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 the use of 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 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 Ilumina and Nanopore technology were conducted. Transmissibility and replicon types of bla NDM-harbouring plasmids were evaluated. Restriction digestion using restriction enzyme, BtsCI was developed to distinguish between bla NDM-1 and 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. bla NDM was detected by PCR, and Sanger sequences of bla NDM showed two peaks at 262 (G & T) and 460 (A & 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 a IncFIA/FII (ST167) and IncC (ST101) plasmids. Digestion with BtsC1 could discriminate bla NDM-1 and bla NDM-5. 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 single isolate. Importance The global dissemination of antimicrobial resistance genes is a serious concern. One such gene, bla NDM, has spread all across the globe via plasmids. bla NDM confers resistance against all β-lactam antibiotics except monobactams. Most of the earlier literature reported presence of single bla NDM variants. 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 E. coli epidemic clones ST167 and ST101; isolated from a septicaemic neonate and a pregnant mother respectively. bla NDM-5 differs from bla NDM-1 due to presence of two point mutations i.e., V88L and M154L. This study detected dual bla NDM-variants through Sanger sequences and further validated through hybrid-genome assembly. Detection of multiple bla NDM-variants in a single isolate remains difficult until genome sequencing or southern blotting are carried out. Hence, a simple restriction digestion method was devised for rapid screening of dual bla NDM-variants containing M154L mutation.