{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T15:36:30Z","timestamp":1774625790970,"version":"3.50.1"},"reference-count":28,"publisher":"American Society for Microbiology","issue":"4","license":[{"start":{"date-parts":[[2025,4,1]],"date-time":"2025-04-01T00:00:00Z","timestamp":1743465600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,4,1]],"date-time":"2025-04-01T00:00:00Z","timestamp":1743465600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100001411","name":"Indian Council of Medical Research","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100001411","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2025,4]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n The concurrent presence of multiple New Delhi metallo-\u03b2-lactamase (\n bla<\/jats:italic>\n NDM<\/jats:sub>\n ) variants within an isolate often goes undetected without next-generation sequencing. This study detects and characterizes dual\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants in\n Escherichia coli<\/jats:italic>\n through Sanger and whole-genome sequencing. Additionally, a rapid identification method utilizing restriction digestion was designed for detecting\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants carrying M154L mutation. Antibiotic susceptibility, minimal inhibitory concentration for meropenem and ertapenem, PCR, and Sanger sequencing of\n bla<\/jats:italic>\n NDM<\/jats:sub>\n along with genome sequencing using Illumina and Nanopore technology were conducted. Transmissibility and replicon types of\n bla<\/jats:italic>\n NDM<\/jats:sub>\n -harboring plasmids were evaluated. Restriction digestion using restriction enzyme, BtsCI was developed to distinguish between\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n and\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants possessing M154L mutation, such as\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n ,\n bla<\/jats:italic>\n NDM-7<\/jats:sub>\n etc. Two isolates belonging to phylogroups A; ST167 and B1; ST101 and resistant to meropenem and ertapenem (\u226516 mg\/L) were recovered from the blood of a neonate and the rectal swab of a pregnant woman, respectively.\n bla<\/jats:italic>\n NDM<\/jats:sub>\n was detected by PCR, and Sanger sequences of\n bla<\/jats:italic>\n NDM<\/jats:sub>\n showed two peaks at 262 (G and T) and 460 (A and C) nucleotide positions indicative of more than one\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variant. Hybrid assembly confirmed co-existence of\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n and\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n in each isolate.\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n was located on IncY (ST167) and IncHI1A\/HI1B (ST101), while\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n was on IncFIA\/FII (ST167) and IncC (ST101) plasmids in the two isolates. Digestion with BtsC1 could discriminate between\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n and\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n . The co-existence of multiple\n bla<\/jats:italic>\n NDMs<\/jats:sub>\n ,\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n , and\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n in epidemic clones of\n E. coli<\/jats:italic>\n is concerning. Restriction digestion method and Sanger sequencing can facilitate quick identification of dual\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants in a single isolate.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n The global dissemination of antimicrobial resistance genes is a serious concern. One such gene,\n bla<\/jats:italic>\n NDM<\/jats:sub>\n , has spread globally via plasmids.\n bla<\/jats:italic>\n NDM<\/jats:sub>\n confers resistance against all \u03b2-lactam antibiotics, except monobactams. Most of the earlier literature reported the presence of single\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variant. However, this study reports the prevalence of dual\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants (\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n and\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n ) located on two separate plasmids identified in two distinct\n Escherichia coli<\/jats:italic>\n epidemic clones ST167 and ST101 isolated from a septicemic neonate and a pregnant mother, respectively.\n bla<\/jats:italic>\n NDM-5<\/jats:sub>\n differs from\n bla<\/jats:italic>\n NDM-1<\/jats:sub>\n due to the presence of two point mutations (i.e., V88L and M154L). This study detected dual\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants through Sanger sequences and further validated them through hybrid-genome assembly. Detection of multiple\n bla<\/jats:italic>\n NDM<\/jats:sub>\n 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\n bla<\/jats:italic>\n NDM<\/jats:sub>\n variants containing M154L mutation.\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/spectrum.00987-24","type":"journal-article","created":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T15:02:07Z","timestamp":1740495727000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Co-existence of plasmid-mediated\n bla<\/i>\n NDM-1<\/sub>\n and\n bla<\/i>\n NDM-5<\/sub>\n in\n Escherichia coli<\/i>\n sequence type 167 and ST101 and their discrimination through restriction digestion"],"prefix":"10.1128","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5613-8963","authenticated-orcid":false,"given":"Amrita","family":"Bhattacharjee","sequence":"first","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections 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India"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6897-7390","authenticated-orcid":true,"given":"Shanta","family":"Dutta","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases)","place":["Kolkata, India"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7811-7140","authenticated-orcid":true,"given":"Sulagna","family":"Basu","sequence":"additional","affiliation":[{"name":"Division of Bacteriology, ICMR-National Institute for Research in Bacterial Infections (Formerly ICMR-National Institute of Cholera and Enteric Diseases)","place":["Kolkata, 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