In vitro antibacterial activity and mechanism of action of J-111,225, a novel 1ß-methylcarbapenem, against transferable IMP-1 metallo-ß-lactamase producers

Rie Nagano*, Yuka Adachi, Terutaka Hashizume and Hajime Morishima

Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-2611, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
IMP-1 ß-lactamase, a class B zinc metallo-enzyme encoded by the transferable blaIMP gene, is known to confer high-level resistance to carbapenems as well as to penicillins and cephalosporins. J-111,225 is a novel 1ß-methylcarbapenem with a structurally unique side chain comprising a trans-3,5-disubstituted pyrrolidinylthio moiety at the C2 position. It inhibited 17 Serratia marcescens and two Pseudomonas aeruginosa IMP-1-producing clinical isolates at a concentration of 32 mg/L (range 4–32 mg/L). It showed synergy with imipenem against IMP-1-producing S. marcescens BB5886 and P. aeruginosa GN17203 with minimal FIC indices of 0.38 and 0.5, respectively. J-111,225 was more resistant than imipenem to hydrolysis by class B metallo-ß-lactamases. In kinetic studies, J-111,225 inhibited the IMP-I enzyme with a Ki of 0.18 µM when imipenem was used as a substrate. In contrast, J-111,225 was the substrate for hydrolysis by other class B ß-lactamases such as Bacteroides fragilis CcrA, Stenotrophomonas maltophilia L1 and Bacillus cereus type II enzyme with respective Km values of 11, 10 and 148 µM. The greater antibacterial activity of J-111,225 against IMP-1-producing bacteria may result from its unique interaction with the ß-lactamase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Carbapenems such as imipenem and meropenem are potent broad-spectrum antibiotics with good stability against bacterial ß-lactamases of molecular classes A and C.1 However, zinc metallo-ß-lactamases (molecular class B) are known for their carbapenem-hydrolysing activity and can also hydrolyse penicillins and cephalosporins.2 Since the first isolation in 1991 of a Pseudomonas aeruginosa strain that produced a plasmid-mediated metallo-ß-lactamase,3 there have been outbreaks in various districts of Japan of carbapenem-resistant Enterobacteriaceae, including Serratia marcescens and P. aeruginosa.3–5 The mechanism of resistance has been ascribed to the production of IMP-1 enzyme encoded by the transferable metallo-ß-lactamase gene, blaIMP.6 Recently, IMP-1-producing organisms have been reported in South Korea7 and Europe.8 A concern is that the blaIMP gene can be transferred among Gram-negative bacteria, and there is no effective inhibitor to date. Thus, the spread of metallo-ß-lactamases is a threat to antimicrobial chemotherapy with ß-lactam antibiotics and carbapenems.9

J-111,225 is a novel trans-3,5-disubstituted pyrrolidinylthio carbapenem with an ultra-broad spectrum covering Gram-positive and Gram-negative bacteria including methicillin-resistant staphylococci and P. aeruginosa.10 In the course of evaluation, J-111,225 was found to have better antibacterial activity than marketed carbapenems against IMP-1 metallo-ß-lactamase-producing organisms. In this paper, we describe the interaction of J-111,225 with ß-lactamases.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotics

J-111,225 and its side chain, as well as meropenem and panipenem, were synthesized at the Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd, Tsukuba, Japan. J-111,225 has two chiral centres, C3 and C5, bearing a sulphide bond and benzene ring, respectively, in its pyrrolidine ring (Figure 1Go). Imipenem and amikacin were the products of Banyu Pharmaceutical Co. Ltd. The following drugs were obtained commercially: ceftazidime (Tanabe Pharmaceutical Co. Ltd, Osaka, Japan), piperacillin (Toyama Chemical Co. Ltd, Tokyo, Japan), aztreonam (Eisai Co. Ltd, Tokyo, Japan), ofloxacin (Sigma Chemical Co., St Louis, MO, USA), cephaloridine (Shionogi Pharmaceutical Co., Osaka, Japan) and nitrocefin (Oxoid, Basingstoke, UK).



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Figure 1. Structure of J-111,225 and its side chain. Two chiral centres of the pyrrolidine ring of J-111,225 are shown in the structure.

 
Bacterial strains

Clinical isolates that produced IMP-1 metallo-ß-lactamase were collected from various districts in Japan since 1994, and the blaIMP gene was detected by PCR.5 P. aeruginosa GN17203, which harbours the blaIMP-carrying plasmid, pMS350, and Bacteroides fragilis GAI30079 were generously given by M. Inoue, Kitasato University, School of Medicine, Kanagawa, Japan, and K. Watanabe, Institute of Anaerobic Bacteriology, Gifu University, School of Medicine, Gifu, Japan, respectively.

Determination of MICs

MICs were determined by the two-fold serial broth microdilution method using Mueller–Hinton broth (Difco Laboratories, Detroit, MI, USA). A culture of test strains grown at 37°C for 6 h in Mueller–Hinton broth was diluted to 107 cfu/mL, and these dilutions were inoculated into the drug-containing broth with an inoculation apparatus (MIC-2000: Dinatech Laboratories Inc., Chantilly, VA, USA) at the final inoculum size of 105 cfu/mL. The MIC was defined as the lowest antibiotic concentration that completely prevented visible growth after incubation at 37°C for 20 h. Synergy of drug combinations was determined by the chequerboard method using S. marcescens BB5886 and P. aeruginosa GN17203, which showed resistance to imipenem with an MIC of 128 mg/L in Mueller–Hinton broth. The fractional inhibitory concentration (FIC) index was determined according to the method of Elion et al.11

ß-Lactamase preparation

The IMP-1 metallo-ß-lactamase was purified from P. aeruginosa GN17203 harbouring the blaIMP-carrying plasmid pMS350. Cells were incubated at 4 °C for 1 h in 10 mM 3-(N-morpholino)propanesulphonic acid (MOPS) buffer (pH 7.0) containing 27% sucrose and 2 mg/mL lysozyme (Sigma Chemical Co.), and were then disrupted by sonication. The cellular debris was removed by centrifugation (16,000g, 15 min, 4°C), and the supernatant was precipitated in an 80% saturated concentration of ammonium sulphate. This fraction was dialysed against 10 mM MOPS buffer (pH 7.0) and applied to a CM-Sephadex C-50 column (Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with 10 mM MOPS buffer (pH 7.0). The enzyme was eluted with a linear NaCl gradient. The active fractions were pooled, dialysed against 10 mM MOPS buffer (pH 7.0) containing 1 µM ZnCl2 and concentrated by ultrafiltration with a UK-10 ultra filter (Advantec Toyo Co., Tokyo, Japan). The purity of the preparation was checked by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and the purified enzyme solution was stored at –80°C.

The CcrA metallo-ß-lactamase was prepared from B. fragilis GAI30079. Cells were suspended in 50 mM sodium phosphate buffer (pH 7.0) and disrupted by sonication. Cellular debris was removed by centrifugation (13,500g, 15 min, 4°C) and the supernatant was dialysed against 50 mM phosphate buffer (pH 7.8). This fraction was applied to a DEAE–Toyopearl 650M column (Toso Co., Tokyo, Japan) equilibrated with 50 mM phosphate buffer (pH 7.8) and the enzyme was eluted with a linear NaCl gradient. The pooled active fractions were dialysed against 50 mM phosphate buffer (pH 7.0), concentrated by ultrafiltration with a UK-10 ultrafilter and rechromatographed with a Sephadex G-100 column (Pharmacia Biotech AB) equilibrated with 50 mM phosphate buffer (pH 7.0). The concentrated crude enzyme preparation was dialysed against 50 mM phosphate buffer (pH 7.0) and stored at –80°C.

The L1 metallo-ß-lactamase was prepared from Stenotrophomonas maltophilia GN12873 as described previously.12 The Bacillus cereus type II metallo-ß-lactamase, Escherichia coli TEM-1 penicillinase and Enterobacter cloacae cephalosporinase were purchased from Sigma Chemical Co.

Determination of ß -lactamase activity

The activity at each step of metallo-ß-lactamase preparation was determined by monitoring the hydrolysis of 100 µM imipenem ({triangleup}{epsilon} = 9.04 mM–1 cm–1 at 299 nm) at 30°C in 10 mM MOPS buffer (pH 7.0) containing 100 µM of ZnCl2. One unit (U) of ß-lactamase activity was defined as the amount of enzyme that hydrolysed 1 µmol of imipenem in 1 min at 30°C.

Determination of IC 50

The 50% inhibitory concentration (IC50) for the IMP-1 metallo-ß-lactamase was determined by measuring the enzymatic hydrolysis of a chromogenic cephalosporin, nitrocefin, in the presence of inhibitors. This automated assay system was a modification of a previously reported method.13 To avoid identifying the metal chelators, 10 mM MOPS buffer (pH 7.0) containing 100 µM of ZnCl2 was used in this microassay. One microlitre of inhibitor and 25 µL of IMP-1 metallo-ß-lactamase (3–6 mU/mL) were mixed in a 98-well microplate, and the assay was initiated within 1 min by the rapid addition of 75 µL of nitrocefin solution to create a final nitrocefin concentration of 72.7 µM. Inhibitors were dissolved in 10 mM MOPS buffer (pH 7.0) or dimethyl sulphoxide (DMSO) and prepared at final concentrations of 0.1, 1 and 10 µM. If needed, a concentration of >100 µM was prepared. Control wells contained reaction mixture without inhibitor, enzyme or substrate. Assay plates were incubated with slow shaking in an M-36 microincubator (Taitec Co., Tokyo, Japan) at 30°C, and the hydrolysis of nitrocefin was determined 15 min after incubation by monitoring the increase in absorbance at 492 nm in an MTP-120 plate reader (Corona Electric Co., Ibaraki, Japan). Three wells were prepared at each inhibitor concentration; the mean values were used for determining IC50s. The initial rates of hydrolysis at each inhibitor concentration were calculated, and the IC50s (µM) were determined by plotting percentage inhibition against inhibitor concentration.

ß -Lactamase assay

Kinetic studies were performed at 30°C in 10 mM MOPS buffer (pH 7.0) and the hydrolysis of the substrate, imipenem and cephaloridine ({triangleup}{epsilon} = 10.2 mM–1 cm–1 at 260 nm), by metallo- and serine-ß-lactamases, respectively, was monitored in a temperature-controlled spectrophotometer, UV-2200 (Shimadzu, Tokyo, Japan). The initial hydrolysis velocity of J-111,225 ({triangleup}{epsilon} = 9.82 mM–1 cm–1 at 298 nm) at a concentration of 100 µM was determined, and was calculated relative to that of imipenem and cephaloridine for metallo- and serine-ß-lactamases, respectively. Kinetic parameters were derived from at least two independent experiments from a Hanes–Woolf plot of the initial velocity of substrate hydrolysis by ß-lactamases. To calculate the Ki values of the inhibitors from a Dixon plot, rates of substrate hydrolysis at concentrations ranging from 10 to 100 µM were determined in the presence of various concentrations of inhibitors. The total reaction volume was 1 mL in all cases.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility

Table IGo presents the antimicrobial activities of J-111,225 and reference antibiotics against metallo-ß-lactamase producers. The IMP-1 metallo-ß-lactamase producers, S. marcescens and P. aeruginosa, were highly resistant to the marketed carbapenems, such as imipenem (MICs from 16 to >128 mg/L) and meropenem (MICs from 32 to >128 mg/L), and to ceftazidime (MICs from 128 to >128 mg/L). The MICs of J-111,225 were four- to eight-fold lower (MICs 4–32 mg/L) than imipenem and meropenem for the IMP-1 metallo-ß-lactamase producers, S. marcescens and P. aeruginosa. Piperacillin showed lower MICs for some of the IMP-1 metallo-ß-lactamase producers and MICs of aztreonam were lowest, but high-level resistance was observed in some isolates. The metallo-ß-lactamase producers tested were often resistant to other classes of antibiotics such as amikacin and ofloxacin, indicating other coexistent resistance mechanisms.


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Table I. Susceptibility of IMP-1 metallo-ß-lactamase producers to J-111,225 and reference antibiotics
 
Inhibitory activity against IMP-1 metallo ß -lactamase

J-111,225 showed inhibitory potential with an IC50 value of 0.7 µM. The IC50s of the side chain of J-111,225, meropenem and panipenem were >10, 25 and >100 µM, respectively. Metal chelators such as dipicolinic acids14 and EDTA showed IC50s ranging from 80 to 200 µM. Aztreonam15 had a low affinity for the IMP-1-enzyme, with an IC50 of >100 µM. The comparative hydrolysis rates of aztreonam and J-111,225 were <0.01 and 4, respectively, taking the hydrolysis rate of imipenem as 100.

As shown in Figure 2Go, the Ki of J-111,225 against the IMP-1 metallo-ß-lactamase was determined to be 0.18 µM from a Dixon plot. The Ki of the cis-counterpart at the C5 position of J-111,225 was 0.12 µM, while the inhibitory activity was markedly reduced by inversion of the C3 chiral centre (IC50 > 10 µM) regardless of C5 stereochemistry.



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Figure 2. Dixon's plot for determination of inhibition of IMP-1 metallo-ß-lactamase by J-111,225. Imipenem was used as the substrate at the indicated concentrations.

 
The stability of J-111,225 in the presence of various types of ß-lactamases and its affinity for them are presented in Table IIGo. In contrast to its inhibitory activity against IMP-1 metallo-ß-lactamases, J-111,225 was the substrate for chromosomal metallo-ß-lactamases of B. fragilis, S. maltophilia and B. cereus with Km values of 11, 10 and 148 µM, respectively. Like imipenem,16,17 J-111,225 was stable against hydrolysis by serine-ß-lactamases of class A (TEM-1 type penicillinase) and class C (chromosomal cephalosporinase from E. cloacae) with Ki values in the micromolar range.


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Table II. Stability in the presence of, and affinity for, various types of ß-lactamases J-111,225 Imipenem
 
Combination effect in vitro

Table IIIGo presents the combination effect of J-111,225 and imipenem against IMP-1 metallo-ß-lactamase producers of S. marcescens BB5886 and P. aeruginosa GN17203. As expected, synergy (FIC <=0.5) was observed between the antibacterial activities of J-111,225 and imipenem.


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Table III. Effects of imipenem and J-111,225 alone and in combination against metallo-ß-lactamase producers
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We found that J-111,225 is resistant to hydrolysis by a transferable IMP-1 metallo-ß-lactamase. J-111,225 is an inhibitor with a Ki of 0.18 µM when imipenem is used as a substrate. Its inhibitory activity was demonstrated in a whole-cell FIC assay which indicated synergy between the two carbapenems, J-111,225 (inhibitor) and imipenem (substrate) against IMP-1-producing S. marcescens and P. aeruginosa. Our studies on the structure and inhibitory activity against the IMP-1 metallo-ß-lactamase indicated that the side chain of J-111,225 itself and the C5 stereochemistry of the pyrrolidine ring do not appear to contribute to the affinity for the IMP-1 enzyme, while C3 stereochemistry is important for the inhibitory activity. In addition, the C3/C5 stereochemistry of the pyrrolidine ring has previously been reported to influence antibacterial activities against methicillin-resistant staphylococci and P. aeruginosa.13

Recent studies on X-ray crystal structures of metallo-ß-lactamases such as B. cereus type II,18,19 Ccr20–22 and L123 enzymes and an enzymatic study on IMP-124 have revealed that there are two zinc atoms at the active site. The occupancy of the zinc atoms and the amino acid alignment vary between these metallo-ß-lactamases and seem to affect the catalytic activities of each enzyme. The fact that J-111,225 is an inhibitor of IMP-1 but a substrate for chromosomal metallo-ß-lactamases such as CcrA, L1 and type II might be ascribed to diversity among the metallo-ß-lactamases.

Thus, J-111,225 is a novel carbapenem antibiotic exhibiting inhibitory activity against the transferable IMP-1 metallo-ß-lactamase as compared with the marketed carbapenems such as imipenem, meropenem and panipenem (available only in Japan). Recent reports on the appearance of the blaIMP-carrying organisms in South Korea7 and Europe8 in addition to Japan have indicated that precautions are needed to prevent further spread of the metallo-ß-lactamases, which are a threat to antimicrobial chemotherapy with ß-lactam antibiotics including carbapenems. Previous reports on metallo-ß-lactamase inhibitors mainly described their inhibitory properties against chromosomal metallo-ß-lactamases.22,25–28 It is important to take steps to cope with the organisms carrying the blaIMP gene which codes for a resistance mechanism that can be transferred among the enteric bacteria and glucosenon-fermentative Gram-negative rods including pseudomonads.


    Notes
 
* Corresponding author. Tel: +81-298-77-2000; Fax: +81-298-77-2029; E-mail: naganori{at}banyu.co.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 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.[ISI][Medline]

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

3 . Watanabe, M., Iyobe, S., Inoue, M. & Mitsuhashi, S. (1991). Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, 147–51.[ISI][Medline]

4 . Ito, H., Arakawa, Y., Ohsuka, S., Wacharotayankun, R., Kato, N. & Ohta, M. (1995). Plasmid-mediated dissemination of the metallo-ß-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrobial Agents and Chemotherapy 39, 824–9.[Abstract]

5 . Senda, K., Arakawa, Y., Ichiyama, S., Nakashima, K., Ito, H., Ohsuka, S. et al. (1996). PCR detection of metallo-ß-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum ß-lactams. Journal of Clinical Microbiology 34, 2909–13.[Abstract]

6 . Arakawa, Y., Murakami, M., Suzuki, K., Ito, H., Wacharotayankun, R., Ohsuka, S. et al. (1995). A novel integron-like element carrying the metallo-ß-lactamase gene blaIMP. Antimicrobial Agents and Chemotherapy 39, 1612–15.[Abstract]

7 . Lee, K., Chong, Y., Shin, H. B. & Yong, D. (1998). Rapid increase of imipenem-hydrolyzing Pseudomonas aeruginosa in a Korean hospital. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract E-85, p.193. American Society for Microbiology, Washington DC.

8 . Cornaglia, G., Riccio, M. L., Mazzariol, A., Lauretti, L., Fontana, R. & Rossolini, G. M. (1999). Appearance of IMP-1 metallo-ß- lactamase in Europe. Lancet 353, 899–900.[ISI][Medline]

9 . Livermore, D. M. (1997). Acquired carbapenemases. Journal of Antimicrobial Chemotherapy 39, 673–6.[Free Full Text]

10 . Shimizu, A., Sugimoto, Y., Sakuraba, S., Imamura, H., Sato, H., Ohtake, R. et al. (1998). Novel trans-3,5-disubsituted pyrrolidinylthio 1ß-methylcarbapenems with potent activity against MRSA and Pseudomonas aeruginosa. In Program and Abstracts of the Thirty-Eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract F-052, p. 246. American Society for Microbiology, Washington DC.

11 . Elion, G. B., Singer, S. & Hitchings, H. (1954). Antagonists of nucleic acid derivatives; synergism in combination of biochemically related antimetabolites. Journal of Biological Chemistry 208, 477–88.[Free Full Text]

12 . Saino, Y., Kobayashi, F., Inoue, M. & Mitsuhashi, S. (1982). Purification and properties of inducible penicillin ß-lactamase isolated from Pseudomonas maltophilia. Antimicrobial Agents and Chemotherapy 22, 564–70.[ISI][Medline]

13 . Payne, D. J., Coleman, K. & Cramp, R. (1991). The automated in-vitro assessment of ß-lactamase inhibitors. Journal of Antimicrobial Chemotherapy 28, 775–6.[ISI][Medline]

14 . Yang, D., Roll, D. M., Wildey, M. J., Lee, M., Greenstein, W. M., Maiese, W. M. et al. (1994). Inhibition of metallo-ß-lactamase by LL-10G568{alpha} and LL-10G568ß. In Program and Abstracts of the Thirty-Fourth Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, FL. Abstract C-056, p. 88. American Society for Microbiology, Washington DC.

15 . Bush, K., Freudenberger, J. S. & Sykes, R. B. (1982). Interaction of aztreonam and related monobactams with ß-lactamases from gram-negative bacteria. Antimicrobial Agents and Chemotherapy 22, 414–20.[ISI][Medline]

16 . Labia, R., Morand, A., Tiwari, K., Sirot, D. & Chanal, C. (1989). Interactions of meropenem with ß-lactamases, including enzymes with extended-spectrum activity against third-generation cephalosporins. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 219–23.[ISI][Medline]

17 . Sotto, A., Brunschwig, C., O'Callaghan, D., Ramuz, M. & Jourdan, J. (1996). In-vitro evaluation of ß-lactamase inhibition by latamoxef and imipenem. Journal of Antimicrobial Chemotherapy 37, 697–701.[Abstract]

18 . Carfi, A., Dunee, E., Galleni, M., Frere, J. M. & Dideberg, O. (1997). Metallo-ß-lactamase from Bacillus cereus 569h9. Brookhaven Protein Data Bank entry 1BME.

19 . Fabiane, S. M., Sohi, M. K., Wan, T., Payne, D. J., Bateson, J. H., Mitchell, T. et al. (1998). Crystal structure of the zinc-dependent ß-lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzyme. Biochemistry 37, 12404–11.[ISI][Medline]

20 . Concha, N. O., Rasmussen, B. A., Bush, K. & Herzberg, O. (1996). Crystal structure of the wide-spectrum binuclear zinc ß- lactamase from Bacteroides fragilis. Structure 4, 823–36.[ISI][Medline]

21 . Fitzgerald, P. M., Wu, J. K. & Toney, J. H. (1998). Unanticipated inhibition of the metallo-ß-lactamase from Bacteroides fragilis by 4-morpholineethanesulfonic acid (MES): a crystallographic study at 1.85-Å resolution. Biochemistry 37, 6791–800.[ISI][Medline]

22 . Toney, J. H., Fitzgerald, P. M., Grover-Sharma, N., Olson, S. H., May, W. J., Sundelof, J. G. et al. (1998). Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-ß-lactamase. Chemistry and Biology 5, 185–96.[ISI][Medline]

23 . Ullah, J. H., Walsh, T. R., Taylor, I. A., Emery, D. C., Verma, C. S., Gamblin, S. J., et al. (1998). The crystal structure of the L1 metallo-ß-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution. Journal of Molecular Biology 284, 125–36.[ISI][Medline]

24 . Laraki, N., Franceschini, N., Rossolini, G. M., Santucci, P., Meunier, C., de Pauw, E. et al. (1999). Biochemical characterization of the Pseudomonas aeruginosa 101/1477 metallo-ß-lactamase IMP-1 produced by Escherichia coli. Antimicrobial Agents and Chemotherapy 43, 902–6.[Abstract/Free Full Text]

25 . Gilpin, M. L., Fulston, M., Payne, D., Cramp, R. & Hood, I. (1995). Isolation and structure determination of two novel phenazines from a Streptomyces with inhibitory activity against metallo-enzymes, including metallo-ß-lactamase. Journal of Antibiotics 48, 1081–5.[ISI][Medline]

26 . Keynan, S., Hooper, N. M., Felici, A., Amicosante, G. & Turner, A. J. (1995). The renal membrane dipeptidase (dehydropeptidase I) inhibitor, cilastatin, inhibits the bacterial metallo-ß-lactamase enzyme CphA. Antimicrobial Agents and Chemotherapy 39, 1629–31.[Abstract]

27 . Payne, D. J., Bateson, J. H., Gasson, B. C., Proctor, D., Khushi, T., Farmer, T. H. et al. (1997). Inhibition of metallo-ß-lactamases by a series of mercaptoacetic acid thiol ester derivatives. Antimicrobial Agents and Chemotherapy 41, 135–40.[Abstract]

28 . Walter, M. W., Felici, A., Galleni, M., Soto, R. P., Adlington, R. M., Baldwin, J. E. et al. (1996). Trifluoromethyl alcohol and ketone inhibitors of metallo-ß-lactamases. Bioorganic and Medicinal Chemistry Letters 6, 2455–8.

Received 19 July 1999; returned 1 October 1999; revised 4 November 1999; accepted 22 November 1999