Molecular and Biochemical Analysis of AST-1, a Class A β-Lactamase from Nocardia asteroides Sensu Stricto

Abstract

A ␤-lactamase gene was cloned from a Nocardia asteroides sensu stricto clinical isolate. A recombinant plasmid, pAST-1, expressed the ␤-lactamase AST-1 in Escherichia coli JM109. Its pI was 4.8, and its relative molecular mass was 31 kDa. E. coli JM109(pAST-1) was resistant to penicillins and narrow-spectrum cephalosporins. The ␤-lactamase AST-1 had a restricted hydrolytic activity spectrum. Its activity was partially inhibited by clavulanic acid but not by sulbactam and tazobactam. AST-1 is an Ambler class A ␤-lactamase sharing 65% amino acid identity with ␤-lactamase FAR-1, the most closely related enzyme.The Nocardia genus includes several species that are opportunistic pathogens in immunocompromised patients (3,13). Species of Nocardia asteroides sensu stricto are the predominant human pathogens and are involved in pulmonary and brain abscesses (13). Since nocardiosis requires a long treatment (6 to 12 months or longer) and may cause a high mortality rate, the choice of the optimal antibiotic treatement is crucial (7).␤-Lactams have been used as first-line treatment with little concern for the ␤-lactam susceptibility of Nocardia sp. isolates (13). Knowledge of the mechanisms of ␤-lactam resistance profiles of Nocardia isolates may be critical for assessing the potential clinical efficacy of ␤-lactams. A study of the antimicrobial susceptibility patterns of 78 clinical isolates belonging to the N. asteroides complex found that 95% of the isolates exhibit one of the four major antibiotic resistance patterns (24). Type I (20% of the isolates) is susceptible to ampicillin and carbenicillin but intermediate in susceptibility to imipenem; type III (18%) is susceptible to ampicillin and erythromycin; type V (17%) is resistant to broad-spectrum cephalosporins; and type VI, the most prevalent group (35%), is resistant to ampicillin but susceptible to extended-spectrum cephalosporins and imipenem. Type II and type IV are extremely rare and not well characterized. Wallace et al. show that drug resistance patterns of type III and type V correlate with taxonomic groups and have been reclassified as Nocardia nova and Nocardia farcinica, respectively (21,22). Isolates belonging to types I, IV, and VI are grouped into the same subspecies, named N. asteroides sensu stricto.Although some nocardial ␤-lactamases have been characterized biochemically in N. asteroides (9, 17), Nocardia brasiliensis (19, 23), and N. farcinica (11, 20), the accurate role of ␤-lactamase in the ␤-lactam resistance pattern has scarcely been explored. Sequences of ␤-lactamase genes are available only for N. farcinica and the nonhuman pathogen Nocardia lactamdurans (5, 11).We report on the molecular and biochemical characterization of a class A ␤-lactamase named AST-1 from a clinical isolate belonging to the most prevalent group of N. asteroides sensu stricto species. Hydrolytic activity of ␤-lactamase AST-1 was compared to that of ␤-lactamase FAR-1. MATERIALS AND METHODSBacterial strains and plasmids. The N. asteroides sensu stricto isolate JPL was fro...

Chromosome-Borne Class A BOR-1 β - Lactamase of Bordetella bronchiseptica and Bordetella parapertussis

“…1). BOR-1 shared 42 to 45% amino acid identity with the most closely related enzymes, the naturally occurring ␤-lactamases L-2 from S. maltophilia, PenA from Burkholderia cepacia, FAR-1 from Nocardia farcinica, AST-1 from Nocardia asteroides, YENT from Yersinia enterocolitica, and SHV-1 from Klebsiella pneumoniae, and the plasmid-mediated ␤-lactamase TEM-1 (3,6,7). The surrounding sequences of the bla BOR-1 gene in B. bronchiseptica and B. parapertussis were identical.…”

“…Selection of recombinant clones was performed on Mueller-Hinton (MH) plates containing 50 g of amoxicillin and 30 g of kanamycin per ml. Once cloned, the PCR product was sequenced on both strands as described previously (6), and the ␤-lactamase gene sequence revealed identity with that found in the databases for B. bronchiseptica RB50. Disk diffusion susceptibility testing performed for E. coli DH10B (pFL-1) suggested that the cloned gene encoded a functional ␤-lactamase named BOR-1.…”

“…Purification of ␤-lactamase BOR-1 was performed with culture of E. coli DH10B harboring recombinant plasmid pFL-1, as described previously (6). Briefly, culture extracts were subjected to two purification steps using ion-exchange chromatography.…”

“…septica DOR was extracted as described previously (6). A PCR experiment was performed using primers PREBOR-1A (5Ј-A TACGCTGACGCAGACATTG-3Ј) and PREBOR-1B (5Ј-A GAAGGCCGTGGTCACCTTC-3Ј), designed according to the database sequence of B. bronchiseptica RB50 to amplify the entire ␤-lactamase gene.…”

Crystal Structure of the Mycobacterium fortuitum Class A β-Lactamase: Structural Basis for Broad Substrate Specificity

“…Cefoxitin, dicloxacillin, imipenem, and aztreonam are poorly recognized by the enzyme, and the protein is inhibited by the penem inhibitor BRL42715 and, to a lesser extent, clavulanic acid. The specificity profile is similar overall to those of CTX-M-type enzymes; some TEM-derived extended-spectrum ␤-lactamases (ESBLs); the chromosomally encoded ESBLs from Citrobacter sedlakii and Yersinia enterocolitica; and ␤-lactamases identified in the genera Nocardia, Kluyvera, and Burkholderia (1,12,31,33,35,37). At the aminoacid level, the mature protein shares 70% identity with the ␤-lactamase of Mycobacterium smegmatis and between 40 and 45% with the aforementioned proteins.…”