{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T08:05:25Z","timestamp":1791273925200,"version":"4.1.0"},"reference-count":31,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[1998,7,1]],"date-time":"1998-07-01T00:00:00Z","timestamp":899251200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Antimicrob Agents Chemother"],"published-print":{"date-parts":[[1998,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>Mechanism-based inactivators of \u03b2-lactamases are used to overcome the resistance of clinical pathogens to \u03b2-lactam antibiotics. This strategy can itself be overcome by mutations of the \u03b2-lactamase that compromise the effectiveness of their inactivation. We used PCR mutagenesis of the TEM-1 \u03b2-lactamase gene and sequenced the genes of 20 mutants that grew in the presence of ampicillin-clavulanate. Eleven different mutant genes from these strains contained from 1 to 10 mutations. Each had a replacement of one of the four residues, Met69, Ser130, Arg244, and Asn276, whose substitutions by themselves had been shown to result in inhibitor resistance. None of the mutant enzymes with multiple amino acid substitutions generated in this study conferred higher levels of resistance to ampicillin alone or ampicillin with \u03b2-lactamase inactivators (clavulanate, sulbactam, or tazobactam) than the levels of resistance conferred by the corresponding single-mutant enzymes. Of the four enzymes with just a single mutation (Ser130Gly, Arg244Cys, Arg244Ser, or Asn276Asp), the Asn276Asp \u03b2-lactamase conferred a wild-type level of ampicillin resistance and the highest levels of resistance to ampicillin in the presence of inhibitors. Site-directed random mutagenesis of the Ser130 codon yielded no other mutant with replacement of Ser130 besides Ser130Gly that produced ampicillin-clavulanate resistance. Thus, despite PCR mutagenesis we found no new mutant TEM \u03b2-lactamase that conferred a level of resistance to ampicillin plus inactivators greater than that produced by the single-mutation enzymes that have already been reported in clinical isolates. Although this is reassuring, one must caution that other combinations of multiple mutations might still produce unexpected resistance.<\/jats:p>","DOI":"10.1128\/aac.42.7.1542","type":"journal-article","created":{"date-parts":[[2018,10,9]],"date-time":"2018-10-09T00:33:03Z","timestamp":1539045183000},"page":"1542-1548","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":55,"title":["Selection and Characterization of \u03b2-Lactam\u2013\u03b2-Lactamase Inactivator-Resistant Mutants following PCR Mutagenesis of the TEM-1 \u03b2-Lactamase Gene"],"prefix":"10.1128","volume":"42","author":[{"given":"Sergei B.","family":"Vakulenko","sequence":"first","affiliation":[{"name":"<!--label omitted: 1-->Departments of Medicine1 and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bruce","family":"Geryk","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Departments of Medicine1 and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lakshmi P.","family":"Kotra","sequence":"additional","affiliation":[{"name":"<!--label omitted: 2-->Department of Chemistry, Wayne State University, Detroit, Michigan 482022"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shahriar","family":"Mobashery","sequence":"additional","affiliation":[{"name":"<!--label omitted: 2-->Department of Chemistry, Wayne State University, Detroit, Michigan 482022"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen A.","family":"Lerner","sequence":"additional","affiliation":[{"name":"<!--label omitted: 1-->Departments of Medicine1 and"},{"name":"<!--label omitted: 3-->Biochemistry and Molecular Biology,3 Wayne State University School of Medicine, Detroit, Michigan 48201, and"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1042\/bj2760269"},{"key":"e_1_3_2_3_2","first-page":"75","article-title":"Nucleotide sequence of the genes coding for the TEM-like \u03b2-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2).","volume":"120","author":"Belaaouaj A.","year":"1994","unstructured":"Belaaouaj A. Lapoumeroulie C. Canica M. M. Vedel G. Nevot P. Krishnamoorthy R. Paul G. Nucleotide sequence of the genes coding for the TEM-like \u03b2-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). FEMS Microbiol. Lett. 120 1994 75 80","journal-title":"FEMS Microbiol. Lett."},{"key":"e_1_3_2_4_2","doi-asserted-by":"crossref","first-page":"2059","DOI":"10.1128\/AAC.37.10.2059","article-title":"Characterization of a new TEM-type \u03b2-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli.","volume":"37","author":"Blasquez J.","year":"1993","unstructured":"Blasquez J. Baquero M.-R. Canton R. Alos I. Baquero F. Characterization of a new TEM-type \u03b2-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. Antimicrob. Agents Chemother. 37 1993 2059 2063","journal-title":"Antimicrob. Agents Chemother."},{"key":"e_1_3_2_5_2","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/0167-4838(94)00187-L","article-title":"Complementary roles of mutations at positions 69 and 242 in a class A \u03b2-lactamase.","volume":"1247","author":"Bonomo R. A.","year":"1995","unstructured":"Bonomo R. A. Dawes C. G. Knox J. R. Shlaes D. M. Complementary roles of mutations at positions 69 and 242 in a class A \u03b2-lactamase. Biochim. Biophys. Acta 1247 1995 113 120","journal-title":"Biochim. Biophys. Acta"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/38.2.183"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi961044g"},{"key":"e_1_3_2_8_2","first-page":"111","article-title":"Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to \u03b2-lactamase inhibitors.","volume":"120","author":"Brun T.","year":"1994","unstructured":"Brun T. Peduzzi J. Canica M. M. Paul G. Nevot P. Barthelemy M. Labia R. Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to \u03b2-lactamase inhibitors. FEMS Microbiol. Lett. 120 1994 111 118","journal-title":"FEMS Microbiol. Lett."},{"key":"e_1_3_2_9_2","doi-asserted-by":"crossref","first-page":"5938","DOI":"10.1021\/ja00127a005","article-title":"Potent mechanism-based inhibition of the TEM-1 \u03b2-lactamase by novel N-sulfonyloxy \u03b2-lactams.","volume":"117","author":"Bulychev A.","year":"1995","unstructured":"Bulychev A. O\u2019Brien M. E. Massova I. Teng M. Gibson T. A. Miller M. J. Mobashery S. Potent mechanism-based inhibition of the TEM-1 \u03b2-lactamase by novel N-sulfonyloxy \u03b2-lactams. J. Am. Chem. Soc. 117 1995 5938 5943","journal-title":"J. Am. Chem. 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