{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T08:24:58Z","timestamp":1774945498414,"version":"3.50.1"},"reference-count":29,"publisher":"Oxford University Press (OUP)","issue":"4","license":[{"start":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T00:00:00Z","timestamp":1774915200000},"content-version":"vor","delay-in-days":27,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005869","name":"University of Fribourg","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100005869","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,3,4]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background and objectives<\/jats:title>\n                    <jats:p>Metallo-\u03b2-lactamase (MBL)\u2013producing Enterobacterales represent a major public health threat because they confer resistance to all \u03b2-lactams except monobactams. The most prevalent MBLs include VIM, IMP, and NDM types, which render all clinically approved \u03b2-lactamase inhibitors ineffective. This study evaluates whether cyanide-containing molecules such as the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP) or the cytochrome C oxidase inhibitor potassium cyanide (KCN) can restore carbapenem susceptibility in MBL\u2013producing strains by disrupting zinc-dependent enzymatic activity.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>A series of isogenic Escherichia coli TOP10 and MG1655 strains carrying the shuttle vector pUCP24 harbouring various MBLs and clinical strains were included in this study. The strains were subjected to antimicrobial susceptibility testing by broth microdilution, checkerboard assays, determination of the 50% inhibitory concentration (IC50) of carbapenemase activity, kinetic assays and time-kill curve assays.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>CCCP, FCCP and KCN restored carbapenem (imipenem) susceptibility in E. coli strains producing VIM-1\u2013like enzymes. Synergy was observed in checkerboard assays, and reduction of bacterial growth was confirmed by time-kill experiments. The addition of Zn2+ reversed this effect, supporting a Zn2+-chelating or Zn2+-displacing mechanism. Non\u2013VIM-1-like MBLs, including VIM-2, IMP-1 and NDM-1, were not affected by the addition of cyanide-containing compounds, indicating strong selectivity of these inhibitory effects. IC50 measurements confirmed the selective activity of CCCP, FCCP and KCN against VIM-1\u2013like MBLs.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>CCCP, FCCP and KCN exhibited inhibitory activity against VIM-1\u2013like MBLs, restoring imipenem susceptibility in E. coli at very low concentrations (0.04\u201319.55\u2005\u00b5M). Thus, certain efflux pump inhibitors, as well as the gaseous biological mediator cyanide, have a novel action as inhibitors of MBL enzymatic activity, which, in turn, renders them potentially useful as adjunct antibacterial agents.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1093\/jac\/dkag122","type":"journal-article","created":{"date-parts":[[2026,3,20]],"date-time":"2026-03-20T12:42:23Z","timestamp":1774010543000},"source":"Crossref","is-referenced-by-count":0,"title":["Metallo-\u03b2-lactamase\u2013producing Enterobacterales: cyanide-containing efflux pump inhibitors as potential dual-activity inhibitors"],"prefix":"10.1093","volume":"81","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7374-0788","authenticated-orcid":false,"given":"Ot\u00e1vio","family":"Hallal Ferreira Raro","sequence":"first","affiliation":[{"name":"Faculty of Science and Medicine, Medical and Molecular Microbiology Unit\u2013Department of Oncology, Microbiology, Immunology (OMI), University of Fribourg , Fribourg CH-1700 ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3110-4235","authenticated-orcid":false,"given":"Csaba","family":"Szabo","sequence":"additional","affiliation":[{"name":"Faculty of Science and Medicine, Pharmacology Unit\u2013Department of Oncology, Microbiology, Immunology (OMI), University of Fribourg , Fribourg ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1343-1622","authenticated-orcid":false,"given":"Patrice","family":"Nordmann","sequence":"additional","affiliation":[{"name":"Faculty of Science and Medicine, Medical and Molecular Microbiology Unit\u2013Department of Oncology, Microbiology, Immunology (OMI), University of Fribourg , Fribourg CH-1700 ,","place":["Switzerland"]},{"name":"Faculty of Science and Medicine, Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), University of Fribourg , Fribourg ,","place":["Switzerland"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2026,3,31]]},"reference":[{"key":"2026033103105579700_dkag122-B1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciae403","article-title":"Infectious Diseases Society of America 2024. Guidance on the treatment of antimicrobial-resistant gram-negative infections","author":"Tamma","year":"2024","journal-title":"Clin Infect Dis"},{"key":"2026033103105579700_dkag122-B2","doi-asserted-by":"publisher","first-page":"321","DOI":"10.1098\/rstb.1980.0049","article-title":"The structure of \u03b2-lactamases","volume":"289","author":"Ambler","year":"1980","journal-title":"Philos Trans R Soc Lond B Biol Sci"},{"key":"2026033103105579700_dkag122-B3","doi-asserted-by":"publisher","first-page":"306","DOI":"10.1128\/CMR.18.2.306-325.2005","article-title":"Metallo-b-lactamases: the quiet before the storm?","volume":"18","author":"Walsh","year":"2005","journal-title":"Clin Microbiol Rev"},{"key":"2026033103105579700_dkag122-B4","doi-asserted-by":"publisher","first-page":"317","DOI":"10.2217\/fmb.11.13","article-title":"Epidemiology and genetics of VIM-type metallo-\u03b2-lactamases in gram-negative bacilli","volume":"6","author":"Zhao","year":"2011","journal-title":"Future Microbiol"},{"key":"2026033103105579700_dkag122-B5","author":"Sakoulas"},{"key":"2026033103105579700_dkag122-B6","doi-asserted-by":"publisher","first-page":"168","DOI":"10.3390\/pathogens14020168","article-title":"\u03b2-lactam\/\u03b2-lactamase inhibitor combination antibiotics under development","volume":"14","author":"Katsarou","year":"2025","journal-title":"Pathogens"},{"key":"2026033103105579700_dkag122-B7","doi-asserted-by":"publisher","first-page":"28047","DOI":"10.1038\/s41598-025-13554-7","article-title":"Efficacy of imipenem combined with dimercaptosuccinic acid in a murine sepsis model using Pseudomonas aeruginosa","volume":"15","author":"Herrera-Espejo","year":"2025","journal-title":"Sci Rep"},{"key":"2026033103105579700_dkag122-B8","doi-asserted-by":"publisher","first-page":"2019","DOI":"10.1093\/jac\/dkad200","article-title":"In vitro activity of cefiderocol against MBL-producing gram-negative bacteria collected in North America and Europe in five consecutive annual multinational SIDERO-WT surveillance studies (2014\u20132019)","volume":"78","author":"Takemura","year":"2023","journal-title":"J Antimicrob Chemother"},{"key":"2026033103105579700_dkag122-B9","doi-asserted-by":"publisher","first-page":"1223","DOI":"10.1093\/jac\/dkl493","article-title":"Antibiotic efflux pumps in gram-negative bacteria: the inhibitor response strategy","volume":"59","author":"Mahamoud","year":"2007","journal-title":"J Antimicrob Chemother"},{"key":"2026033103105579700_dkag122-B10","doi-asserted-by":"publisher","first-page":"179","DOI":"10.1007\/BF01868942","article-title":"The kinetic mechanism by which CCCP (carbonyl cyanide m-chlorophenylhydrazone) transports protons across membranes","volume":"82","author":"Kasianowicz","year":"1984","journal-title":"J Membr Biol"},{"key":"2026033103105579700_dkag122-B11","doi-asserted-by":"publisher","first-page":"381","DOI":"10.1016\/S0006-3495(83)84449-X","article-title":"The molecular mechanism of action of the proton ionophore FCCP (carbonylcyanide p-trifluoromethoxy-phenylhydrazone)","volume":"41","author":"Benz","year":"1983","journal-title":"Biophys J"},{"key":"2026033103105579700_dkag122-B12","doi-asserted-by":"publisher","first-page":"100","DOI":"10.1016\/j.bcp.2017.12.018","article-title":"Current mechanistic insights into the CCCP-induced cell survival response","volume":"148","author":"Kane","year":"2018","journal-title":"Biochem Pharmacol"},{"key":"2026033103105579700_dkag122-B13","doi-asserted-by":"publisher","first-page":"e0003922","DOI":"10.1128\/aac.00039-22","article-title":"Impact of acquired broad-spectrum \u03b2-lactamases on susceptibility to cefiderocol and newly developed \u03b2-lactam\/\u03b2-lactamase inhibitor combinations in Escherichia coli and Pseudomonas aeruginosa","volume":"66","author":"Poirel","year":"2022","journal-title":"Antimicrob Agents Chemother"},{"key":"2026033103105579700_dkag122-B14","doi-asserted-by":"publisher","first-page":"e0157023","DOI":"10.1128\/aac.01570-23","article-title":"Relative inhibitory activities of the broad-spectrum \u03b2-lactamase inhibitor xeruborbactam in comparison with taniborbactam against metallo-\u03b2-lactamases produced in Escherichia coli and Pseudomonas aeruginosa","volume":"68","author":"Le Terrier","year":"2024","journal-title":"Antimicrob Agents Chemother"},{"key":"2026033103105579700_dkag122-B15","author":"EUCAST"},{"key":"2026033103105579700_dkag122-B16","doi-asserted-by":"publisher","first-page":"e45","DOI":"10.1093\/nar\/gkm047","article-title":"A versatile ligation-independent cloning method suitable for high-throughput expression screening applications","volume":"35","author":"Berrow","year":"2007","journal-title":"Nucleic Acids Res"},{"key":"2026033103105579700_dkag122-B17","first-page":"1321","article-title":"Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent","volume":"6","author":"Hochuli","year":"1988","journal-title":"Bio\/Technology"},{"key":"2026033103105579700_dkag122-B18","doi-asserted-by":"publisher","first-page":"245","DOI":"10.1016\/S0076-6879(00)26058-8","article-title":"Purification of proteins using polyhistidine affinity tags","volume":"326","author":"Bornhorst","year":"2000","journal-title":"Methods Enzymol"},{"key":"2026033103105579700_dkag122-B19","doi-asserted-by":"publisher","first-page":"10634","DOI":"10.1021\/bi0342822","article-title":"Crystal structure of extended-spectrum \u03b2-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion","volume":"42","author":"Ibuka","year":"2003","journal-title":"Biochemistry"},{"key":"2026033103105579700_dkag122-B20","doi-asserted-by":"publisher","first-page":"684","DOI":"10.1016\/j.bbapap.2009.10.023","article-title":"The catalytic efficiency (kcat\/km) of the class A beta-lactamase toho-1 correlates with the thermal stability of its catalytic intermediate analog","volume":"1804","author":"Nitanai","year":"2010","journal-title":"Biochim Biophys Acta"},{"key":"2026033103105579700_dkag122-B21","doi-asserted-by":"publisher","first-page":"573","DOI":"10.1093\/jac\/dki477","article-title":"In vitro antibacterial activities of tigecycline in combination with other antimicrobial agents determined by chequerboard and time-kill kinetic analysis","volume":"57","author":"Petersen","year":"2006","journal-title":"J Antimicrob Chemother"},{"key":"2026033103105579700_dkag122-B22","doi-asserted-by":"publisher","first-page":"56","DOI":"10.3390\/molecules27010056","article-title":"Zinc-chelating compounds as inhibitors of human and bacterial zinc metalloproteases","volume":"27","author":"Rahman","year":"2022","journal-title":"Molecules"},{"key":"2026033103105579700_dkag122-B23","doi-asserted-by":"publisher","first-page":"2481","DOI":"10.1111\/febs.16135","article-title":"The two faces of cyanide: an environmental toxin and a potential novel mammalian gasotransmitter","volume":"289","author":"Zuhra","year":"2022","journal-title":"FEBS J"},{"key":"2026033103105579700_dkag122-B24","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1111\/j.1432-1033.1973.tb02735.x","article-title":"Cyanide binding to carbonic anhydrase. A 13 C-nuclear-magnetic-resonance study.","volume":"34","author":"Feeney","year":"1973","journal-title":"Eur J Biochem"},{"key":"2026033103105579700_dkag122-B25","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1111\/febs.14695","article-title":"Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-\u03b2-lactamases","volume":"286","author":"Salimraj","year":"2019","journal-title":"FEBS J"},{"key":"2026033103105579700_dkag122-B26","doi-asserted-by":"publisher","first-page":"486","DOI":"10.1002\/stem.590","article-title":"Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells","volume":"29","author":"Mandal","year":"2011","journal-title":"Stem Cells"},{"key":"2026033103105579700_dkag122-B27","volume-title":"StatPearls [Internet]","author":"Schaffer","year":"2025"},{"key":"2026033103105579700_dkag122-B28","doi-asserted-by":"publisher","first-page":"e2026245118","DOI":"10.1073\/pnas.2026245118","article-title":"Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics","volume":"118","author":"Randi","year":"2021","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2026033103105579700_dkag122-B29","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1038\/s42255-025-01225-w","article-title":"Regulation of mammalian cellular metabolism by endogenous cyanide production","volume":"7","author":"Zuhra","year":"2025","journal-title":"Nat Metab"}],"container-title":["Journal of Antimicrobial Chemotherapy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/jac\/article-pdf\/81\/4\/dkag122\/67680732\/dkag122.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jac\/article-pdf\/81\/4\/dkag122\/67680732\/dkag122.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T07:11:04Z","timestamp":1774941064000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jac\/article\/doi\/10.1093\/jac\/dkag122\/8565928"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,4]]},"references-count":29,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2026,3,4]]}},"URL":"https:\/\/doi.org\/10.1093\/jac\/dkag122","relation":{},"ISSN":["0305-7453","1460-2091"],"issn-type":[{"value":"0305-7453","type":"print"},{"value":"1460-2091","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,4]]},"published":{"date-parts":[[2026,3,4]]},"article-number":"dkag122"}}