a Dipartimento di Scienze e Tecnologie Biomediche, Università dellAquila, Via Vetoio, 67100 LAquila; b Medical Department AstraZeneca, Milan, Italy
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Abstract |
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Introduction |
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Imipenem and meropenem are currently used clinically. Other carbapenems have shown interesting features: panipenem has been registered in Japan, whilst biapenem has not yet fulfilled registration requirements.5 Despite the clinically well-established antimicrobial efficacy of carbapenems, few studies have tried to elucidate some of the kinetic aspects of their interaction with different classes of ß-lactamases. The inhibitory activity of these molecules seems to be related to the high affinity for the ß-lactamases characterized by a poor turnover due to a slow deacylation process.6
Our report focuses mainly on the kinetics of the interaction of meropenem with active-site serine ß-lactamases and comparative in vitro susceptibility with other carbapenems, imipenem, biapenem and panipenem, against strains producing known ß-lactamases.
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Materials and methods |
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Providencia stuartii (TEM-60); Citrobacter diversus ULA-27, Proteus mirabilis (TEM-2, TEM-52); Chryseobacterium meningosepticum (CME-1, blaB); Aeromonas hydrophila AE036, Acinetobacter baumannii (blaIMP-2); P. aeruginosa (blaVIM-1); A. baumannii 187, Escherichia coli J53 (OXA-1, OXA-2, OXA-3, OXA-5, OXA-7); Serratia marcescens (TEM-AQ); and Morganella morganii MN-89 were used to produce the purified enzymes indicated, and for susceptibility tests.
Antibiotics
Meropenem, biapenem, imipenem and panipenem were from AstraZeneca (Milan, Italy), Cyanamid (Catania, Italy), Merck Sharp & Dohme (Rome, Italy) and Sankyo Co. Ltd Biological Research Laboratories (Tokyo, Japan), respectively. Tetracycline, chloramphenicol and other chemicals were from SigmaAldrich (Milan, Italy). Ceftazidime was a gift from GlaxoWellcome (Verona, Italy). Nitrocefin was purchased from Unipath (Milan, Italy).
Enzyme purification
Class A ß-lactamases from C. diversus ULA-27, P. stuartii (TEM-60), TEM-52, TEM-AQ, SHV-12 and C. meningosepticum CME-1; and class C ß-lactamases from A. baumannii 187, M. morganii MN-89 and the the metallo-ß-lactamases CphA from E. coli BL21(DE3)pLysS carrying the pET-CphA expression vector, BlaB, VIM-1 and IMP-2 were purified in our laboratory as reported previously. Bacillus cereus 5/B/6 and IMP-1 were a gift from Dr M. Galleni (CIP, University of Liege, Belgium). The oxacillinases OXA-3 and OXA-7, used for kinetic measurements, were only partially purified.
Susceptibility testing
MICs were determined by the two-fold serial broth microdilution method. Cultures were grown overnight at 37°C in MuellerHinton broth. Each dilution was inoculated into drug-containing media with a multi-point inoculator. The final inoculum was 105 cfu/mL.
ß-Lactamase activity
The metallo-ß-lactamase from A. hydrophila was assayed using 30 mM sodium cacodylate buffer pH 6.5 without zinc addition. The other metallo-ß-lactamases were assayed in 50 mM HEPES pH 7.2 containing 50 µM ZnCl2. Class A and C enzymes were assayed in 50 mM sodium phosphate buffer pH 7, whereas 0.1 M Tris-sulphate pH 7 was used for class D enzymes. All the measurements were made at 30°C with a 2 spectrophotometer (Perkin-Elmer, Rahway, NJ, USA) equipped with a thermostatic cell holder. Kinetic parameters were derived from at least three different measurements by the HanesWolf plot of the initial rate of substrate hydrolysis.
Transient inactivation parameters for class A, D and C ß-lactamases
Nitrocefin or oxacillin were used as reporter substrates for OXA-3 and OXA-7. The interaction of carbapenems with active-site serine ß-lactamases was characterized by the accumulation of a fairly stable acyl-enzyme that could be studied on the basis of the following model:
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Competitive inhibition
The Ki value was determined by plotting Vo/Vi against I, which gives a line with a slope of KSm/( KSm + S)*Ki, where Vo and Vi are the initial rates in the absence or presence of inhibitor respectively, I is the inhibitor concentration, S is the reporter substrate concentration, and KSm is the Michaelis constant of the enzyme for the reporter substrate.8
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Results and discussion |
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The interaction between meropenem and class A, C and D ß-lactamases was characterized by a transient inactivation followed by a slow deacylation process. The acylation efficiency (k+2/K) ranged from 450/Ms for the cephalosporinase produced by A. baumannii 187 to >2 x 105/Ms for OXA-7. The turn-over of the acyl-enzyme complex was always present with all the ß-lactamases tested. The k+3 values ranged from 5.6 x 10-4/s for Cme-1 to 8.7 x 10-3/s for the class C enzyme from M. morganii MN-89 (Table 1). A comparative analysis between different carbapenems with some selected class A enzymes, namely TEM-2, TEM-60 and PER-1, confirmed the behaviour shown by other ß-lactamases with meropenem (data not shown). The only exception was PER-1, which showed a pattern of competitive inhibition with all carbapenems. The Ki values computed for PER-1 were very similar for all the carbapenems and lay in the range 0.361.4 µM (data not shown).
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Class B ß-lactamases
The metallo-ß-lactamases were able to hydrolyse all the carbapenems with high catalytic efficiency. Differences were found with regard to affinity constant Km and especially for the turnover constant (kcat). The specific parameters calculated for meropenem are reported in Table 1. MIC testing showed that in E. coli, VIM-1, IMP-2 and BlaB were not able to confer resistance to meropenem or imipenem. Susceptibility was reduced after the introduction of the plasmid-encoding gene, but the MICs remained below the breakpoint. Stenotrophomonas maltophilia enzyme expressed in E. coli did not confer resistance to meropenem or biapenem (Table 2
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In addition to the favourable kinetic profile of meropenem and the active-site serine ß-lactamases, other features of the microbiological properties of this carbapenem should be taken into account. Enterobacteriaceae produce an AmpC ß-lactamase that is inducible in the presence of periplasmic concentrations of ß-lactams. Meropenem is a weaker inducer9 of this enzyme than are the other carbapenems and cephamycins. Moreover, meropenem diffuses through the outer membrane of Gram-negative bacteria at a rate comparable to that of cephaloridine, albeit to a lesser extent than imipenem.10 A further factor that should be considered is the high affinity of meropenem for PBPs, in particular PBP2, PBP3 and PBP4. All these factors, and the high stability to hydrolytic activity, make meropenem an excellent antibiotic for all Enterobacteriaceae producing ß-lactamases. However, in order to limit the spread of emerging clinical isolates that produce metallo-ß-lactamases, such as VIM-1 and IMP-1, a rational approach to the use of carbapenems needs to be taken.
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Acknowledgements |
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Notes |
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References |
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Received 10 July 2001; returned 15 October 2001; revised 15 November 2001; accepted 21 November 2001