Co-existence of plasmid-mediated
bla
NDM-1
and
bla
NDM-5
in
Escherichia coli
sequence type 167 and ST101 and their discrimination through restriction digestion
Authors: Bhattacharjee Amrita , Basak Priyanka , Mitra Shravani , Sarkar Jagannath , Dutta Shanta , Basu Sulagna 
Published: February 2025

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.

IMPORTANCE

The 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.