Original articles. Rie Nagano*, Yuka Adachi, Terutaka Hashizume and Hajime Morishima

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1 Journal of Antimicrobial Chemotherapy (2000) 45, Original articles JAC In vitro antibacterial activity and mechanism of action of J-111,225, a novel 1 -methylcarbapenem, against transferable IMP-1 metallo- -lactamase producers Rie Nagano*, Yuka Adachi, Terutaka Hashizume and Hajime Morishima Banyu Tsukuba Research Institute, Okubo 3, Tsukuba , Japan IMP-1 -lactamase, a class B zinc metallo-enzyme encoded by the transferable bla IMP gene, is known to confer high-level resistance to carbapenems as well as to penicillins and cephalosporins. J-111,225 is a novel 1 -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- -lactamases. In kinetic studies, J-111,225 inhibited the IMP-I enzyme with a K i of 0.18 M when imipenem was used as a substrate. In contrast, J-111,225 was the substrate for hydrolysis by other class B -lactamases such as Bacteroides fragilis CcrA, Stenotrophomonas maltophilia L1 and Bacillus cereus type II enzyme with respective K m values of 11, 10 and 148 M. The greater antibacterial activity of J-111,225 against IMP-1-producing bacteria may result from its unique interaction with the -lactamase. Introduction Carbapenems such as imipenem and meropenem are potent broad-spectrum antibiotics with good stability against bacterial -lactamases of molecular classes A and C. 1 However, zinc metallo- -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 aerugi - nosa strain that produced a plasmid-mediated metallo- - 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- 6 -lactamase gene, bla IMP. Recently, IMP-1-producing organisms have been reported in South Korea 7 and Europe. 8 A concern is that the bla IMP gene can be transferred among Gram-negative bacteria, and there is no effective inhibitor to date. Thus, the spread of metallo- - lactamases is a threat to antimicrobial chemotherapy with -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- -lactamase-producing organisms. In this paper, we describe the interaction of J-111,225 with -lactamases. Materials and methods 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 1). Imipenem and amikacin were the products of Banyu Pharmaceutical Co. Ltd. The following drugs were obtained commercially: ceftazidime (Tanabe *Corresponding author. Tel: ; Fax: ; naganori@banyu.co.jp 2000 The British Society for Antimicrobial Chemotherapy 271

2 R. Nagano et al. 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. 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). Bacterial strains Clinical isolates that produced IMP-1 metallo- -lactamase were collected from various districts in Japan since 1994, and the bla IMP gene was detected by PCR. 5 P. aeruginosa GN17203, which harbours the bla IMP -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 10 7 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 10 5 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 -Lactamase preparation The IMP-1 metallo- -lactamase was purified from P. aeru - ginosa GN17203 harbouring the bla IMP -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 M ZnCl 2 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- -lactamase was prepared from B. fragilis GAI 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- -lactamase was prepared from Steno - trophomonas maltophilia GN12873 as described previously. 12 The Bacillus cereus type II metallo- -lactamase, Escherichia coli TEM-1 penicillinase and Enterobacter cloacae cephalosporinase were purchased from Sigma Chemical Co. Determination of -lactamase activity The activity at each step of metallo- -lactamase preparation was determined by monitoring the hydrolysis of 100 M imipenem ( 9.04 mm 1 cm 1 at 299 nm) at 30 C in 10 mm MOPS buffer (ph 7.0) containing 100 M of ZnCl 2. One unit (U) of -lactamase activity was defined as the amount of enzyme that hydrolysed 1 mol of imipenem in 1 min at 30 C. 272

3 IMP-1 -lactamase inhibition by J-111,225 Determination of IC 50 The 50% inhibitory concentration (IC 50 ) for the IMP-1 metallo- -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 M of ZnCl 2 was used in this microassay. One microlitre of inhibitor and 25 L of IMP-1 metallo- -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 L of nitrocefin solution to create a final nitrocefin concentration of 72.7 M. 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 M. If needed, a concentration of 100 M 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 IC 50 s. The initial rates of hydrolysis at each inhibitor concentration were calculated, and the IC 50 s ( M) were determined by plotting percentage inhibition against inhibitor concentration. -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 ( 10.2 mm 1 cm 1 at 260 nm), by metallo- and serine- -lactamases, respectively, was Figure 2. Dixon s plot for determination of inhibition of IMP-1 metallo- -lactamase by J-111,225. Imipenem was used as the substrate at the indicated concentrations. monitored in a temperature-controlled spectrophotometer, UV-2200 (Shimadzu, Tokyo, Japan). The initial hydrolysis velocity of J-111,225 ( 9.82 mm 1 cm 1 at 298 nm) at a concentration of 100 M was determined, and was calculated relative to that of imipenem and cephaloridine for metallo- and serine- -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 -lactamases. To calculate the K i values of the inhibitors from a Dixon plot, rates of substrate hydrolysis at concentrations ranging from 10 to 100 M were determined in the presence of various concentrations of inhibitors. The total reaction volume was 1 ml in all cases. Results Susceptibility Table I presents the antimicrobial activities of J-111,225 and reference antibiotics against metallo- -lactamase producers. The IMP-1 metallo- -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- -lactamase producers, S. marcescens and P. aeruginosa. Piperacillin showed lower MICs for some of the IMP-1 metallo- -lactamase producers and MICs of aztreonam were lowest, but high-level resistance was observed in some isolates. The metallo- -lactamase producers tested were often resistant to other classes of antibiotics such as amikacin and ofloxacin, indicating other coexistent resistance mechanisms. Inhibitory activity against IMP-1 metallo- -lactamase J-111,225 showed inhibitory potential with an IC 50 value of 0.7 M. The IC 50 s of the side chain of J-111,225, meropenem and panipenem were 10, 25 and 100 M, respectively. Metal chelators such as dipicolinic acids 14 and EDTA showed IC 50 s ranging from 80 to 200 M. Aztreonam 15 had a low affinity for the IMP-1-enzyme, with an IC 50 of 100 M. 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 2, the K i of J-111,225 against the IMP-1 metallo- -lactamase was determined to be 0.18 M from a Dixon plot. The K i of the cis-counterpart at the C5 position of J-111,225 was 0.12 M, while the inhibitory activity was markedly reduced by inversion of the C3 chiral centre (IC M) regardless of C5 stereochemistry. The stability of J-111,225 in the presence of various types of -lactamases and its affinity for them are presented in 273

4 R. Nagano et al. Table II. In contrast to its inhibitory activity against IMP-1 m e t a l l o - -lactamases, J-111,225 was the substrate for chromosomal metallo- -lactamases of B. fragilis, S. malto - philia a n d B. cereus with K m values of 11, 10 and 148 M, respectively. Like imipenem, 1 6, 1 7 J-111,225 was stable against hydrolysis by serine- -lactamases of class A (TEM-1 type penicillinase) and class C (chromosomal cephalosporinase from E. cloacae) with K i values in the micromolar range. Combination effect in vitro Table III presents the combination effect of J-111,225 and imipenem against IMP-1 metallo- -lactamase producers of S. marcescens BB5886 and P. aeruginosa GN As expected, synergy (FIC 0.5) was observed between the antibacterial activities of J-111,225 and imipenem. Table I. Susceptibility of IMP-1 metallo- -lactamase producers to J-111,225 and reference antibiotics MIC (mg/l) b IMP-1-producing organism a J-111,225 imipenem meropenem ceftazidime piperacillin aztreonam amikacin ofloxacin S. marcescens BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB BB P. aeruginosa BB GN a bla IMP gene was detected by PCR. b Broth microdilution method. Table II. Stability in the presence of, and affinity for, various types of -lactamases J-111,225 Imipenem -Lactamase hydrolysis b K m b K i hydrolysis K m K i (original host) (relative %) a ( M) ( M) (relative %) a ( M) ( M) IMP-1 (P. aeruginosa) CcrA (B. fragilis) L1 (S. maltophilia) Type II (B. cereus) Class A, TEM-1 (E. coli) Class C (E. cloacae) a Taking the hydrolosis rate of imipenem and cephaloridine as 100% for metallo- and serine- -lactamases, respectively. b Imipenem and cephaloridine were used as the substrate for metallo- and serine- -lactamases, respectively. 274

5 IMP-1 -lactamase inhibition by J-111,225 Table III. Effects of imipenem and J-111,225 alone and in combination against metallo- -lactamase producers MIC (mg/l) a imipenem J-111,225 Strain with J-111,225 alone with imipenem alone FIC index b S. marcescens BB P. aeruginosa GN a Chequerboard method. b FIC index FIC imipenem FIC J-111,225 (MIC imipenem combine )/(MIC imipenem alone ) (MIC J-111,225 combine )/(MIC J-111,225 alone ). A FIC index of 0.5 indicates synergy; 1.0; additivity; 2.0, indifference, antagonism. Discussion We found that J-111,225 is resistant to hydrolysis by a transferable IMP-1 metallo- -lactamase. J-111,225 is an inhibitor with a K i of 0.18 M 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- -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- - lactamases such as B. cereus type II, 18,19 Ccr and L1 23 enzymes and an enzymatic study on IMP-1 24 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- -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- -lactamases such as CcrA, L1 and type II might be ascribed to diversity among the metallo- - lactamases. Thus, J-111,225 is a novel carbapenem antibiotic exhibiting inhibitory activity against the transferable IMP-1 m e t a l l o - -lactamase as compared with the marketed carbapenems such as imipenem, meropenem and panipenem (available only in Japan). Recent reports on the appearance of the bla IMP -carrying organisms in South Korea 7 and Europe 8 in addition to Japan have indicated that precautions are needed to prevent further spread of the metallo- -lactamases, which are a threat to antimicrobial chemotherapy with -lactam antibiotics including carbapenems. Previous reports on metallo- -lactamase inhibitors mainly described their inhibitory properties against chromosomal metallo- -lactamases. 22,25 28 It is important to take steps to cope with the organisms carrying the bla IMP gene which codes for a resistance mechanism that can be transferred among the enteric bacteria and glucosenon-fermentative Gram-negative rods including pseudomonads. References 1. Ambler, R. P. (1980). The structure of -lactamases. Philosophical Transactions of the Royal Society of London, Series B: Biological Sciences 289, Rasmussen, B. A. & Bush, K. (1997). Carbapenem-hydrolyzing -lactamases. Antimicrobial Agents and Chemotherapy 41, Watanabe, M., Iyobe, S., Inoue, M. & Mitsuhashi, S. (1991). Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 35, Ito, H., Arakawa, Y., Ohsuka, S., Wacharotayankun, R., Kato, N. & Ohta, M. (1995). Plasmid-mediated dissemination of the metallo- -lactamase gene bla IMP among clinically isolated strains of Serratia marcescens. Antimicrobial Agents and Chemotherapy 39, Senda, K., Arakawa, Y., Ichiyama, S., Nakashima, K., Ito, H., Ohsuka, S. et al. (1996). PCR detection of metallo- -lactamase gene (bla IMP ) in gram-negative rods resistant to broad-spectrum -lactams. Journal of Clinical Microbiology 34, Arakawa, Y., Murakami, M., Suzuki, K., Ito, H., Wacharotayankun, R., Ohsuka, S. et al. (1995). A novel integron-like element carrying the metallo- -lactamase gene bla IMP. Antimicrobial Agents and Chemotherapy 39, 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. 275

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