{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:08:19Z","timestamp":1789391299890,"version":"build-2803163510"},"reference-count":45,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32-GM008336"],"award-info":[{"award-number":["T32-GM008336"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Appl Environ Microbiol"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Antibiotic-resistant bacteria are a global public health threat that is becoming increasingly difficult to address with conventional therapeutics. Consequently, there is much interest in studying alternative biologics that circumvent antibiotic resistance. The Brevinin-2 family of antimicrobial peptides (AMPs) is a group of naturally occurring molecules that have the potential for high activity and low toxicity. Herein, we investigated the potential of a consensus-sequence-driven approach to Brevinin-2 peptide synthesis and evaluated their action against a panel of multidrug-resistant (MDR) bacteria, including carbapenemase-resistant\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      . We used the positional frequency of amino acids to generate novel synthetic peptides representative of the Brevinin-2 family. Four templates\u2014G30, G33, S33, and G37\u2014were synthesized by standard fluorenylmethyloxycarbonyl (FMOC) chemistry, purified by reverse-phase fast protein liquid chromatography (RP-FPLC), and tested for antibacterial activity and hemolytic toxicity. The results demonstrated a broadly applicable chemical peptide synthesis pipeline for the Brevinin-2 family with a high degree of purity (&gt;90%). Two peptides\u2014S33 and G33\u2014exhibited activity consistent with potential selectivity toward gram-positive and -negative bacteria, respectively, while G37 displays broad-spectrum activity, with growth of a Class B carbapenemase-resistant\n                      <jats:italic toggle=\"yes\">E. coli<\/jats:italic>\n                      inhibited at 16 \u00b5M and a Class A carbapenemase-resistant\n                      <jats:italic toggle=\"yes\">K. pneumoniae<\/jats:italic>\n                      inhibited at 64 \u00b5M. G37 acts rapidly, slowing growth within 30 min and fully killing targeted bacteria within 150 min. Although moderate levels of hemolytic toxicity pose a challenge for future development, the consensus-sequence approach toward novel Brevinin-2 AMP discovery via alignment combined with\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      antimicrobial analysis shows promise to test and initially validate other peptides.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      The escalating threat of antimicrobial resistance (AMR) demands innovative therapeutic strategies beyond traditional antibiotics. This study demonstrates a systematic, consensus-sequence-driven approach to designing antimicrobial peptides (AMPs) from the naturally occurring Brevinin-2 family, offering a replicable framework for accelerated drug discovery. Our novel peptide G37 exhibits bactericidal activity against carbapenemase-resistant\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      within 150 min while maintaining low hemolytic activity at therapeutic concentrations. The potential preferential activity shown by peptides S33 and G33 for gram-positive versus gram-negative bacteria provides prospective valuable insights into structure-activity relationships that can guide further peptide optimization. By combining computational sequence analysis, standardized solid-phase peptide synthesis, and comprehensive\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      validation, this work establishes a streamlined pipeline for Brevinin-2 peptide development. This methodology addresses the urgent need for alternative antimicrobials while providing a scalable approach to combat multidrug-resistant (MDR) pathogens.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/aem.01553-26","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:01:16Z","timestamp":1789390876000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["<i>In vitro<\/i>\n                    evaluation of novel consensus-sequence-generated Brevinin-2 antimicrobial peptides"],"prefix":"10.1128","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9499-0436","authenticated-orcid":true,"given":"Colin M.","family":"McDowell","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dk0ce17","id-type":"ROR","asserted-by":"publisher"}],"name":"Paul G. Allen School for Global Health, College of Veterinary Medicine, Washington State University","place":["Pullman, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jessica","family":"Carder","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dk0ce17","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Washington State University","place":["Pullman, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Brozik","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dk0ce17","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Chemistry, Washington State University","place":["Pullman, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Horton","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dk0ce17","id-type":"ROR","asserted-by":"publisher"}],"name":"Paul G. Allen School for Global Health, College of Veterinary Medicine, Washington State University","place":["Pullman, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6791-055X","authenticated-orcid":true,"given":"Douglas R.","family":"Call","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05dk0ce17","id-type":"ROR","asserted-by":"publisher"}],"name":"Paul G. Allen School for Global Health, College of Veterinary Medicine, Washington State University","place":["Pullman, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"key":"e_1_3_3_2_2","unstructured":"Centers for Disease Control and Prevention. 2019. Antibiotic resistance threats in the United States. Atlanta GA U.S. Department of Health and Human Services"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/healthcare11131946"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(24)01867-1"},{"key":"e_1_3_3_5_2","volume-title":"Antibacterial agents in clinical and preclinical development: an overview and analysis","author":"World Health Organization","year":"2021","unstructured":"World Health Organization. 2021. Antibacterial agents in clinical and preclinical development: an overview and analysis. World Health Organization."},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1002\/phar.1868"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.3109\/07388551.2011.594423"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.drup.2023.100954"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40779-021-00343-2"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bsheal.2022.02.003"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4471-6458-6_10"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.regpep.2003.12.003"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv1278"},{"key":"e_1_3_3_14_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa991"},{"key":"e_1_3_3_15_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41597-019-0154-y"},{"key":"e_1_3_3_16_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts141"},{"key":"e_1_3_3_17_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv1051"},{"key":"e_1_3_3_18_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3714"},{"key":"e_1_3_3_19_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1816707116"},{"key":"e_1_3_3_20_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-020-65688-5"},{"key":"e_1_3_3_21_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics10080895"},{"key":"e_1_3_3_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.regpep.2008.04.002"},{"key":"e_1_3_3_23_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btm404"},{"key":"e_1_3_3_24_2","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btn392"},{"key":"e_1_3_3_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3213"},{"key":"e_1_3_3_26_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2010.5"},{"key":"e_1_3_3_27_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-9-40"},{"key":"e_1_3_3_28_2","series-title":"CLSI Supplement M100","volume-title":"Performance standards for antimicrobial susceptibility testing","author":"CLSI","year":"2018","unstructured":"CLSI. 2018. Performance standards for antimicrobial susceptibility testing. 28th ed. CLSI Supplement M100. Clinical and Laboratory Standards Institute, Wayne, PA.","edition":"28"},{"key":"e_1_3_3_29_2","unstructured":"UK Health Security Agency. 2017. NCTC antimicrobial resistance reference strains and antimicrobial susceptibility testing control strains. Culture Collections Porton Down Salisbury UK. Available from: https:\/\/www.culturecollections.org.uk\/media\/1267\/nctc-amr_ams-booklet.pdf"},{"key":"e_1_3_3_30_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.44.9.2382-2388.2000"},{"key":"e_1_3_3_31_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkn396"},{"key":"e_1_3_3_32_2","doi-asserted-by":"publisher","DOI":"10.1128\/jcm.13.4.754-759.1981"},{"key":"e_1_3_3_33_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms24032914"},{"key":"e_1_3_3_34_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics9020085"},{"key":"e_1_3_3_35_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.42.4.857"},{"key":"e_1_3_3_36_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.nbt.2019.02.004"},{"key":"e_1_3_3_37_2","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202300472"},{"key":"e_1_3_3_38_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2019.00128"},{"key":"e_1_3_3_39_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-81140-8"},{"key":"e_1_3_3_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jgar.2020.05.013"},{"key":"e_1_3_3_41_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0210508"},{"key":"e_1_3_3_42_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.02823-14"},{"key":"e_1_3_3_43_2","doi-asserted-by":"publisher","DOI":"10.1128\/cmr.00135-23"},{"key":"e_1_3_3_44_2","doi-asserted-by":"publisher","DOI":"10.3390\/jof7020079"},{"key":"e_1_3_3_45_2","first-page":"107","volume-title":"Current topics in microbiology and immunology","author":"Lewis K","year":"2008","unstructured":"Lewis K. 2008. Multidrug tolerance of biofilms and persister cells, p 107\u2013131. In Current topics in microbiology and immunology. Vol. 322. Springer. https:\/\/doi.org\/10.1007\/978-3-540-75418-3_6."},{"key":"e_1_3_3_46_2","doi-asserted-by":"publisher","DOI":"10.1007\/s12551-021-00784-y"}],"container-title":["Applied and Environmental Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/aem.01553-26","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/aem.01553-26","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:01:28Z","timestamp":1789390888000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/aem.01553-26"}},"subtitle":[],"editor":[{"given":"Gemma","family":"Reguera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,14]]},"references-count":45,"alternative-id":["10.1128\/aem.01553-26"],"URL":"https:\/\/doi.org\/10.1128\/aem.01553-26","relation":{},"ISSN":["0099-2240","1098-5336"],"issn-type":[{"value":"0099-2240","type":"print"},{"value":"1098-5336","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,14]]},"assertion":[{"value":"2026-08-06","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-26","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-14","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e01553-26"}}