{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:01:10Z","timestamp":1785420070559,"version":"3.56.0"},"reference-count":63,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T00:00:00Z","timestamp":1780358400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T00:00:00Z","timestamp":1780358400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"name":"The project Research Grant of Toho University School of Medicine","award":["23-3"],"award-info":[{"award-number":["23-3"]}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP21K08497"],"award-info":[{"award-number":["JP21K08497"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,6,2]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Traditionally, the impact of the substrate specificity of \u03b2-lactamase produced by bacteria on \u03b2-lactam resistance (\u03b2-lactamase-mediated resistance) has been assessed by performing antimicrobial susceptibility testing (AST) on transformants carrying the cloned gene on arbitrarily chosen \u03b2-lactamase-free plasmid vectors. However, because the promoter strength and plasmid copy number of these vectors are not standardized, the resulting \u03b2-lactamase expression levels vary, further hampering cross-study comparisons. We constructed pMiniF-1 by inserting a\n                      <jats:italic toggle=\"yes\">tac<\/jats:italic>\n                      promoter, a ribosome-binding site, and transcriptional terminators into the mini F plasmid, which is maintained at one to two copies per cell. This design enables controlled \u03b2-lactamase expression for accurate evaluation of \u03b2-lactamase-mediated resistance via AST. This approach enables the determination and comparison of MICs with \u03b2-lactamase expression levels controlled to be as nearly identical as possible. We performed AST on\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      DH5\u03b1\u0394\n                      <jats:italic toggle=\"yes\">bla<\/jats:italic>\n                      <jats:sub>EC-like<\/jats:sub>\n                      transformants expressing 22 \u03b2-lactamase genes (Ambler classes A\u2013D), including carbapenemases that were cloned into pMiniF-1. The resulting antimicrobial susceptibility profiles and MIC differences did not contradict the known substrate specificities of the enzymes. Meropenem and imipenem MICs for the IMP-6 producer were fourfold higher and eightfold lower, respectively, than IMP-1. Aztreonam MICs were similar between transformants lacking \u03b2-lactamase genes and those with class B \u03b2-lactamases. Imipenem MIC for the OXA-48 producer was \u22658-fold higher than for OXA-1, whereas cefepime MICs were 16-fold lower. We demonstrate that pMiniF-1 enables the comparison of \u03b2-lactamase substrate specificity via AST. The pMiniF-1 plasmid (Catalog No. RDB21122) is available from the RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan) (\n                      <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"https:\/\/web.brc.riken.jp\/en\/\" xlink:type=\"simple\">https:\/\/web.brc.riken.jp\/en\/<\/jats:ext-link>\n                      ).\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\u03b2-lactamases play a central role in bacterial resistance to \u03b2-lactam antibiotics, and novel variants continue to emerge. However, the impact of individual \u03b2-lactamase variants on \u03b2-lactam resistance across diverse enzyme-substrate combinations remains insufficiently characterized. Traditional kinetic analyses require enzyme purification and multiple reaction assays, which hinder comprehensive experimental evaluation. Expression-based approaches have also been used to assess \u03b2-lactamase function, but conventional systems depend on unstable high-copy plasmids, raising concerns about reproducibility due to variable plasmid copy numbers. In this study, we developed pMiniF-1, a stable, low-copy plasmid that enables consistent gene expression without antibiotic selection, thereby maintaining stable copy numbers under drug-free conditions. Using 22 \u03b2-lactamase genes, we demonstrated that susceptibility testing with pMiniF-1 adequately reflects their known substrate specificity profiles. This system provides a reliable method for assessing \u03b2-lactamase substrate specificity and will facilitate comparative studies on the functional diversity and evolution of emerging \u03b2-lactamase variants.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.03360-25","type":"journal-article","created":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T13:00:07Z","timestamp":1776344407000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Development of a stable low-copy mini F plasmid derivative for evaluating \u03b2-lactamase substrate specificity through antimicrobial susceptibility testing"],"prefix":"10.1128","volume":"14","author":[{"given":"Haruka","family":"Nakagawa Kamura","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03t78wx29","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for the Planetary Health and Innovation Science (PHIS), Hiroshima University, The IDEC Institute","place":["Higashi-Hiroshima, Japan"]},{"name":"Department of Microbiology and Infectious Diseases, Toho University School of Medicine","place":["Tokyo, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9237-1217","authenticated-orcid":true,"given":"Kotaro","family":"Aoki","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Toho University School of Medicine","place":["Tokyo, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kohji","family":"Komori","sequence":"additional","affiliation":[{"name":"Division of Collaborative Regional Infection Control, Department of Community Well-being, Toho University School of Medicine","place":["Tokyo, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiro","family":"Tateda","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, Toho University School of Medicine","place":["Tokyo, Japan"]},{"name":"Division of Collaborative Regional Infection Control, Department of Community Well-being, Toho University School of Medicine","place":["Tokyo, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1943-4648","authenticated-orcid":true,"given":"Yoshikazu","family":"Ishii","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03t78wx29","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for the Planetary Health and Innovation Science (PHIS), Hiroshima University, The IDEC Institute","place":["Higashi-Hiroshima, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00079-17"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00047-19"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1128\/aac.00333-22"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.45.5.1343-1348.2001"},{"key":"e_1_3_4_6_2","unstructured":"National Center for Biotechnology Information. 2025. 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