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

The concurrent presence of multiple New Delhi metallo-β-lactamase (blaNDM) variants within an isolate often goes undetected without the use of next-generation sequencing. This study detects and characterizes dual blaNDM-variants in Escherichia coli through Sanger and whole-genome sequencing. Additionally, a rapid identification method utilizing restriction digestion was designed for detecting variants carrying M154L mutation. Antibiotic susceptibility, minimal inhibitory concentration for meropenem and ertapenem, PCR and Sanger sequencing of blaNDMalong with genome sequencing using Ilumina and Nanopore technology were conducted. Transmissibility and replicon types of blaNDM-harbouring plasmids were evaluated. Restriction digestion using restriction enzyme, BtsCI was developed to distinguish between blaNDM-1and variants possessing M154L mutations. Two isolates belonging to phylogroups A; ST167 and B1; ST101 and resistant to meropenem and ertapenem (≥16mg/L) were recovered from the blood of a neonate and the rectal swab of a pregnant woman respectively. blaNDMwas detected by PCR, and Sanger sequences of blaNDMshowed two peaks at 262 (G & T) and 460 (A & C) nucleotide positions indicative of more than one blaNDMvariant. Hybrid assembly confirmed co-existence of blaNDM-1and blaNDM-5in each isolate. blaNDM-1was located on IncY (ST167) and IncHI1A/HI1B (ST101), while blaNDM-5was on a IncFIA/FII (ST167) and IncC (ST101) plasmids. Digestion with BtsC1 could discriminate blaNDM-1and blaNDM-5. Co-existence of multiple blaNDMS, blaNDM-1andblaNDM-5in epidemic clones of E. coli is concerning. Restriction digestion method and Sanger sequencing can facilitate quick identification of dual blaNDMvariants in single isolate.