M154L mutation.