Role of the 7a-Methoxy and Side-Chain Carboxyl of

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1981, p /81/1-1/7$./ Vol. 19, No. 1 Role of the 7a-Methoxy and Side-Chain Carboxyl of Moxalactam in,8-lactamase Stability and Antibacterial Activity KAZUHISA MURAKAMI* AND TADASHI YOSHIDA Shionogi Research Laboratories, Shionogi & Co., Ltd., Fukushima-ku, Osaka 553, Japan The effects of the a-carboxyl of the phenyinalonyl side chain and the 7amethoxy group in moxalactam (659-S) {7,8-[-carboxy--(4-hydroxyphenyl) acetamido]-7a-methoxy-3-[[(1-methyl-lh-tetrazol-5-yl)thio] methyl]-1-oxa-1-dethia-3-cephem-4-carboxylic acid) and in the 1-sulfur congener on the stability to fl-lactamase were investigated by spectrophotometric and microbiological assays. The 7a-methoxy substituent stabilized the compounds against penicillinase hydrolysis, and the a-carboxyl group stabilized them against cephalosporinase. An exception is the f6-lactamase produced by Proteus vulgaris, an inducible cephalosporinase, which hydrolyzed compounds having the a-carboxyl group but not those having the 7a-methoxy group. Both substituents exerted their stabilizing effects independently, and compounds with both substituents, e.g., moxalactam (659-S) and its 1-sulfur congener, were resistant to both penicillinases and cephalosporinases. The stabilization of the compounds to,b-lactamase hydrolysis improved their antibacterial activity against 8-lactamase-producing strains. Bacterial resistance to 83-lactam antibiotics, penicillins and cephalosporins, can be ascribed to one or more of the following: (i) production of B-lactamase (EC ), (ii) existence of a permeability barrier in the outer membrane, and (iii) alteration of the properties of the target enzymes (1). Among them, the production of,8-lactamase is thought to be the main mechanism of resistance (14,, 7). Although the,blactamases produced by gram-negative bacteria are diverse with respect to their physicochemical and enzymological properties, they can be classified according to their substrate specificity as follows: (i) cephalosporinases, predominantly active against cephalosporins, (ii) penicillinases, predominantly active against penicillins, and (iii) an intermediate type, equally active against both cephalosporins and pencillins (7). To enhance the antibacterial activity of,8- lactam antibiotics against resistant strains, two approaches have been employed in the past two decades. One was to seek an agent which inhibits Bl-lactamase activity (15, ). Such inhibitors were expected to have a synergistic effect on resistant strains when used together with other /1-lactam antibiotics sensitive to,8-lactamase hydrolysis. The second approach to this problem was to create fl-lactam antibiotics resistant to B8-lactamase hydrolysis (4, 17, 18). There has been great interest in structures which stabilize antibiotics against /8-lactamases (, 6, 15), and among them the cephamycins produced by 1 [[(1-methyl-1H-tetrazol-5-yl) Streptomyces sp. and their derivatives were shown to be extremely stable due to the 7amethoxy substituent present (1, 18, 6). 1-Oxacephalosporins have decreased stability against,8-lactamases compared with the 1-sulfur congeners, although they have enhanced intrinsic antibacterial activity (K. Murakami, M. Takasuka, K. Motokawa, and T. Yoshida, J. Med. Chem., in press). However, the 1-oxacephalosporin of moxalactam (659-S) (7,--[-carboxy-- (4-hydroxyphenyl)acetamido]-7a-methoxy-3- thio]-methyl]-loxa-l-dethia-3-cephem-4-carboxylic acid) (13), was demonstrated to be very stable to every type of fl-lactamase (31). In this study, the effects of the 7a-methoxy substituent and of the a-carboxyl group of the phenylmalonyl moiety in moxalactam on stability to ft-lactamases were investigated. (Part of this study was reported at the Royal Society Meeting, Penicillin-5 years from Fleming, in London [3]). MATERIALS AND METHODS P-Lactam compounds. All compounds except cephaloridine, cephalexin, and penicillin G were synthesized in Shionogi Research Laboratories (M. Narisada, T. Yoshida, M. Ohtani, K. Ezumi, and M. Takasuka, manuscript in preparation). The structures of the compounds related to moxalactam (659-S) are shown in Fig. 1 and Table 1. Cephaloridine, cephalexin, and penicillin G were obtained from commercial sources. Downloaded from on December 15, 18 by guest

2 MURAKAMI AND YOSHIDA CH~~CCONH8 N-N E X CHSStt NA COONa CH3 FIG. 1. Substitutions of cephalosporin used in the present study. TABLE 1. Structure of /-lactam compounds and differences in their extinction coefficients upon hydrolysis Structure' Waveno. X length Compound Aeb 1 O H H 7 5, S H H 75 7,5 3 H OCH3 7 8,8 4 S H OCH3 75 7,4 5 COONa H 7 5, 6 S COONa H 75 7,7 7 (659-S) COONa OCH3 7 9,7 8 S COONa OCH3 76 6,8 asee Fig. 1. bdifference in extinction coefficient of f.-lactam compound upon hydrolysis. Bacterial strains, The cultures of Escherichia coli W311 carrying R-plasmid RTEM (3), Enterobacter cloacae 53 (8), and E. cloacae 14 (5) were kindly supplied by M. H. Richmond (University of Bristol, Bristol, England), and cultures of E. coli ML141 carrying R-plasmid RGN38 (9) and Klebsiella pneumoniae GN69 (5) were from T. Sawai (Chiba University, Chiba, Japan). E. coli NIHJ JC- was a laboratory standard strain. Other strains were clinical isolates that had been selected for this study on the basis of their resistance to one or more commercially available fi-lactam antibiotics and their production of fb-lactamase. Of these bacterial strains, E. coli ML141 (RGN38), E. coli W311 (RTEM), and E. coli 73 produced an R-plasmid,B-lactamase. The substrate profiles for ampicillin, cephaloridine, and cefazolin of f8-lactamases produced by the bacterial strains described above were reported previously (31). Preparation of crude extracts. The medium used for fl-lactamase preparations was nutrient phosphate broth (Nissui, Tokyo, Japan). Overnight cultures of bacteria which produced inducible,8-lactamase were diluted 1-fold with fresh medium and incubated at 37 C for h with shaking. Penicillin G was added as an inducer to give a final concentration of 1 mg/nil for Pseudomonas aeruginosa and 1 ytg/ ml for the other bacteria. Incubations were continued for h. Bacteria which produced constitutive fl-lactamase were grown without shaking overnight at 37C. After incubation, the cells were harvested by centrifugation at 3, x g for 15 min at 4 C, washed once, and suspended in.1 M potassium phosphate buffer (ph 7.). The bacteria were sonically disrupted with a Sonicator-15 (Ohtake, Tokyo, Japan) in an ice bath. Cell debris was removed by centrifugation at 33, ANTIMICROB. AGENTS CHEMOTHER. x g for 3 min at 4 C, and the supernatant was filtered through a membrane filter (. um; Millipore Corp.) and stored at -78 C. Purification of fi-lactamase. The,B-lactamases produced by E. coli W311 (RTEM) and Klebsiella sp. 363 were partially purified by column chromatography. Crude extracts were dialyzed against.1 M NaHPO4-KHPO4 (ph 8.) and loaded onto a diethylaminoethyl-sephadex A-5 column equilibrated against the same buffer. A gradient was constructed by dropping the buffer (.5 M, ph 6.)) into a mixing chamber containing 18 mi, of the buffer (.1 M, ph 8.) Ṫhe f,-lactamases from E. coli 6, E. cloacae 14, E. cloacae 53, Proteus vulgaris 31, and E. coli ML141 (RGN38) were partially purified with carboxymethyl-sephadex C-5 as described previously (5). Active fractions were pooled, dialyzed against.1 M potassium phosphate buffer (ph 7.), and stored at -78 C until used. Assay of /8-lactamase. Potassium phosphate buffer (.1 M, ph 7.) was used throughout the,blactamase assay. (i) Microbiological assay. The microbiological assay method was described previously (3). Substrate solutions of.5 ml (5 ug/ml) were added to.5 ml of a twofold dilution of the enzyme on a microtiter plate (Cooke Engineering Co.). After incubation at 37C for h, residual antibiotic activity was determined by the agar well assay using E. coli B. The relative hydrolysis rate of cephaloridine was set at a value of 1, and those of the other compounds were calculated from the ratios of the enzyme concentrations at which 5% of the compounds added were inactivated. (ii) Spectrophotometric assay. The spectrophotometric assay method was based on the decrease of ultraviolet absorption of the,8-lactam ring upon hydrolysis (9, 16). The extinction coefficient for each f,- lactam was obtained from differential spectra before and after complete hydrolysis (Table 1). The hydrolysis was carried out at 3 C with. N NaOH ( equivalents) for 6 min and 1 min for compounds 6 and 8, respectively. The hydrolysis of compound 7 (moxalactam, 659-S) was described previously (31). The other,b-lactam compounds were hydrolyzed by partially purified,8-lactamase preparations. The enzyme preparations were diluted with buffer containing.1% gelatin. The enzyme solution (. ml) was added to ml of the buffer containing the substrate at 3 C, and the reaction mixture was incubated in a euvette in a spectrophotometer (Hitachi model -) with circulation of water at 3C. The hydrolysis rate of the,b-lactam ring was calculated from the decrease in optical density. The kinetic parameters V. and Km were calculated from Lineweaver-Burk plots (11), and K, was obtained from plots of apparent Km against concentrations of an inhibitor. Susceptibility test. Minimum inhibitory concentration was determined by the agar dilution method with sensitivity test agar (Eiken, Tokyo, Japan). Overnight cultures of bacteria in trypto-soy broth (Eiken, Tokyo, Japan) were diluted to about 16 cells per ml with the same broth, and.5-il samples of the bacterial suspension were inoculated with a multiloop inoculat- Downloaded from on December 15, 18 by guest

3 VOL. 19, 1981 ing device onto plating agar containing serial twofold dilutions of the compound. All cultures were incubated at 37 C for 18 to h. The minimum inhibitory concentration of the compound was defined as the lowest concentration that inhibited visible growth. RESULTS The stability to 16-lactamase of the 1-oxacephalosporin analogs (Fig. 1 and Table 1) was measured by the microbiological method. The importance of the -7a-methoxy substituent and the a-carboxyl group of the p-hydroxyphenylmalonyl side chain was demonstrated in Table. Compound 1 was easily hydrolyzed by every type of 83-lactamase. Compound 3, having the 7a-methoxy substituent, was completely stable against the penicillinases tested, but hydrolysis was observed with some of the cephalosporinases. On the other hand, compound 5, having the phenylmalonylamino side chain, was not hydrolyzed by the cephalosporinases, except for the one from P. vulgaris, whereas it was hydrolyzed by the penicillinases. Although the f3-lactamase produced by P. vulgaris was classified as an inducible cephalosporinase, it could not hydrolyze compound 3, but it could hydrolyze compound 5. The,8-lactamase from Proteus inconstans, which was also a cephalosporinase, hydrolyzed neither compound 3 nor 5 at a detectable rate. STABILIZATION OF 1-OXACEPHEM TO /l-lactamase None of the enzymes hydrolyzed both compounds 3 and 5. Both of the substituents played independent roles to protect the molecule against fl-lactamases. Consequently, compound 7 (moxalactam, 659-S) was very stable to every type of enzyme. As far as examined, there were no 8-lactamases which could hydrolyze 7 at a detectable rate. To elucidate the effects of the two types of substituents upon the interaction of the 83-lactams with,8-lactamases in more detail, the kinetic parameters Vmax, Ki, and Ki were determined by spectrophotometric assay with three cephalosporinases, from E. coli 6, E. cloacae 14, and P. vulgaris 31, and three penicillinases, from E. coli W311 (RTEM), E. cloacae 53, and Klebsiella sp. 363, which were partially purified. The effects of both substituents, 7a-methoxy and a-carboxyl, on the Vmax values of 1-oxacephems were essentially the same as those in Table (Table 3). Furthermore, the substituents also exhibited similar effects on 1-sulfur congeners. The a-carboxyl group exclusively protected the 83-lactam ring from the cephalosporinases, but had little effect on penicillinase activity, as indicated by Vmax ratios between the compounds substituted with a-carboxyl and the nonsubstituted counterparts (5/1 and 6/ in Table 4). In fact, the reduction of Vmax values was only 1 to 57% for penicillinases and the P. vulgaris en- TABLE. Effects of substituents upon stability to /3-lactamase Relative hydrolysis rate of: Sub-,8-Lactamase source' strate Comspecific- Compound pound 3 Compound 7 copu poundr7 ityh 1 (7a-meth- 5 (x-car- boxyl-7aoxy) methoxy) Escherichia coli 6 C <1 <1 Proteus morganii 8 C 1 3 <1 <1 Proteus inconstans 31 C 11 <1 <1 <1 Enterobacter aerogenes 1 C 5 15 <1 <1 Enterobacter cloacae 9 C 3 <1 <1 Serratia marcescens HIG C 6 <1 <1 Citrobacter freundii 7 C 14 5 <1 <1 Pseudomonas aeruginosa 3 C 87 <1 <1 Proteus vulgaris 31 C 49 <1 14 <1 Escherichia coli W311 (RTEM)d P 35 <1 18 <1 Klebsiella pneumoniae GN69 P 1 <1 76 <1 Klebsiella sp. 363 P 37 <1 87 <1 Enterobacter cloacae 53 P 1 <1 71 <1 Escherichia coli ML141 (RGN38)d P 87 <3 6 <3 a Partially purified enzyme preparations were used for E. coli W311 (RTEM), E. coli ML141 (RGN38), and E. cloacae 53, and crude extracts were used for the other bacterial strains. hc and P indicate cephalosporinase and penicillinase, respectively. The substrate profiles for ampicillin, cephaloridine, and cefazolin were previously reported (31). 'Hydrolysis rate was determined by microbiological assay and is expressed relative to an arbitrary value of 1 for cephaloridine. d fl-lactamase produced was mediated by an R-plasmid. 3 Downloaded from on December 15, 18 by guest

4 _> 4 MURAKAMI AND YOSHIDA ANTIMICROB. AGENTS CHEMOTHER. TABLE 4. Stabilizing effect of substituents against E- f3-lactamase *5.E eq eq Ratios of V,,,,,' A A Source of fl-lactamase 3/1 4/ 5/1 6/ v v vv v v Escherichia coli Nil Nil o- co a4) 4)V o Enterobacter cloacae.79.9 Nil Nil 14 Proteus vulgaris 31 Nil Nil.44.1 of A Escherichia coli W311 Nil Nil (RTEM) a,e Enterobacter cloacae 53 Nil Nil v v vv v v 16 4) Klebsiella sp. 363 Nil Nil.5.57 a -C E- = Calculated from the Vmax data shown in Table 3. co Compound numbers are as given in Table 1. o to 4 C6 CO6 4)VQ* A Lt w, > I 6 P- U' :r cn _ LO - ulc v.o Ca4 zyme, and in the case of E. cloacae 53 enzyme even a twofold increase of Vmax was seen. Thus, la Q 4) ~ the protective function of the a-carboxyl seemed 6 to be restricted solely to cephalosporinases except for the P. vulgaris enzyme. On the other -Zu 8. I\ A CO P.) S ~ hand, the 7a-methoxy substitution completely Co E, 4) - CO 46) blocked the activity of penicillinases and the P. _ o t3 a11 vulgaris enzyme and concomitantly led to a C4 C.).9.8 decrease of the hydrolysis rate with cephalosporinases (Table 4). Accordingly, the protective I _ C ~ * VV eq he o ~4ie function of the 7a-methoxy was not greatly affected by the type of f8-lactamase, although the rt E. c...t- n..g V V CO v v substituent stabilized the 83-lactam ring preferentially against penicillinases and P. vulgaris C.)- - eqo 4 enzyme. Furthermore, the stabilization against Q cephalosporinases by 7a-methoxy was more efficient for cephalosporin than for its 1-oxa con- o V V o C gener, as shown by the values of 3/1 and 4/ in cs t)-4 C C-O _4 Table 4. In r F any case, the hydrolysis of L. cephalosporin was slower than that of the _ eq.q a 4)4 QH corresponding _-- t- H* 1ME 1-oxa congener (Table 3). 4 C3 cq. The 7a-methoxy substituent increased the affinity of the compounds for the P. vulgaris CIDO enzyme to some extent (low value of Ks), whereas,ai coi it had no extreme effects upon the affinity to the other enzymes (Table 3). On the other hand, the.a.a- 4) a-carboxyl group always gave the compounds an Fe increased affinity for the cephalosporinases (low Q. ~! values of Ki in Table 3). Conversely, this substitution decreased the affinity of the,8-lactams for - Go the penicillinases and the P. vulgaris enzyme sr U.m (high value of Km in Table 3). > x ca >, x d: Minimum inhibitory concentrations of the cjaz Co LO compounds against 83-lactamase-producing and g co w zd nonproducing strains were determined by the Q co aa agar dilution method (Table 5). The nonproducing strain, E. coli NIHJ JC-, was susceptible CO. co C e to all of the compounds. The cephalosporinaseproducing strain, E. cloacae 33, was susceptible '64 to the analogs having the a-carboxyl group; the penicillinase-producing strains, Klebsiella sp. Downloaded from on December 15, 18 by guest

5 VOL. 19, 1981 STABILIZATION OF 1-OXACEPHEM TO fl-lactamase 5 TABLE 5. Minimum inhibitory concentrations of /8-lactam compounds,8-lacta- Minimum inhibitory concn (,ug/ml) of Organismmae compound': Organmmase Escherichia coli NIHJ JC- -c Enterobacter cloacae 33 C >1 >1 1 > Klebsiella sp. 363 P > Escherichia coli 73 P > a Substrate specificity of f8-lactamases produced are expressed as C and P for cephalosporinase and penicillinase, b respectively. Compound numbers are as given in Table 1. fi8-lactamase was not produced. 363 and E. coli 73, were susceptible to the compounds substituted with the 7a-methoxy group. Thus, the analogs having both substituents, compounds 7 (moxalactam, 659-S) and 8, were active against all strains. Clear correlation between minimum inhibitory concentration and stability to /3-lactamases was observed. Furthermore, 1-oxacephalosporins had stronger antibacterial activities than did the corresponding 1-sulfur congeners when the compounds were not hydrolyzed by bacterial,8-lactamase. DISCUSSION Substituents at the a-position of the acylamino side chain of penicillins and cephalosporins are known to play important roles in changing the antibacterial spectrum against gram-positive and -negative bacteria (19, 8). This study demonstrated that the a-carboxyl group of the phenylmalonylamino moiety of the C-7 side chain protected the amide bond of /3-lactam from cephalosporinase hydrolysis (Tables and 3). The compounds having the a-carboxyl group showed very high affinity for cephalosporinases (low values of Ki shown in Table 3), except for the P. vulgaris enzyme. This fact suggested that the negatively charged a-carboxyl substituent exerted a protective effect by strong interaction with an amino group or positive charge in the active site of the cephalosporinase. On the other hand, the affinity of the compounds for penicillinases and the P. vulgaris enzyme was decreased by the introduction of the a-carboxyl group, indicating that there were no such interactions, and, as a consequence, the a-carboxyl did not stabilize the compound against these enzymes. 7a-Methoxycephalosporins, cephamycins, found in fermentation broth have been shown to be resistant to 83-lactamase hydrolysis (1, 6). Onishi et al. reported that cephamycin C was hydrolyzed by 8 out of 91 fl-lactamases, and cefoxitin, a derivative of cephamycin, was hydrolyzed by 7 out of 91 enzymes (18). However, the correlation between the ability of a ft-lactamase to hydrolyze 7a-methoxycephalosporins and the type of enzyme had not been clarified. We were able to correlate this for the first time in the present study. Tables and 3 clearly show that only cephalosporinases, except for the P. vulgaris enzyme, were capable of hydrolyzing the 7a-methoxy derivatives. Thus, 7a-methoxy substitution is sufficient for the protection of the,8-lactam rings against penicillinases, but not against cephalosporinases. The 7a-methoxy substituent had less effect on the affinity for the fl-lactamases, except for the P. vulgaris enzyme, than did an a-carboxyl group for cephalosporinases produced by E. coli 6 and E. cloacae 14 (Table 3). This fact indicates that the 7a-methoxy is not so important for the interaction between the compound and,8-lactamase as a-carboxyl group. Indelicato and Wilham reported that the 6a-substituent in penicillins provided steric hindrance to nucleophilic attack of 8-lactams in free solution and that, in contrast, the 7a-methoxy substituent in cephalosporins had no such steric effect (7). The nucleophilic attack by an amino acid residue in the active site of the enzyme can be considered to be much more sterically restricted. Indeed, on the basis of the three-dimensional structure of the fl-lactam compound, Boyd suggested that the 8-lactam ring was attacked at its a-face in the active site of the enzyme (1). Thus even 7amethoxy in cephalosporins seems to provide steric hindrance to penicillinases and, to a lesser extent, to cephalosporinases in the active site. This is in agreement with the observations that 7a-methoxy stabilized the,8-lactam against both types of 18-lactamase, completely against penicillinases, and partially against cephalosporinases, in contrast with the a-carboxyl, which protected the compound exclusively against cephalosporinases (Tables 3 and 4). The cephalosporinases from P. inconstans 31 and P. vulgaris 31 were exceptional in their behavior in the substitution effect (Tables and 3). For the enzyme from P. inconstans 31, enzymic hydrolysis of compound 3 might have occurred, but too slowly to be detected. On the Downloaded from on December 15, 18 by guest

6 6 MURAKAMI AND YOSHIDA other hand, the enzyme from P. vulgaris 31 was an exception. P. vulgaris is closely related taxonomically to P. mirabilis (1), which is the only chromosomal penicillinase-producing species in the genus Proteus (4). Indeed, the cephalosporinase from P. vulgaris showed a substrate profile with properties somewhat similar to those of penicilhinase. Thus, the P. vulgaris enzyme might be considered an intermediate between a pencillinase and a cephalosporinase. All f-lactamases, except the one from P. inconstans, were found to hydrolyze either 7amethoxy or a-carboxyl derivatives. No enzymes hydrolyzed both compounds (Tables and 3). Thus, fi-lactamases can be divided into two groups with respect to the substitution effect, and enzymes in the same group appear to be similar in their other properties. Enzymes of one group, i.e., those which could not hydrolyze malonylamido compounds, are predominantly active against cephalosporins and remarkably inhibited by cloxacillin. Enzymes of the other group, i.e., those which could not hydrolyze 7amethoxy compounds, are predominantly active against penicillins (the P. vulgaris enzyme is an exception) and poorly inhibited by cloxacillin. These two groups of,b-lactamases may have different origins. There is an obvious correlation between,blactamase stability of the compounds and their antibacterial activity (Table 5). Since f8-lactamases mediated by an R-plasmid or produced by Klebsiella sp. are penicillinases, the 7a-methoxy substitution is effective for killing these bacteria in spite of,b-lactamase activity. Since chromosomal fi-lactamases from many gram-negative species are cephalosporinases, a-carboxyl substitution in the side chain is in turn effective for killing these bacteria. Both functional groups work together for complete protection from /Blactamase attack and contribute to the extensive improvement of the antibacterial activity. Consequently, compound 7, moxalactam (659-S), has an expanded antibacterial spectrum as reported previously (31). The 1-oxacephalosporins are generally more hydrolyzable by,-lactamases than are the corresponding 1-sulfur congeners (Table 3). This can be ascribed to the enhancement of the reactivity of the,f-lactam rings by 1-oxa replacement (Murakami et al., in press). In connection with this, the enhanced reactivity might explain the lesser effectiveness of steric hindrance of the 7amethoxy in 1-oxa congeners upon cephalosporinases (Table 4). Furthermore, the enhancement of the fl-lactam reactivity might contribute to the stronger antibacterial activities of 1-oxacephalosporins (Table 5). The results presented in this paper imply that ANTIMICROB. AGENTS CHEMOTHER. one of the main problems in bacterial resistance, production of f8-lactamase, has been solved by introduction of both a 7a-methoxy group and a side-chain carboxyl into 1-oxacephalosporins and cephalosporins. ACKNOWLEDGMENTS We are grateful to K. Motokawa and M. Doi for their technical assistance. Thanks are also due to W. Nagata for his helpful discussion and encouragement throughout this work. LITERATURE CITED 1. Boyd, D. B Transition state structures of a dipeptide related to the mode of action offl-lactam antibiotics. Proc. Natl. Acad. Sci. U.S.A. 74: Crompton, B., M. Jago, K. Crawford, G. G. F. Newton, and E. P. Abraham Behaviour of some derivatives of 7-aminocephalosporanic acid and 6-aminopenicillanic acid as substrates, inhibitors, and inducers of penicillinases. Biochem. J. 83: Datta, N., and M. H. Richmond The purification and properties of a penicillinase whose synthesis is mediated by an R-factor in Escherichia coli. Biochem. J. 98: Fu. K. P., and H. C. Neu Beta-lactamase stability of HR756, a novel cephalosporin, compared to that of cefuroxime and cefoxitin. Antimicrob. Agents Chemother. 14: Hennessey, T. D., and M. H. Richmond The purification and some properties of a fi-lactamase (cephalosporinase) synthesized by Enterobacter cloacae. Biochem. J. 19: Hou, J. P., and J. W. Poole Kinetics of /8-lactamase inactivation of penicillins I: effect of side-chain structure, ionic strength, ph, and temperature. J. Pharm. Sci. 6: Indelicato, J. M., and W. L. Wilham Effect of 6- a substitution in penicillins and 7-a substitution in cephalosporins upon f-lactam reactivity. J. Med. Chem. 17: Jack, G. W., and M. H. Richmond A comparative study of eight distinct f?-lactamases synthesized by gram-negative bacteria. J. Gen. Microbiol. 61: Jansson, J. A. T A direct spectrophotometric assay for penicillin,-lactamase (penicillinase). Biochim. Biophys. Acta 99: Lautrop, H Genus X. Proteus Hauser 1885, 1, p In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 11. Lineweaver, H., and D. Burk The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: Nagarajan, R., L. D. Boeck, M. Gorman, R. L. Hamill, C. E. Higgens, M. M. Hoehn, W. M. Stark, and J. G. Whitney ,8-Lactam antibiotics from Streptomyces. J. Am. Chem. Soc. 93: Narisada, M., T. Yoshida, H. Onoue, M. Ohtani, T. Okada, T. Tsuji, I. Kikkawa, N. Haga, H. Satoh, H. Itani, and W. Nagata Synthetic studies on f?- lactam antibiotics. Part 1. Synthesis of 7fi-[-carboxy- - (4-hydroxyphenyl)acetamido]-7a-methoxy-3-[[(1- methyl- 1H-tetrazol-5-yl)thio]-methyl]-1-oxa- 1-dethia- 3-cephem-4-carboxylic acid disodium salt (659-S) and its related 1-oxacephems. J. Med. Chem.; : Neu, H. C The role of,b-lactamases in the resistance of Gram-negative bacteria to penicillin and cephalosporin derivatives. Infect. Dis. Rev. 3: O'Callaghan, C. H., P. W. Muggleton, S. M. Kirby, and D. M. 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7 VOL. 19? 1981 cephalosporins, p Antimicrob. Agents Chemother O'Callaghan, C. H., P. W. Muggleton, and G. W. Ross Effects of B-lactamase from gram-negative organisms on cephalosporins and penicillins, p Antimicrob. Agents Chemother O'Callaghan, C. IL, R. B. Sykes, D. M. Ryan, R. D. Foord, and P. W. Muggleton Cefuroxime-a new cephalosporin antibiotic. J. Antibiot. 9: Onishi, H. R., D. R. Daoust, S. B. Zimmerman, D. Hendlin, and E.. Stapley Cefoxitin, a semisynthetic cephamycin antibiotic: resistance to beta-lactamase inactivation. Antimicrob. Agents Chemother. 5: Price, K. E Structure-activity relationships of semisynthetic penicillins, p In D. Perlman (ed.), Structure-activity relationships among the semisynthetic antibiotics. Academic Press, Inc., New York.. Reading, C., and M. Cole Clavulanic acid: a betalactamase-inhibiting beta-lactam from Streptomyces clavuligerus. Antimicrob. Agents Chemother. 11: Richmond, M. H Factors influencing the antibacterial action of,b-lactam antibiotics. J. Antimicrob. Chemother. 4(Suppl. B): Richmond, M. H., and R. B. Sykes The,B-lactamases of Gram-negative bacteria and their possible physiological role. Adv. Microbiol. Physiol. 9: Ross, G. W., K. V. Chanter, A. M. Harris, S. M. Kirby, M. J. Marshall, and C. H. O'Callaghan Comparison of assay techniques for fb-lactamase activity. Anal. Biochem. 54: Sawai, T., S. Mitauhashi, and S. Yamagishi Drug resistance of enteric bacteria. XIV. Comparison of fl-lactamases in Gram-negative rod bacteria resistant STABILIZATION OF 1-OXACEPHEM TO fi-lactamase to a-aminobenzylpenicillin. Jpn. J. Microbiol. 1: Sawai, T. S., Yamagishi, and S. Mitsuhashi Penicillinases of Klebsiella pneumoniae and their phylogenetic relationship to penicillinases mediated by R factors. J. Bacteriol. 115: Stapley, E. O., M. Jackson, S. Hernandez, S. B. Zimmerman, S. A. Currie, S. Mochales, J. M. Mata, H. B. Woodruff, and D. Hendlin Cephamycins, a new family of,8-lactam antibiotics. I. Production by Actinomycetes, including Streptomyces lactamdurans sp. n. Antimicrob. Agents Chemother. : Sykes, R. B., and M. Matthew The #t-lactamases of Gram-negative bacteria and their role in resistance to B-lactam antibiotics. J. Antimicrob. Chemother. : Webber, J. A., and J. L. Ott Structure-activity relationships in the cephalosporins. II. Recent developments, p In D. Perlman (ed.), Structureactivity relationships among the semisynthetic antibiotics. Academic Press, Inc., New York. 9. Yamagishi, S., K. O'Hara, T. Sawai, and S. Mitsuhashi The purification and properties of penicillinf,-lactamases mediated by transiissible R factors in Escherichia coli. J. Biochem. 66: Yoshida, T Structural requirements for antibacterial activity and fl-lactamase stability of 7fl-arylmalonylamino-7a-methoxy-1-oxacephems. Philos. Trans. R. Soc. London Ser. B 89: Yoshida, T., S. Matsuura, M. Mayama, Y. Kameda, and S. Kuwahara Moxalactam (659-S), a novel 1-oxa-f,-lactam with an expanded antibacterial spectrum: laboratory evaluation. Antimicrob. Agents Chemother. 17: Downloaded from on December 15, 18 by guest