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Antimicrobial Agents and Chemotherapy, May 1998, p. 1181-1186, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Sequencing of the Gene Encoding Toho-2, a Class A beta -Lactamase Preferentially Inhibited by Tazobactam

Ling Ma,1 Yoshikazu Ishii,1,* Masaji Ishiguro,2 Hiroshi Matsuzawa,3 and Keizo Yamaguchi1

Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540,1 Suntory Institute for Bioorganic Research, 1-1 Wakayamadai, Shimamoto, Osaka 618-8503,2 and Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,3 Japan

Received 9 October 1997/Returned for modification 15 January 1998/Accepted 9 March 1998

Escherichia coli TUM1083, which is resistant to ampicillin, carbenicillin, cephaloridine, cephalothin, piperacillin, cefuzonam, and aztreonam while being sensitive to cefoxitin, moxalactam, cefmetazole, ceftazidime, and imipenem, was isolated from the urine of a patient treated with beta -lactam antibiotics. The beta -lactamase (Toho-2) purified from the bacteria hydrolyzed beta -lactam antibiotics such as penicillin G, carbenicillin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam and especially had increased relative hydrolysis rates for cephalothin, cephaloridine, cefotaxime, and ceftizoxime. Different from other extended-spectrum beta -lactamases, Toho-2 was inhibited 16-fold better by the beta -lactamase inhibitor tazobactam than by clavulanic acid. Resistance to beta -lactams was transferred by conjugation from E. coli TUM1083 to E. coli ML4909, and the transferred plasmid was about 54.4 kbp, belonging to the incompatibility group IncFII. The cefotaxime resistance gene for Toho-2 was subcloned from the 54.4-kbp plasmid. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 981 bases. The nucleotide sequence of the gene (DDBJ accession no. D89862) designated as blatoho was found to have 76.3% identity to class A beta -lactamase CTX-M-2 and 76.2% identity to Toho-1. It has 55.9% identity to SHV-1 beta -lactamase and 47.5% identity to TEM-1 beta -lactamase. Therefore, the newly isolated beta -lactamase designated as Toho-2 produced by E. coli TUM1083 is categorized as an enzyme similar to Toho-1 group beta -lactamases rather than to mutants of TEM or SHV enzymes. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 327 amino acid residues. Comparison of Toho-2 with other beta -lactamase (non-Toho-1 group) suggests that the substitutions of threonine for Arg-244 and arginine for Asn-276 are important for the extension of the substrate specificity.

* Corresponding author. Mailing address: Department of Microbiology, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 1438540, Japan. Phone: 81-3-3762-4151. Fax: 81-3-5493-5415. E-mail: yoishii{at}sirius.med.toho-u.ac.jp.

Antimicrobial Agents and Chemotherapy, May 1998, p. 1181-1186, Vol. 42, No. 5
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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