Hydrolysis of cefazolin by enzymes produced by Pseudomonas aeruginosa after exposure to ceftazidime in vitro

Abstract

Sometimes resistance of Pseudomonas aeruginosa (Ps. aeruginosa) is developed during antibiotic treatment, in spite of the initial susceptibility in vitro. The aim of this study was to use an in vitro model for the study of the development of resistant strains of Ps. aeruginosa after a short exposure to ceftazidime, and to study the hydrolysing capacity of beta-lactamases produced by the resistant strains. Among 563 clinical strains of Ps. aeruginosa, 37 multisensitive strains were collected for the study. After being identified, strains with simultaneous sensitivity to 5 expanded spectrum cephalosporins were chosen. For each strain, the minimal inhibitory concentration (MIC) of the 5 expanded spectrum cephalosporins was determined, and the production of extended spectrum beta-lactamases (ESBL) was excluded by the double-disc synergy diffusion test. Strains non producing ESBL were cultivated in concentrations of ceftazidime equal to MICx2 and MICx4. After 24 hours of culture, the development of resistant strains was estimated and the cephalosporinase activity of the produced beta-lactamases was determined by their ability to hydrolyse cefazolin. Hydrolysis of cefazolin was studied by measuring the change of its absorbance on 272 nm using a Shimadzu 160A spectrophotometer. The hydrolyzing capacity of the enzymes was expressed as the percentage of the antibiotic, which was hydrolysed in 10 sec. A total of 60% and 50% of strains developed resistant strains after exposure to ceftazidime in concentration MICx2 and MICx4, respectively. The hydrolyzing capacity of the original strains was 15-36% while the hydrolyzing capacity of the resistant strains was 10-73%. Totally 64% of the resistant strains expressed higher hydrolyzing capacity than the original strains. Regardless of the susceptibility test results, Ps. aeruginosa presented a high tendency to develop resistant strains after a short exposure to ceftazidime in vitro. In most cases the resistant strains expressed higher cephalosporinase activity than the original strains, suggesting derepression of chromosomal beta-lactamases. Our model offers a simple, inexpensive and rapid method for detecting resistance of Ps. aeruginosa developed due to derepression of beta-lactamases, and for discriminating resistant strains with derepressed beta-lactamases from strains that developed other mechanisms of resistance.