Inhibitors and Inactivators of Beta-Lactamase from Mycobacterium fortuitum

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...

Section: Resultssupporting

confidence: 80%

“…As assessed by IC 50 s, the activity of inhibitors was weak, especially for sulbactam and tazobactam (Table 3). Similar results were obtained for ␤-lactamases extracted from N. asteroides, M. fortuitum, and N. farcinica (FAR-1) isolates (2,6,9,11). Thus, it may be hypothesized that ␤-lactamases of Nocardia spp.…”

Section: Resultssupporting

confidence: 72%

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

Poirel

Laurent

Naas

et al. 2001

“…Purified P-lactamase of M. fortuitum hydrolyzes both penicillins and cephalosporins (1). Furthermore, this enzyme was inhibited by clavulanic acid and inactivated by isoxazolylpenicillins (10).…”

mentioning

confidence: 99%

Beta-lactamase of Mycobacterium fortuitum: kinetics of production and relationship with resistance to beta-lactam antibiotics

Fattorini

Scardaci

Jin

et al. 1991

The kinetics of both intracellular and extracellular beta-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacterium fortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in beta-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of beta-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. However, for a mutant of M. fortuitum (gamma 27) with virtually nonexistent beta-lactamase production, the antibiotics still had relatively high MICs (for instance, benzylpenicillin and cephaloridine had MICs of 64 and 32 micrograms/ml, respectively). This suggests that, aside from beta-lactamase production, other mechanisms such as cell wall permeability and/or affinity for penicillin-binding proteins could coexist in M. fortuitum and explain its natural resistance to beta-lactam antibiotics.

“…Resistance to ␤-lactam antibiotics has been attributed to an interplay between ␤-lactamase activity and cell permeability as well as a low affinity to penicillin-binding proteins (12,15). ␤-Lactamase production by mycobacteria is likely to be an important factor in the expression of resistance to ␤-lactam antibiotics (11,12). Indeed, ␤-lactams in combination with either clavulanic acid or other ␤-lactamase inhibitors have in vitro activity against mycobacteria not observed with either agent alone (5,7,8,31).…”

mentioning

confidence: 99%

Susceptibilities of nontuberculosis mycobacterial species to amoxicillin-clavulanic acid alone and in combination with antimycobacterial agents

Utrup

Moore

Actor

et al. 1995

Neither amoxicillin nor clavulanic acid used alone was active at the highest level tested, i.e., 256.0 g/ml, in vitro against 24 isolates of Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium marinum. However, the MIC of an amoxicillin-clavulanic acid combination of 2:1 was <8.0/4.0 g/ml for 50 percent of the isolates tested, with all isolates being inhibited in the range of 4.0/2.0 to 32.0/16.0 g/ml, respectively. Titration of amoxicillin-clavulanic acid with a fixed 2-g/ml concentration of ethambutol resulted in synergistic activity against 3 of 9 isolates of M. fortuitum, 10 of 10 isolates of M. kansasii, and 5 of 5 isolates of M. marinum. This observation was confirmed in a checkerboard analysis in which fractional inhibitory concentrations were <0.5 for 20 of the 24 isolates. Synergistic activity was observed against the other four isolates in one of two trials. On the other hand, titration of amoxicillin-clavulanic acid in the presence of either one or two fixed concentrations of isoniazid, rifampin, cycloserine, tetracycline, or amikacin failed to result in synergism.There has been a resurgence of mycobacterial infections in the United States mainly attributed to the spread of AIDS in large metropolitan areas (4). A significant number of the mycobacterial isolates causing disease are nontuberculosis mycobacteria (NTM) (29,32). Of these organisms, Mycobacterium avium complex, which represents more than 60% of the NTM isolates, is by far the most common cause of opportunistic NTM infection in patients with AIDS (23, 32). Among other NTM isolates capable of causing infection are M. fortuitum complex (19%) and M. kansasii (10%), with the remaining 10% being other mycobacterial species (13,21). In addition to disseminated infections in patients with AIDS, NTM cause pulmonary, postoperative, soft tissue, and bone and joint infections (29,30).NTM tend to be resistant to a wide variety of antimicrobial agents including many of the ␤-lactam antibiotics (2). Resistance to ␤-lactam antibiotics has been attributed to an interplay between ␤-lactamase activity and cell permeability as well as a low affinity to penicillin-binding proteins (12,15). ␤-Lactamase production by mycobacteria is likely to be an important factor in the expression of resistance to ␤-lactam antibiotics (11, 12). Indeed, ␤-lactams in combination with either clavulanic acid or other ␤-lactamase inhibitors have in vitro activity against mycobacteria not observed with either agent alone (5,7,8,31).Amoxicillin-clavulanic acid in combination with antimycobacterial agents has been used successfully to treat two patients with multiple-drug-resistant M. tuberculosis infections (18). Preliminary data demonstrate that amoxicillin-clavulanic acid MICs for M. tuberculosis obtained with the BACTEC system were 8/4 or 16/8 g/ml (17), and for 20 M. avium complex isolates screened, MICs were Յ 8/4 g/ml for 10 isolates, and MICs were Յ 4/2 g/ml for 8 isolates (28).Since antimycobacterial agents are usually used in combination for the treatment of my...