Substrate specificity of HMRZ-86 for beta-lactamases, including extended-spectrum beta-lactamases (ESBLs).
ABSTRACT HMRZ-86 was designed as a new chromogenic cephalosporin to detect extended-spectrum beta-lactamases (ESBLs) and similar evolved beta-lactamases, such as metallo-beta-lactamases, derepressed AmpC, and extended oxacillinase. We report here our investigation of the kinetic parameters of several types of beta-lactamases to show the enzymatic characteristics of HMRZ-86. The Michaelis constant (Km values of HMRZ-86 for ESBLs were twice to three and half times as high as those of nitrocefin, and the maximum velocity (Vmax) was one-fifth that of nitrocefin. The Km and Vmax of HMRZ-86 for AmpC were both smaller than those of nitrocefin. The kinetic parameters of HMRZ-86 for metallo beta-lactamase (MBL) were very variable, depending on the type of buffer solution used and the concentration of zinc ions. For MBL, the Km values of HMRZ-86 were higher than those of nitrocefin, but the Vmax values were almost the same as those of nitrocefin. Although the chemical structure of HMRZ-86 is similar to that of nitrocefin, we think the enzymatic reactivities of the two entities for beta-lactamases are very different.
- SourceAvailable from: Hayao Taguchi[show abstract] [hide abstract]
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 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.Antimicrobial Agents and Chemotherapy 11/1995; 39(10):2269-75. · 4.57 Impact Factor
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ABSTRACT: Conventional detection of extended-spectrum beta-lactamase (ESBL) producers in blood cultures usually requires overnight incubation. This could delay the prescribing of appropriate therapy. We evaluated whether the chromogenic cephalosporin HMRZ-86, which is hydrolysed by ESBLs, could be used for the rapid detection of ESBL producers directly in blood culture broths. The HMRZ-86 test was first applied to broth cultures of isolates producing known beta-lactamases. A colour change indicating hydrolysis, which was inhibited by clavulanic acid, was taken as an indication of ESBL production. A similar method was used for testing blood culture supernatants and broth subcultures of blood cultures. The HMRZ-86 test detected all the ESBL producers among 83 clinical isolates and control strains. Only one false positive was seen. The usefulness of HMRZ-86 for the direct testing of blood culture broths was limited by the presence of lysed blood. However, by using a 2 h broth subculture of the blood culture broths, the HMRZ-86 test was able to detect all those blood cultures containing an ESBL producer. No false positive or negative tests occurred according to the results of our standard phenotypic tests. The HMRZ-86 test is a simple and rapid method that can be used for detecting ESBL producers in blood cultures.Journal of Antimicrobial Chemotherapy 10/2007; 60(3):652-4. · 5.34 Impact Factor
- Antimicrobial Agents and Chemotherapy 01/2001; 45(3):660-663. · 4.57 Impact Factor