Some Molecular Properties of Citrobacter diversus Beta-Lactamases

Summary

Citrobacter diversus ULA-27, a clinical isolate showing a broad resistance pattern towards both penicillins and cephalosporins, produces chromosome encoded β-lactamases. However, the strain remains susceptible to some cephamycins, imipenem, ceftazidime and tetracyclines.

Crude bacterial extracts analyzed by isoelectric focusing on polyacrylamide gels, revealed the presence of two main isoforms and some «satellite» bands focusing in the pH range 5.7-7.2.

The isoform showing the pIs 6.8 and 6.2 were characterized as class «A» β-lactamases (according to Ambler's classification) based on the rate of interaction of β-iodopenicillanate and the aminoacid sequence around the active site serine. The substrate specificity of the Citrobacter diversus β-lactamases explains the resistance phenomenon of this bacterium to penicillins and cephalosporins.     

The beta-lactamases of Citrobacter diversus and their hydrolysis kinetics for some structurally-related cephalosporins

We measured the kinetics of hydrolysis of various cephalosporins by the chromosomally-encoded beta-lactamases of Citrobacter diversus ULA-27. Cefonicid, cefamandole, cefatrizine and cefoperazone were all hydrolyzed but these antibiotics showed a different feature in their kinetic parameters. Moreover, cefoperazone was a non-competitive inhibitor of this type of enzyme. Cefotetan was stable to hydrolysis and behaved like a progressive inactivator. The ability of these enzymes to inactivate the reported antibiotics contributes largely to the resistance of the studied strain. We conclude that hydrolysis is the main mechanism of resistance of this strain to the new cephalosporins.     

Beta-lactamase induction antagonizes beta-lactam susceptibilities in Citrobacter diversus and Enterobacter cloacae clinical isolates.

Inducible beta-lactamases were obtained after exposure to several beta-lactams in clinical isolates of Enterobacter cloacae and Citrobacter diversus. Enzyme production was related to the inducer and medium composition. beta-lactamase is able to inactivate only labile compounds, thus generating minimum inhibitory concentrations higher than in the absence of the inducer; imipenem susceptibilities usually were not changed.     

A retrospective view of beta-lactamases

The discovery of a penicillinase (later shown be a beta-lactamase) 50 years ago in Oxford came from the thought that the resistance of many Gram-negative bacteria to Fleming's penicillinase might be due to their production of a penicillin-destroying enzyme. The emergence of penicillinase-producing staphylococci in the early 1950s, particularly in hospitals, raised the question whether the medical value of penicillin would decline. The introduction of new semi-synthetic penicillins and cephalosporins in the 1960s began to reveal many beta-lactamases distinguishable by their different substrate profiles. In this period it was established that genes encoding beta-lactamases from Gram-negative bacilli could be carried from one organism to another on plasmids and also that penicillin inhibited a transpeptidase involved in bacterial cell wall synthesis. During the last two decades a number of these enzymes have been purified and the genes encoding them have been cloned. Much has now been learned, with the aid of powerful modern techniques, about their structures, their active sites, their relationship to penicillin-sensitive proteins in bacteria and to their likely evolution. Further knowledge may contribute to a more rational approach to chemotherapy in this area. Experience suggests that a need for new substances will continue.     

结直肠癌分子靶向治疗面临的若干问题

目前,结直肠癌的多学科综合治疗已经得到广泛的认同,但手术仍然是转移性结直肠癌的主要治疗手段.随着外科技术的进步以及临床医生对肝内、肝外转移病灶手术观念的变化,结直肠癌远处转移灶的可切除率显著提高,患者术后的5年生存率也提高到27%～41%[1].由于可切除的结直肠癌     

Comparison of five different methods for detection of SHV extended-spectrum beta-lactamases

Five different methods for detection of different types of SHV extended-spectrum beta-lactamases (ESBL) were compared: minimum inhibitory concentration (MIC) determination of beta-lactam with and without clavulanic acid, double-disk synergy test (DDST), inhibitor potentiated disk diffusion test (IPDDT), three-dimensional test (TDT) and PCR/Nhe I test. MIC determination of beta-lactam with and without clavulanic acid was the most sensitive method regardless of the type of beta-lactamase. However the specificity of this method was a little above 90%. IPDDT turned out to be a very sensitive method too but it lacks specificity because 26.9% of ceftazidime sensitive strains (putative ESBL negative), gave a positive result. It is important to put all four disks on the plate because ceftazidime and aztreonam were more sensitive indicators for SHV-5 and SHV-12 beta-lactamase producers while cefotaxime and ceftriaxone were more reliable in detecting SHV-2 beta-lactamase producers. The DDST detected all SHV-5 and SHV-12 beta-lactamase producers and 95.2% of SHV-2, so it was less sensitive than MIC determination but was highly specific, since there were no false negative results observed. The sensitivity of DDST can be improved by using all four disks and placing them at the smaller distance from the central disk (2.5 cm). The TDT was the least sensitive method, particularly for SHV-5 and SHV-12 beta-lactamase producers. The PCR/Nhe I test for detection of ESBL blaSHV genes is a highly sensitive and specific method but it is rather laborious and thus not very practical for use in routine clinical laboratories. Nevertheless it has potential to serve as the gold standard in epidemiological investigations on ESBLs. According to the results of this investigation MIC determination of beta-lactam with and without clavulanic acid, even if only one antibiotic is used and the PCR/Nhe I tests are the most reliable methods for detection of SHV ESBLs.     

Detection of extended spectrum beta-lactamases in Escherichia coli and Klebsiella pneumoniae.

The presence of extended spectrum beta-lactamases (ESbetaLs) in 129 Escherichia coli and 128 Klebsiella pneumoniae strains was determined with the E test. beta-lactamases were detected in 66 (51.2%) of E.coli and 107 (83.6%) of K. pneumoniae by the nitrocefin disk method. Putative ESbetaL production was observed in 15.5% of E. coli and 55.5% of K. pneumoniae with the E test. Among the putative ESbetaL-producer strains 11 were found to be susceptible to ceftizoxime, 6 to ceftazidime, 2 to ceftriaxone, cefotaxime and aztreonam, and one to cefoperazone according to the disk diffusion test. All putative ESbetaL-producers were resistant to cefodizime, thus it may be a good indicator of the presence of mainly TEM-type ESbetaL. The E test was also found to be practicable for the detection of ESbetaLs in clinical laboratory. Further studies are needed to clarify whether ESbetaL can be identified reliably in species that, in addition to plasmid mediated ESbetaL production, chromosomally encoded AmpC beta-lactamases and other class A ESbetaLs by disk diffusion test.