Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli

Abstract

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 ␤-lactam antibiotics. The ␤-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed ␤-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by ␤-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to ␤-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. KTG). Toho-1 was about 83% homologous to the ␤-lactamase mediated by the chromosome of K. oxytoca D488 and the ␤-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated ␤-lactamase Toho-1 produced by E. coli TUH12191 is similar to ␤-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 ␤-lactamase. Detailed comparison of Toho-1 with other ␤-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.Expanded-spectrum cephalosporins have chemical structures which confer stability to many ␤-lactamases from gram-negative bacteria. However, many members of the family Enterobacteriaceae other than Escherichia coli developed resistance to the expanded-spectrum cephems (40). The primary mechanism of this resistance was demonstrated to be excessive production of a chromosomal ␤-lactamase (AmpC) (23). However, bacteria that show resistance mediated by other ␤-lactamases appeared in 1984 (8). Species of the Enterobacteriaceae such as Klebsiella pneumoniae, Klebsiella oxytoca, and E. coli acquired resistance against expanded-spectrum cephem antibiotics by producing extended-spectrum ␤-lactamase. The extended spectrum of the ␤-lactamase was often acquired by the variation of ␤-lactamase genes on transmissible plasmids (43,44). Under the influence of antimicrobial agents, bacteria producing primarily TEM-type or SHV-type ␤-lactamases developed point mutations in structural genes which served to extend the substrate specificity of the enzymes (44). These TEM-type and SHV-type ␤-lactamases show about 65% amino acid sequence homology, with isoelectric points of 5.5 to 6.3 and 7.0 to 8.2, respectively (8, 9).

Atomic Resolution Structures of CTX-M β-Lactamases: Extended Spectrum Activities from Increased Mobility and Decreased Stability

“…Also, the activity spectrum among CTX-M enzymes is unusually heterogeneous. The enzyme Toho-1, which belongs to the CTX-M-2 cluster, is more susceptible to inhibition by clavulanate than tazobactam (K i , 0.6 versus 5.3 mM), 14 whereas most other CTX-M enzymes exhibit an opposite behavior. Toho-1 has a lower hydrolytic activity against penicillins and higher hydrolytic activity against ceftazidime than the "typical" CTX-M enzymes.…”

Extended-spectrumβ-Lactamases: Implications for the Clinical Laboratory and Therapy

“…In the same year, a new plasmid-mediated cefotaximase, designated CTX-M-2, with an isoelectric point different from that of CTX-M-1, was described from multidrug-resistant Salmonella enterica serovar Typhimurium [38]. In 1995, Ishii et al [39] reported a novel enzyme, Toho-1 (renamed as CTX-M-44 later), which was highly homologous to MEN-1. In the following year, nucleotide sequencing studies concluded that the deduced amino acid sequence of CTX-M-1 was identical to the reported sequence of MEN-1, and the amino acid sequence of CTX-M-2 was 84% identical to that of CTX-M-1 [40].…”

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

“…However, based on sequences and structures, there appears to be conservation of positive charge in this region that results from arginine extending from positions 220, 244, or 276 as has previously been noted. [14][15][16][17] Thus, a…”