Ishii Y, Ohno A, Taguchi H, Imajo S, Ishiguro M, Matsuzawa H
Department of Microbiology, Toho University School of Medicine, Tokyo, Japan.
Antimicrob Agents Chemother. 1995 Oct;39(10):2269-75. doi: 10.1128/AAC.39.10.2269.
Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A beta-lactamase. Detailed comparison of Toho-1 with other beta-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.
从一名接受β-内酰胺类抗生素治疗的患者尿液中分离出大肠杆菌TUH12191，它对哌拉西林、头孢唑林、头孢替安、头孢唑肟、头孢米诺和氨曲南耐药，但对头孢西丁、拉氧头孢、氟氧头孢和亚胺培南敏感。从该细菌中纯化得到的β-内酰胺酶（Toho-1）的等电点为7.8，分子量约为29,000，可水解β-内酰胺类抗生素，如青霉素G、氨苄西林、苯唑西林、羧苄西林、哌拉西林、头孢噻吩、头孢西丁、头孢噻肟、头孢他啶和氨曲南。Toho-1受到β-内酰胺酶抑制剂如克拉维酸和他唑巴坦的显著抑制。对β-内酰胺类、链霉素、大观霉素、磺胺甲恶唑和甲氧苄啶的耐药性通过接合转移从大肠杆菌TUH12191转移至大肠杆菌ML4903，转移的质粒约为58 kbp，属于不相容群M。通过转化大肠杆菌MV1184从58-kbp质粒中克隆出Toho-1的头孢噻肟耐药基因。测定了Toho-1基因的序列，该基因的开放阅读框由873或876个碱基组成（起始序列，ATGATG）。发现该基因的核苷酸序列（DDBJ登录号D37830）与产酸克雷伯菌E23004产生的A类β-内酰胺酶基因序列约73%同源。根据从DNA序列推导的氨基酸序列，前体由290或291个氨基酸残基组成，其中包含A类β-内酰胺酶共有的氨基酸基序（70SXXK、130SDN和234KTG）。Toho-1与产酸克雷伯菌D488染色体介导的β-内酰胺酶以及大肠杆菌MEN-1质粒介导的β-内酰胺酶约83%同源。因此，新分离的大肠杆菌TUH12191产生的β-内酰胺酶Toho-1与产酸克雷伯菌D488、产酸克雷伯菌E23004和大肠杆菌MEN-1产生的β-内酰胺酶相似，而与TEM或SHV酶的突变体不同。Toho-1与产酸克雷伯菌A类β-内酰胺酶的相似性最高。Toho-1与其他β-内酰胺酶的详细比较表明，Asn-276被Arg取代并伴随Thr取代Arg-244是底物特异性扩展中的一个重要突变。
