Meropenem stability to ß-lactamase hydrolysis and comparative in vitro activity against several ß-lactamase-producing Gram-negative strains

N. Franceschinia, B. Segatorea, M. Perillia, S. Vessilliera, L. Franchinob and G. Amicosantea,*

a Dipartimento di Scienze e Tecnologie Biomediche, Università dell’Aquila, Via Vetoio, 67100 L’Aquila; b Medical Department AstraZeneca, Milan, Italy


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The interaction between meropenem and class A, B, C and D ß-lactamases was studied by a spectrophotometric method. Class A, C and D ß-lactamases were unable to confer in vitro resistance to carbapenems. Surprisingly, several class B metallo-ß-lactamases expressed in Escherichia coli failed to confer resistance when a conventional inoculum (105 cfu/mL) was used.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
ß-Lactamases are the primary cause of clinical failure of ß-lactam therapy. Of the four classes of ß-lactamase,1 classes A and C are the most common in pathogenic bacteria, with the emergence of extended spectrum class D enzymes (oxacillinases) only recently being reported.2 These enzymes possess serine residues that undergo acylation by the ß-lactam antibiotics, forming an acyl-enzyme intermediate, the fate of which is hydrolysis, and opening of the ß-lactam ring. Carbapenems are resistant to these enzymes, but are susceptible to class B metallo-ß-lactamases and a few other active-site serine enzymes such as IMI-1, Sme-1 and NmcA.3 Resistance to carbapenems may also be due to the loss of an outer membrane porin (D2), as seen in multidrug-resistant Pseudomonas aeruginosa or occasional de-repression in some Enterobacteriaceae harbouring chromosomal class C ß-lactamases.4

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains

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 Sigma–Aldrich (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 Mueller–Hinton 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 {lambda}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 Hanes–Wolf 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:


(1)
where E, C, EC and P are, respectively, free enzyme, inhibitor, Henri–Michaelis complex and the hydrolysis product. The single kinetic parameters k+2 and k+3 represent the acylation and deacylation rate constants, and K is the dissociation constant of the Henri–Michaelis complex. These parameters were derived from the first order rate constant (Ki), characterizing the EC* accumulation and monitoring the hydrolysis of nitrocefin or cefazolin according to the methods of Frère et al.7 and Galleni et al.8

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


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Class A, C and D ß-lactamases

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/M•s for the cephalosporinase produced by A. baumannii 187 to >2 x 105/M•s 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 1Go). 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 0.36–1.4 µM (data not shown).


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Table 1. Kinetic parameters relative to the interaction between meropenem and some selected ß-lactamases
 
The kinetic analysis of interaction of oxacillinases with carbapenems was difficult because of the biphasic kinetics that characterize these enzymes; OXA-3 and OXA-7 were the only oxacillinases tested in this study. Using oxacillin as reporter substrate, inhibition experiments were performed with all the carbapenems. Complete inactivation of both enzymes was achieved using a carbapenem concentration <10 µM for all the compounds tested (data not shown). A detailed analysis of the inactivation was performed with OXA-7 and meropenem, revealing a high value of catalytic efficiency: k+2/K >2 x 105/M•s (Table 1Go). Ki values of 67, 114, 128 and 1500 nM were computed for meropenem, panipenem, imipenem and biapenem, respectively, with OXA-3. The E. coli producing the OXA enzyme showed susceptibility to all carbapenems and ceftazidime (Table 2Go).


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Table 2. Antimicrobial susceptibility [MIC (mg/L)] of some strains producing ß-lactamases
 
MIC testing confirmed the ability of carbapenems to inhibit growth, at an inoculum size of 105 cfu/mL, for all strains producing active-site serine ß-lactamases (Table 2Go) The only exception was P. mirabilis producing the TEM-52 that showed resistance to biapenem, due to the presence of other ß-lactamases. Also for TEM-60, both in the original strain and E. coli, the MICs of panipenem and biapenem were 16- to 32-fold higher with respect to meropenem. In all cases the MICs were two- to four-fold lower for meropenem than for imipenem.

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 1Go. 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 2Go).

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work was supported by the (MURST) PRIN 99 to G.A. and by a grant from AstraZeneca, Milan, Italy.


    Notes
 
* Corresponding author. Tel: +39-0862-433455; Fax: +39-0862-433433; E-mail: amicosante{at}cc.univaq.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Ambler, R. P. (1980). The structure of ß-lactamases. Philosophical Transactions of the Royal Society of London Series B, Biological Sciences 289, 321–31.[Medline]

2 . Naas, T. & Nordmann, P. (1999). OXA-type ß-lactamases. Current Pharmaceutical Design 5, 865–79.[ISI][Medline]

3 . Rasmussen, B. A. & Bush, K. (1997). Carbapenem-hydrolyzing ß-lactamases. Antimicrobial Agents and Chemotherapy 41, 223–32.[Free Full Text]

4 . Lee, E. H., Nicolas, M. H., Kitzis, M. D., Pialoux, G., Collatz, E. & Gutman, L. (1991). Association of two resistant mechanisms in a clinical isolate of Enterobacter cloacae with high-level resistance to imipenem. Antimicrobial Agents and Chemotherapy 35, 1093–8.[ISI][Medline]

5 . Edwards, J. R. & Betts, M. J. (2000). Carbapenems: the pinnacle of the ß-lactam antibiotics or room for improvements? Journal of Antimicrobial Chemotherapy 45, 1–4.[Free Full Text]

6 . Felici, A., Perilli, M., Segatore, B., Franceschini, N., Setacci, D., Oratore, A. et al. (1995). Interactions of biapenem with active-site serine and metallo-ß-lactamases. Antimicrobial Agents and Chemotherapy 39, 1300–5.[Abstract]

7 . Frère, J. M., Dormans, C., Duychaerts, C. & De Graeve, J. (1982). Interaction of ß-iodopenicillanate with the ß-lactamases of Streptomyces albus G and Actinomadura R39. Biochemical Journal 207, 437–44.[ISI][Medline]

8 . Galleni, M., Franceschini, N., Fattorini, L., Orefici, G., Oratore, A., Frère, J. M. et al. (1994). Use of chromosomal class A ß-lactamase of Mycobacterium fortuitum D316 to study potentially poor substrates and inhibitory compounds. Antimicrobial Agents and Chemotherapy 38, 1608–14.[Abstract]

9 . Chen, H. Y. & Livermore, D. M. (1994). In-vitro activity of biapenem, compared with imipenem and meropenem against Pseudomonas aeruginosa strains and mutants with known resistance mechanisms. Journal of Antimicrobial Chemotherapy 33, 949–58.[Abstract]

10 . Yang, Y., Bhachech, N. & Bush, K. (1995). Biochemical comparison of imipenem, meropenem, and biapenem: permeability, binding to penicillin-binding proteins, and stability to hydrolysis by ß-lactamases. Journal of Antimicrobial Chemotherapy 35, 75–84.[Abstract]

Received 10 July 2001; returned 15 October 2001; revised 15 November 2001; accepted 21 November 2001





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