Abstract
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.