Biochemical characterization of chromosomal cephalosporinases from isolates belonging to the Acinetobactet baumannii complex

Abstract

Members of the Acinetobacter baumannii complex have emerged as some of the most important opportunistic pathogens within the hospital environment, being able to colonize and produce infections in most immunocompromised patients, especially in intensive care units (ICUs). Such infections are difficult to treat due to their multiple resistance to the antibiotics currently available for the treatment of nosocomial infections [1]. Various mechanisms of resistance to b-lactams have been identified in this genus, including b-lactamase production, alteration of penicillin-binding proteins, and reduced levels of penetration across the outer membrane. b-Lactamase production is one of the main mechanisms of resistance to b-lactams in Acinetobacter spp. [2,3]. Such enzymes can be either plasmid-encoded penicillinases (TEM and CARB type) or chromosomal cephalosporinases. The exact nature and character of these latter enzymes are not fully clarified for Acinetobacter spp. They belong to the group 1 b-lactamases of the classification of Bush et al [4], but several authors have found heterogeneity among the Acinetobacter cephalosporinases [5,6]. In the present work, the cephalosporinases produced by 125 strains belonging to the A. baumannii complex were characterized by determination of their pI values, b-lactamase inhibition profiles and substrate profiles. Clinical isolates (N1⁄4 125) belonging to the A. baumannii complex, obtained in the Hospital de la Princesa (Madrid, Spain) from January 1995 to December 1997, were studied. The isolates were identified by the API 20NE system (BioMerieux, Lyon, France) and growth at 44 8C [7]. Minimal inhibitory concentrations (MICs) and susceptibility percentages of 15 b-lactam agents were determined by an agar dilution method, as recommended by the National Committee for Clinical Laboratory Standards [8]. A. baumannii ATCC 19606, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains. b-Lactamase activity was detected in crude enzymatic extracts by the hydrolysis of nitrocefin (0.5 mg/mL). The pI values were determined by isoelectric focusing (IEF) (Multhiphor II Electrophoresis System; Amersham Pharmacia Biotech, Uppsala, Sweden), on wide-range ampholine (pH range 3.5–9.5) polyacrylamide gels; in some cases a narrow-range ampholine (pH 5.5–8.5) was used. The percentage of inhibition of the following inhibitors was studied at fixed concentrations for 43 enzymatic extracts: clavulanic acid (10 mM), sulbactam (20 mM), cloxacillin (4 mg/mL), aztreonam (1.148 mM), NaCl (140 mM) and EDTA (0.1 mM). This assay was performed as described by Papanicolaou et al [9] with several modifications. After incubating the extracts with nitrocefin solution, the changes in optical density at a wavelength of 492 nm were measured at 2, 5 and 10 min, and subsequently at 10-min intervals for 40 min. Duplicate determinations were performed for each extract. Sixteen crude enzymatic extracts were selected to study the substrate profile of the b-lactamases. Two methods were used. First, hydrolysis of the following b-lactams by the crude extracts was studied with a microbiological assay as described by Paton et al [10]: ampicillin, cefazolin, oxacillin, carbenicillin, cefuroxime, cefotaxime, ceftazidime, imipenem and meropenem. OXA-2 and TEM-1 were used as control enzymatic extracts. Staphylococcus aureus ATCC 25923 and E. coli ATCC 25922 were used as hydrolysis control strains depending on the antibiotic tested. Second, assay UV spectrophotometry was used to study the hydrolysis rate of cephaloridine, benzylpenicillin, cefotaxime, ceftazidime, imipenem, meropenem, carbenicillin and oxacillin. Duplicate determinations were performed for each enzymatic extract, with concentrations of either 100 mM or 1 mM. Hydrolysis rates were expressed as a percentage of hydrolysis with respect to cephaloridine. A high percentage of susceptibility was found to imipenem, meropenem and ampicillin–sulbactam (100%, 100% and 84%, respectively); however, low susceptibility was shown to most penicillins (piperacillin, 8%; ticarcillin, 18.4%), and cephalosporins (cefotaxime, 4.8%; ceftazidime, 11.2%; cefepime, 12.8%). Isoelectric focusing showed a unique band of pI> 8