Vol. 34, No. 2
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 349-354
0066-4804/90/020349-06$02.00/0 Copyright © 1990, American Society for Microbiology
In Vitro Investigation of BK-218, a New Oral and Parenteral Cephalosporin SZAB6,1* J. BARABAS,1 A. TAR,2 L. KISS,3 M. FILEP,4 T.
SCHMIDT,4 K. MAROSSY, B. TOTH-MARTINEZ,2 GY. BARABAS,1 AND F. HERNADI2 Institute of Biology1 and Department of Chemotherapy, Institute of Pharmacology,2 University Medical School, and Institute of Biochemistry, L. Kossuth University,3 H4012 Debrecen, and BIOGAL Pharmaceutical Works, H4042 Debrecen,4 Hungary
I.
Received 2 June 1989/Accepted 26 October 1989 The antibacterial activity of BK-218 was similar to that of cefamandole when it was tested against several laboratory strains. The inhibiting effect of BK-218 was greater than that of cephalexin and cefoxitin on penicillin-binding proteins of Escherichia col HB101. This result was in close correlation with the relative inhibition of radiolabeled glucosamine incorporation (greatest with BK-218) and with the lytic effect (most intensive with BK-218). BK-218 proved to be a good inhibitor for all five of the ,-lactamases that were investigated, although two enzymes (Enterobacter cloacae P99 and Pseudomonas aeruginosa Cilote) hydrolyzed it to some extent.
Bacterial strains for susceptibility testing. OKI-marked standard strains of bacteria were obtained from the Hungarian National Collection of Medical Bacteria, Budapest. Enterobacter cloacae P99 was kindly supplied by I. N. Simpson, Glaxo Group Research, Greenford, United Kingdom; methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates were supplied by F. Rozgonyi, Institute of Microbiology, University Medical School, Debrecen, Hungary; and E. coli DCO and DC2 were supplied by Elmar Schrimmer, Abteilung fur Chemoterapie, Hoechst Aktiengesellschaft, Frankfurt am Main, Federal Republic of Germany. Antimicrobial susceptibility tests. Antimicrobial activity was measured by the standard agar dilution method (20) with Mueller-Hinton agar (Oxoid Ltd., Basingstoke, United Kingdom). Mueller-Hinton agar was supplemented with 5% sheep blood when the nonenterococcal streptococci were examined. Susceptibility of the methicillin-resistant S. aureus was determined on Mueller-Hinton agar supplemented with 2% NaCl. The medium for preculture was nutrient broth (Oxoid). From a diluted fresh overnight culture, 105 CFU was applied to the agar plate with a replicatory spot device. Broth macrodilution tests were made in calcium- and magnesium-supplemented Mueller-Hinton broth with an inoculum of about 105 CFU/ml. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth in agar or in broth after 18 h of incubation at 37°C. P-Lactamases and carrier strains. The enzymes used in the kinetic studies were purified from known strains as follows. Bacillus cereus 569/H/9 and S. aureus PC1 1711E ,B-lactamases were prepared with N-acetyl-D-penicillamine-Sepharose 4B affinity chromatography by the method of Clarke et al. (10), as modified in our laboratory (15). B. cereus 569/H/9 was kindly provided by S. Fleming, Department of Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom; S. aureus PC1 1711E was a gift from I. N. Simpson; Pseudomonas aeruginosa Cilote was an isolate from A. M. Philippon, Service de Microbiologie, Centre Hospitalier Universitaire Cochin, Paris, France, and its CARB-3-type enzyme (17) was isolated also by affinity
Cephalexin (4), cefadroxil (7), cefaclor (4, 21), cefroxadine (34), and cefatrizine (22) have been widely used for clinical purposes as oral cephalosporins, but their usage is limited because they have relatively narrow spectra against various strains of pathogenic bacteria and are hydrolyzed by 1lactamases. BK-218, 5-thia-1-azabicyclo[4,2,0]oct-2-en,-7[(3 - chloro - izoxazol - 5 - yl) - acetamido] - 3[[(1 - methyl) - 1Htetrazol - 5 - yl]thiomethyl] - 8 - oxo - 2 - carboxylic acid sodium salt (Fig. 1.), is a new oral cephalosporin. It is well-known that beta-lactam antibiotics first bind to their targets, the so-called penicillin-binding proteins (PBPs). The inhibition of these enzymes suspends peptidoglycan synthesis, and as a consequence the autolytic enzymes are triggered with a mechanism whose details are not completely understood (16). The purpose of this study was to report the results of preliminary investigations concerning the in vitro activity of BK-218 against a limited number of gram-positive and gramnegative bacteria. The MICs of BK-218 were compared with those of other parenteral (cefuroxime, cefamandole, and cefoxitin) and oral (cephalexin) antibiotics. The in vitro effect on the targets was measured by (i) affinity to PBPs, (ii) incorporation of labeled glucosamine into the cell wall of Escherichia coli, and (iii) release of labeled glucosamine-containing fragments from prelabeled cell wall of E. coli to assess the lysis capability of BK-218. The susceptibility of BK-218 to hydrolysis by 1-lactamases was also investigated. MATERIALS AND METHODS Antibiotics. BK-218 was synthesized at the Chemical Research Laboratory of BIOGAL Pharmaceutical Works, Debrecen, Hungary. The other compounds were obtained as follows: cefuroxime, Glaxo Research Ltd., Greenford, United Kingdom; cefoxitin, Merck Sharp & Dohme, Paris, France; and cephalexin and cefamandole, Eli Lilly & Co., Geneva, Switzerland. Solutions of antimicrobial agents were prepared on the day of use as directed by the manufacturers. *
Corresponding author. 349
350
SZAB6
ANTIMICROB. AGENTS CHEMOTHER.
ET AL.
C
2H20
o
CS NN N;CH2 I COONa CH3
FIG. 1. Chemical structure of BK-218.
chromatography on m-aminophenylboronic acid-agarose (hydrophobic spacer arm, type B) (9). Chromatofocusing was used to purify 1-lactamases of E. coli RTEM+ and E. cloacae P99. The sources of these strains are described in detail elsewhere (30, 32). Isolation of cytoplasmic membrane from Enterococcus faecalis ATCC 9790. Cytoplasmic membranes from E. faecalis were prepared as described by Coyette et al. (11) and were put in 1 mM phosphate buffer (pH 7) which contained 1 mM MgCl2. The protein content of membrane preparations was adjusted to 20 mg/ml. Isolation of cytoplasmic membrane from E. coli HB101 (3). Exponentially growing cells of E. coli were harvested by centrifugation at 6,000 x g for 20 min. The cells were suspended in 10 mM phosphate buffer (pH 7) which contained 0.14 M mercaptoethanol. After 3 min of ultrasonication (1.5 W; MSE, Ltd., London, United Kingdom), the intact cells were removed by centrifugation at 6,000 x g for 20 min. The supernatant was spun down at 36,000 x g for 30 min to isolate the membrane. The membrane was resuspended in 10 mM phosphate buffer (pH 7) and washed twice with the same buffer and in the same volume. Labeling of PBPs with radioactive penicillin. Membrane samples (containing about 200 ,ug of protein) were preincubated with different concentrations of cephalosporins for 10 min at 37°C and then incubated with 50 ,uM [3H]benzylpenicillin (specific activity, 773.3 GBq/mmol; Institute of Isotopes, Hungarian Academy of Sciences, Budapest) in a total volume of 21 p.l at 37°C for 10 min and subsequently supplemented with excess nonradioactive benzylpenicillin (BIOGAL Pharmaceutical Works) to a final concentration of 10 mM in a 5-pA volume. One microliter of 20% Sarkosyl-NL 97 was added to the membrane preparations of E. coli. Subsequently, they were incubated for 20 min at room temperature. The supernatant obtained at 40,000 x g for 30 min contained the inner-membrane fraction. Denaturing buffer (25 p.J) containing 10% (wt/vol) glycerol, 0.5 M Tris hydrochloride, 2% (wt/vol) sodium dodecyl sulfate (SDS), 0.01% (wt/vol) bromophenol blue, and 10% (vol/vol) 2mercaptoethanol was added. The suspension was boiled for 2 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis and fluorography. SDS-polyacrylamide gel electrophoresis was done as described by Laemmli and Favre (19) with acrylamide contents of 4 and 10% (wt/vol of each) in the stacking and running gels, respectively. After separation of the proteins at a constant 360 V, the gels were fixed in a solution containing 5% (vol/vol) methanol and 7.5% (vol/vol) acetic acid. Gels were placed in dimethyl sulfoxide to remove water, followed by 2 h of incubation in a 20% (wt/vol) solution of 2,5diphenyloxazole in dimethyl sulfoxide. Following rehydration, gels were dried and exposed to Medifort RP X-ray film for 7 to 10 days at -70°C (5). The band intensities were measured by densitometry (Kipp and Zonen densitometer
DD2). The half-saturation cephalosporin concentration (I50 value) for PBPs was calculated from the optical density of a cephalosporin-inhibited PBP band and from the optical density of the control band which was not treated with betalactam antibiotics. Incorporation of labeled glucosamine into cell wall of E. coli HB101. A 500-,ul sample of an exponentially growing culture (optical density, 0.5 at 550 nm) was pretreated with 3.8 x 10-5 M cephalosporin for 10 min (at 100 rpm and 37°C) in a rotatory shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.). After the addition of 9 kBq of N-acetyl-D-[1-3H]glucosamine (specific activity, 105 GBq/mmol; Amersham International plc, Amersham, United Kingdom), the culture was incubated for 15 min, the radioactivity was chased for 10 min with cold glucosamine (final concentration, 10 mM), and counts and disintegrations per minute retained in boiling SDS-precipitable material were measured (filtered on Millipore membrane; pore size, 0.45 ,um; Millipore Corp., Bedford, Mass.). The final concentration of SDS was 5%, and each precipitate was washed with 10 ml of the same SDS solution. The radioactivity was measured with a scintillation counter (Isocap/300; Nuclear-Chicago Corp., Des Plaines, Ill.). Release of cell wall fragments from E. coli HB101. NAcetyl-D-[1-3H]glucosamine (55.5 kBq) (specific activity, 105 GBq/mmol) was added to 3 ml of an exponentially growing culture of E. coli at 37°C (optical density, 0.5). After 90 min of incubation at 37°C and 200 rpm in a rotatory shaker (New Brunswick Scientific), the culture was chased with cold glucosamine. The cells were washed three times with fresh medium, centrifuged at 10,000 x g for 5 min, and suspended in a fresh culture medium containing 3.8 x 10-5 M antibiotic. The cultivation was continued under the same conditions as before. Samples were taken at different times, and the radioactivity of the supernatant (centrifugation at 10,000 x g for 5 min) was measured with the same scintillation counter as described above. Determination of Km, Vmax, and Vrel values. The kinetic experiments were done in a final volume of 3 ml of incubation mixture at 37°C in 0.05 M phosphate buffer, pH 7 (23, 25, 33). In the case of significant hydrolysis of some of the cephalosporins susceptible to certain 3-lactamases, evaluation of the kinetic parameters was made in the 0.5- to 5.0Km substrate concentration interval (33). Assessment of Ki values for enzymes. The inhibition studies were done under conditions similar to those described above. The enzyme samples were preincubated for 5 min, and the reaction was started with nitrocefin (Oxoid) as the substrate in each mixture (Km values were 56 p.M for P99 [33], 136 ,uM for TEM-1 [23], 15 p.M for the Cilote enzyme, 5 ,uM for PC1, and 0.1 ,uM for ,-lactamase I from B. cereus 569/H/9; unpublished data). The results were evaluated for two substrate concentrations (20 and 30 p,M) and for five inhibitor concentrations in the 0.5- to 5.0-Ki interval. 1BLactamase activity was assayed by measuring the A486 of nitrocefin in a cell of 1-cm pathlength, as described by O'Callaghan et al. (23). One unit is that amount of enzyme which is able to hydrolyze 1 ,umol of nitrocefin in 1 min at 37°C. Evaluation of the Ki values was made by the method of Dixon with a simple computer program.
351
IN VITRO INVESTIGATION OF BK-218, A NEW CEPHALOSPORIN
VOL. 34, 1990
TABLE 1. Comparative in vitro activity of BK-218, cefuroxime, cefamandole, cefoxitin, and cephalexin against gram-positive and gram-negative bacteria MIC (,ug/ml) Organism
Cefuroxime
Cefamandole
Cefoxitin
Cephalexin
s0.12 64 -O.12 0.5 -O.12 32 64 0.5 s0.12 s0.12
0.5 >128 1.0 4.0 1.0 >128 >128 0.25 128 2.0 s0.12 -O.12
2.0 >128 2.0 4.0 2.0 >128 32 1.0 0.5 0.25
2.0 >128 2.0 16.0 1.0 >128 >128 4.0 0.5 s0.12
0.25 s0.12 2.0 0.25 8.0 0.25 0.5
4.0 0.25 4.0 .0.12 4.0 4.0 2.0 2.0 4.0 >128 4.0 2.0 64 1.0 64 0.12 2.0 >128 >128 1.0 >128 >128
0.5 s0.12 1.0 0.12 4.0 0.5 0.5 0.25 1.0 >128 1.0 0.5 64 0.5 >128 0.12 1.0 64 >128 4.0 >128 >128
4.0 0.5 2.0 1.0 2.0 8.0 4.0 2.0 4.0 128 128 2.0 16 4.0 8.0 0.5 4.0 >128 >128 8.0 >128 128
8.0 4.0 8.0 4.0 8.0 8.0 16.0 4.0 8.0 >128 128 2.0 >128 32 >128 2.0 16.0 >128 >128 >128 >128 >128
BK-218
Gram-positive Staphylococcus aureus ATCC 25923 Staphylococcus aureus 4916 (methicillin resistant) Staphylococcus aureus 4916 (methicillin susceptible) Staphylococcus aureus NCTC 9789 (P-lactamase producing) Staphylococcus epidermidis OKI 110001 Enterococcus faecalis OKI 80171 Enterococcus faecalis ATCC 9790 Streptococcus pneumoniae OKI 84503 Streptococcus pyogenes OKI 80001 Micrococcus sp. strain OKI 117001 Gram-negative Escherichia coli HB101 Escherichia coli OKI 35034 Escherichia coli DCO Escherichia coli DC2 Yersinia enterocolitica OKI 98001 Shigellaflexneri OKI 20119 Shigella sonnei OKI 20045 Salmonella typhi OKI 10084 Enterobacter cloacae OKI 53001 Enterobacter cloacae P99 Citrobacterfreundii OKI 47001 Klebsiella pneumoniae ATCC 1200 Serratia marcescens OKI 56501 Proteus vulgaris OKI 60002 Morganella morganii OKI 63001 Providencia rettgeri OKI 65005 Proteus mirabilis OKI 61370 Bordetella bronchiseptica OKI 92003 Acinetobacter anitratus OKI 150001 Aeromonas hydrophila OKI 186001 Pseudomonas aeruginosa ATCC 27853 Xanthomonas maltophilia OKI 173006
.0.12 1.0 128 8.0 0.5 >128 2.0 >128 0.12 2.0 >128 >128 >128 >128 >128
BK-218 did not inhibit Bordetella bronchiseptica, Acine-
RESULTS Antibacterial activity. To assess the antibacterial spectrum of BK-218, we compared its activities against typical laboratory strains with those of cefuroxime, cefamandole, cefoxitin, and cephalexin (Table 1). BK-218 had excellent activity against gram-positive bacteria except methicillin-resistant S. aureus and E. faecalis. The MICs were lower than MICs of other beta-lactams. The MICs of BK-218 ranged from 0.12 to 8 ,ug/ml against most strains of the family Enterobacteriaceae, except cephalosporinase-producing E. cloacae, Serratia marcescens, and Morganella morganii strains.
tobacter anitratus, Aeromonas hydrophila, P. aeruginosa, or Xanthomonas maltophilia at 128 jig/ml. Affinity for PBPs of E. faecalis ATCC 9790 and E. coli HB101. The affinities for PBPs of BK-218, cefoxitin, and cephalexin were examined by measuring the competition of the investigated beta-lactam antibiotics with '4C-labeled penicillin G. Table 2 shows the results of the competition
quantitatively. BK-218, cefoxitin, and cephalexin had very high affinities for PBP 2 of E. faecalis (Table 2), but cefoxitin was a better inhibitor of PBP 1 than was BK-218. Cephalexin had mod-
TABLE 2. Iso values of BK-218, cefoxitin, and cephalexin for PBPs of E. faecalis ATCC 9790 and E. coli HB101 150' (mmol/ml) for PBP:
Organism and agent
E. faecalis BK-218
Cefoxitin Cephalexin E. coli BK-218
Cefoxitin Cephalexin a Data are means
+
1
2
3
4
5
6
0.3 ± 0.02 0.1 ± 0.02 24.7 + 3.50
0.3 ± 0.05 0.3 ± 0.04 0.6 ± 0.1
10.5 ± 2.0 50.7 ± 4.7 18.9 ± 1.5
12.2 + 1.9 >100 >100
ND ND ND
0.3 ± 0.04 20.6 ± 3.9 >100
0.3 ± 0.03 0.3 ± 0.03 >100
0.3 ± 0.03 >100 >100
0.63 ± 0.15 2.5 ± 0.1 15.0 ± 1.8
4.2 ± 0.6 6.0 ± 0.5 18.7 ± 3.2
standard deviations for three independent experiments. ND, Not determined.
5/6
3.5 ± 0.6 0.3 ± 0.04
>100
352
SZAB6
ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Comparison of Ki values of BK-218 with those of three laboratory-standard cephalosporins as measured with 3-lactamases of gram-negative and gram-positive origins
K; (,uM)
Source of enzyme
E. P. S. B. E.
l
25 Time [min]
40
FIG. 2. Release of labeled glucosamine from prelabeled cell wall of E. coli HB101.
erate affinity toward all the other PBPs of E. faecalis, including PBP 3. As shown in Table 2, BK-218 had higher or at least equal affinity for the lethal targets (PBP 1, PBP 2, and PBP 3) of E. coli (13) than cefoxitin and had significantly higher affinity than cephalexin. Incorporation of labeled glucosamine. An exponentially growing culture of E. coli HB101 was pretreated with 3.8 x 10' M cephalosporin. After 10 min of pretreatment, tritiated N-acetyl glucosamine (which incorporates predominantly into the cell wall; 12, 24) was added to the culture. Glucosamine incorporation was about 50% of the control (7,600 dpm) when BK-218 was added (3,600 dpm). Cefoxitin had less of an inhibitory effect (5,600 dpm) and cephalexin had essentially no effect (7,400 dpm) on the incorporation. Release of cell wall fragments. The cell wall of E. coli HB101 was labeled with [3H]glucosamine, after which the release of the radioactivity was measured under the influence of different cephalosporins. The control culture, which was not treated with any antibiotic, showed a low but continuous release of radioactivity because of the turnover of cell wall material (Fig. 2). The release of radioactivity was extensive in the culture treated with BK-218, while the effect of cefoxitin on release was moderate. Cephalexin did not cause significant release above that seen in untreated controls. P-Lactamase stability of BK-218. ,B-Lactamase stability of BK-218 was investigated with five beta-lactam-hydrolyzing enzymes. Two of the enzymes were of gram-positive-bacterial origin: S. aureus PC1 and B. cereus 569/H/9 P-lactamase I. The gram-negative strains and their enzymes were as follows: E. cloacae P99, P. aeruginosa Cilote CARB-3 type, and E. coli RTEM+ TEM-1. We found that only the P99 and Cilote enzymes were able to hydrolyze BK-218 to a significant extent (Vmax values, 130 and 100 ,umol/min, respectively), while BK-218 did not prove to be a substrate for the rest of the investigated enzymes (Vmax values, '10 nmol/min). To characterize these results, we correlated the data with the P-lactamase stabilities of cefuroxime, cefoxitin, and cephalexin. Of these
cloacae P99
aeruginosa Cilote aureus PC1 cereus 5691H/9 coli RTEM+
BK-218
Cefuroxime
80 160 7 40 670
1,200 5,000
35
290
3,500
Cefoxitin
Cephalexin
95 70 80 5,000 1,700
1,290 2,000
185 915
2,000
beta-lactams, only cephalexin was hydrolyzed by TEM-1 (120 ,umol/min), while its breakdown with the P99 enzyme was small (40 nmol/min). The compounds were not hydrolyzed by any other enzyme. Inhibition kinetic studies (Table 3) showed that BK-218 was the best inhibitor of PC1 and weakest against TEM-1. The standard cephalosporins were not as good inhibitors as BK-218 in most cases, except cefuroxime for P99. Cefoxitin and BK-218 inhibited P99 comparably, but BK-218 was less netive a%,tivv, npninqt araill3L Cilntf.
DISCUSSION BK-218 is a new cephalosporin antibiotic for oral use. It was very active against the 10 investigated gram-positive bacteria, except methicillin-resistant S. aureus and E. faecalis strains, and it showed good activity against numerous gram-negative laboratory strains, especially members of the Enterobacteriaceae. MICs of beta-lactam antibiotics alone do not elucidate characteristics such as binding to target enzymes, penetration of the outer membrane, effect of cell wall synthesis, and lysis. For example, the permeability barrier may prevent the beta-lactam from reaching the target enzyme even in the case of a compound which has high affinity for PBPs. The investigation of the latter by the use of isolated cytoplasmic membranes containing PBPs may help to clear up the problem. On the basis of these results, however, the rate of lysis and cell wall inhibition cannot be predicted, since several PBPs take part in the process. The inhibition of cell wall synthesis and the lysis of the cell wall can be measured by the use of labeled glucosamine which can incorporate into the peptidoglycan. In an investigation of the inhibition of PBPs of E. faecalis by BK-218, cefoxitin, and cephalexin, it was shown that BK-218 had lower affinity for PBP 1 than cefoxitin and higher affinity for PBP 3 than cefoxitin and cephalexin. PBP 3 is considered to be the major lethal target of E. faecalis (13). BK-218 was an excellent inhibitor of PBP 6, which is known to be a nonlethal target. As seen with carboxypeptidases of different strains, inactivation does not influence the MICs. BK-218 was very effective against numerous gram-negative strains. The effect of BK-218 on E. coli HB101 was investigated in detail. When the PBP-inhibiting effects of BK-218, cephalexin, and cefoxitin were compared, BK-218 proved to be the best inhibitor of the lethal targets of E. coli (28). The MIC (0.25 ,ug/ml) of BK-218 against E. coli HB101 is not significantly higher than the 150S for PBP 1 and PBP 2. This suggests that BK-218 may penetrate the outer membrane of E. coli well. The experiments on cell wall synthesis and lysis were done with concentrations of BK-218, cefoxitin, and cephal-
VOL. 34, 1990
IN VITRO INVESTIGATION OF BK-218, A NEW CEPHALOSPORIN
exin that were two magnitudes higher than the MIC of BK-218. This proved to be the optimal concentration (data obtained with other concentrations are not given here) for demonstrating the differences among the three investigated cephalosporins. This concentration promoted the quick and thus easily measurable inhibition of cell wall synthesis and lysis of labeled cell wall. The difference between the effects of BK-218 and cefoxitin on cell wall synthesis and lysis could not be predicted from the 150 values (Table 3) because it is not known how much the three lethal targets (PBP 1, PBP 2, and PBP 3) are involved quantitatively in these processes. We believe that the results given above on cell wall synthesis and lysis demonstrate more precisely the effect of beta-lactams on bacteria than do mere MICs and MBCs. BK-218 proved to be the best inhibitor of the S. aureus PC1 enzyme. Cefuroxime and cephalexin did not inhibit this enzyme significantly. Our results showed BK-218 to be a much better inhibitor of P-lactamase I from B. cereus 569/H/9 than the standard cephalosporins used in these experiments. Reference Ki data are available mainly for methicillin (14) and isoxazolyl penicillins (2) but not for cephalosporins, with the exception of cephalosporin C (1). Therefore, our values can be informative for research purposes. As to the Ki values of BK-218 compared with those of the standards for enzymes of gram-negative bacteria, one may observe a diverse picture. P99 was less inhibited by BK-218 than by cefuroxime and was inhibited to about the same extent as by cefoxitin and cephalexin. It is known that moxalactam is a good inhibitor of this enzyme (Ki, 62 nM; 8). The P. aeruginosa Cilote ,-lactamase was approximately equally inhibited by BK-218 and cefoxitin, but cefuroxime and cephalexin proved to be poor inhibitors. Comparative data from references show that cephaloridine (29) and cephalothin (3, 18) are not stable against P-lactamases of different P. aeruginosa strains and are also poor inhibitors. However, cefotaxime and ceftriaxone (31) are stable inhibitors for many enzymes of these strains. E. coli has not been extensively investigated, but the available Ki values are consistent with our findings (26). Ki values of cefuroxime (650 ,uM) and moxalactam (810 ,uM) (8) were found to be in the same range as that of BK-218. In sum, we regard BK-218 as a promising candidate for gram-negative-bacterial screening despite the fact that two investigated enzymes hydrolyzed it to some extent. LITERATURE CITED 1. Abraham, E. P., and G. G. F. Newton. 1956. Experiments on the degradation of cephalosporin C. Biochem. J. 62:658-665. 2. Abraham, E. P., and S. G. Waley. 1979. Beta-lactamases from Bacillus cereus, p. 311-338. In J. M. T. Hamilton-Miller and J. T. Smith (ed.), Beta-lactamases. Academic Press, Inc. (London), Ltd., London. 3. Berks, M., K. Redhead, and E. P. Abraham. 1982. Isolation and properties of an inducible and a constitutive beta-lactamase from Pseudomonas aeruginosa. J. Gen. Microbiol. 128:155-159. 4. Bill, N. J., and J. A. Washington II. 1977. Comparison of in vitro activity of cephalexin, cephadrine, and cefaclor. Antimicrob. Agents Chemother. 11:470-474. 5. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 6. Boyer, H. W., and D. Rouliand-Dunioix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472. 7. Buck, R. E., and K. E. Price. 1977. Cefadroxil, a new broadspectrum cephalosporin. Antimicrob. Agents Chemother. 11: 324-330.
353
8. Cartwright, S. J., and S. G. Waley. 1983. Beta-lactamase inhibitors, p. 341-382. In Medical research news, vol. 3, no. 4. John Wiley & Sons, Inc., New York. 9. Cartwright, S. J., and S. G. Waley. 1984. Purification of lactamases by affinity chromatography on phenylboronic acid agarose. Biochem. J. 221:505-512. 10. Clarke, A. J., P. S. F. Mezes, and T. Viswanatha. 1980. A rapid procedure for the purification of Bacillus cereus 569/H lactamase. J. Appl. Biochem. 2:183-189. 11. Coyette, J., J.-M. Ghuysen, F. Binot, P. Adriaens, B. Meesschaert, and H. Venderhaeghe. 1977. Interaction between ,Blactam antibiotics and isolated membranes of Streptococcus faecalis ATCC 9790. Eur. J. Biochem. 75:231-239. 12. De Boer, W. R., F. J. Kruyssen, and J. T. M. Wouters. 1981. Cell wall turnover in batch and chemostat cultures of Bacillus subtilis. J. Bacteriol. 145:50-60. 13. Fontana, R., P. Canepari, G. Satta, and J. Coyette. 1980. Identification of the lethal target of benzylpenicillin in Streptococcus faecalis by in vivo penicillin binding studies. Nature (London) 287:70-72. 14. Kiener, P. A., and S. G. Waley. 1978. Reversible inhibitors of penicillinases. Biochem. J. 169:197-204. 15. Kiss, L., A. Tar, S. Gal, B. L. T6th-Martinez, and F. J. Hernadi. 1988. Modified general affinity adsorbent for large-scale purification of penicillinases. J. Chromatogr. 448:109-116. 16. Kitano, K., and A. Tomasz. 1979. Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics. Antimicrob. Agents Chemother. 16:838-848. 17. Labia, R., M. Guionie, M. Barthelemy, and A. Philippon. 1981. Properties of three carbenicillin-hydrolysing 13-lactamases (CARB) from Pseudomonas aeruginosa: identification of a new enzyme. J. Antimicrob. Chemother. 7:49-56. 18. Labia, R., M. Guionie, J.-M. Masson, A. Philippon, and M. Barthelemy. 1977. Beta-lactamases produced by a Pseudomonas aeruginosa strain highly resistant to carbenicillin. Antimicrob. Agents Chemother. 11:785-790. 19. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packing events. J. Mol. Biol. 80: 575-599. 20. National Committee for Clinical Laboratory Standards. 1985. Approved standard M7-A. Standard methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 21. Neu, H. C., and K. P. Fu. 1978. Cefaclor: in vitro spectrum of activity and beta-lactamase stability. Antimicrob. Agents Che-
mother. 13:584-588. 22. Neu, H. C., and K. P. Fu. 1979. Cefatrizine activity compared with that of other cephalosporins. Antimicrob. Agents Chemother. 15:209-212. 23. O'Callaghan, C. H., A. Morris, S. Kirby, and A. H. Shingler. 1972. Novel method for detection of P-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 24. Pooley, H. M. 1976. Layered distribution, according to age, within the cell wall of Bacillus subtilis. J. Bacteriol. 125: 1139-1147. 25. Samuni, A. 1975. A direct spectrophotometric assay and determination of Michaelis constants for the beta-lactamase reaction. Anal. Biochem. 63:17-26. 26. Sawai, T., M. Kanno, and K. Tsukamoto. 1982. Characterization of eight ,B-lactamases of gram-negative bacteria. J. Bacteriol. 152:567-571. 27. Simpson, I. N., S. J. Plested, and P. B. Harper. 1982. Investigation of the beta-lactamase stability of ceftazidime and eight other new cephalosporin antibiotics. J. Antimicrob. Chemother. 9:357-368. 28. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem. 72:341-352. 29. Sykes, R. B. 1982. The classification and terminology of enzymes that hydrolyze beta-lactam antibiotics. J. Infect. Dis.
145:762-765. 30. Tar, A., S. Gal, B. L. T6th-Martinez, F. J. Hernaidi, and L. Kiss.
354
SZABO ET AL.
1986. Use of chromatofocusing. for separation of P-lactamases. VII. Analytical and medium scale preparative chromatofocusing of the constitutive chromosomal cephalosporinase P99 from Enterobacter dloacae. J. Chromatogr. 368:427-432. 31. Then, R. L., and P. Angehrui. 1982. Trapping of nonhydrolyzable cephalosporins by cephalosporinases in Enterobacter cloacae and Pseudomonas aeruginosa as a possible resistance mechanism. Antimicrob. Agents Chemother. 21:711-721. 32. T6th-Martinez, B. L., S. Gil, and L. Kiss. 1983. Chromatofo-
ANTIMICROB. AGENTS CHEMOTHER.
cusing for separation of ,B-lactamases. I. Microscale separation of RTEM- and chromosomally mediated P-lactamases of Escherichia coli J6-2. J. Chromatogr. 262:373-378. 33. Waley, S. G. 1974. A spectrophotometric assay of beta-lactamase action on penicillins. Biochem. J. 139:789-790. 34. Zak, O., W. A. Vischer, C. Schenk, W. Tosch, W. Zimmerman, J. Regos, E. R. Suter, F. Kredolier, and J. Gelzer. 1976. CGP9000: a new orally active, broad-spectrum cephalosporin. J. Antibiot. 29:635455.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 349-354
0066-4804/90/020349-06$02.00/0 Copyright © 1990, American Society for Microbiology
In Vitro Investigation of BK-218, a New Oral and Parenteral Cephalosporin SZAB6,1* J. BARABAS,1 A. TAR,2 L. KISS,3 M. FILEP,4 T.
SCHMIDT,4 K. MAROSSY, B. TOTH-MARTINEZ,2 GY. BARABAS,1 AND F. HERNADI2 Institute of Biology1 and Department of Chemotherapy, Institute of Pharmacology,2 University Medical School, and Institute of Biochemistry, L. Kossuth University,3 H4012 Debrecen, and BIOGAL Pharmaceutical Works, H4042 Debrecen,4 Hungary
I.
Received 2 June 1989/Accepted 26 October 1989 The antibacterial activity of BK-218 was similar to that of cefamandole when it was tested against several laboratory strains. The inhibiting effect of BK-218 was greater than that of cephalexin and cefoxitin on penicillin-binding proteins of Escherichia col HB101. This result was in close correlation with the relative inhibition of radiolabeled glucosamine incorporation (greatest with BK-218) and with the lytic effect (most intensive with BK-218). BK-218 proved to be a good inhibitor for all five of the ,-lactamases that were investigated, although two enzymes (Enterobacter cloacae P99 and Pseudomonas aeruginosa Cilote) hydrolyzed it to some extent.
Bacterial strains for susceptibility testing. OKI-marked standard strains of bacteria were obtained from the Hungarian National Collection of Medical Bacteria, Budapest. Enterobacter cloacae P99 was kindly supplied by I. N. Simpson, Glaxo Group Research, Greenford, United Kingdom; methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates were supplied by F. Rozgonyi, Institute of Microbiology, University Medical School, Debrecen, Hungary; and E. coli DCO and DC2 were supplied by Elmar Schrimmer, Abteilung fur Chemoterapie, Hoechst Aktiengesellschaft, Frankfurt am Main, Federal Republic of Germany. Antimicrobial susceptibility tests. Antimicrobial activity was measured by the standard agar dilution method (20) with Mueller-Hinton agar (Oxoid Ltd., Basingstoke, United Kingdom). Mueller-Hinton agar was supplemented with 5% sheep blood when the nonenterococcal streptococci were examined. Susceptibility of the methicillin-resistant S. aureus was determined on Mueller-Hinton agar supplemented with 2% NaCl. The medium for preculture was nutrient broth (Oxoid). From a diluted fresh overnight culture, 105 CFU was applied to the agar plate with a replicatory spot device. Broth macrodilution tests were made in calcium- and magnesium-supplemented Mueller-Hinton broth with an inoculum of about 105 CFU/ml. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth in agar or in broth after 18 h of incubation at 37°C. P-Lactamases and carrier strains. The enzymes used in the kinetic studies were purified from known strains as follows. Bacillus cereus 569/H/9 and S. aureus PC1 1711E ,B-lactamases were prepared with N-acetyl-D-penicillamine-Sepharose 4B affinity chromatography by the method of Clarke et al. (10), as modified in our laboratory (15). B. cereus 569/H/9 was kindly provided by S. Fleming, Department of Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom; S. aureus PC1 1711E was a gift from I. N. Simpson; Pseudomonas aeruginosa Cilote was an isolate from A. M. Philippon, Service de Microbiologie, Centre Hospitalier Universitaire Cochin, Paris, France, and its CARB-3-type enzyme (17) was isolated also by affinity
Cephalexin (4), cefadroxil (7), cefaclor (4, 21), cefroxadine (34), and cefatrizine (22) have been widely used for clinical purposes as oral cephalosporins, but their usage is limited because they have relatively narrow spectra against various strains of pathogenic bacteria and are hydrolyzed by 1lactamases. BK-218, 5-thia-1-azabicyclo[4,2,0]oct-2-en,-7[(3 - chloro - izoxazol - 5 - yl) - acetamido] - 3[[(1 - methyl) - 1Htetrazol - 5 - yl]thiomethyl] - 8 - oxo - 2 - carboxylic acid sodium salt (Fig. 1.), is a new oral cephalosporin. It is well-known that beta-lactam antibiotics first bind to their targets, the so-called penicillin-binding proteins (PBPs). The inhibition of these enzymes suspends peptidoglycan synthesis, and as a consequence the autolytic enzymes are triggered with a mechanism whose details are not completely understood (16). The purpose of this study was to report the results of preliminary investigations concerning the in vitro activity of BK-218 against a limited number of gram-positive and gramnegative bacteria. The MICs of BK-218 were compared with those of other parenteral (cefuroxime, cefamandole, and cefoxitin) and oral (cephalexin) antibiotics. The in vitro effect on the targets was measured by (i) affinity to PBPs, (ii) incorporation of labeled glucosamine into the cell wall of Escherichia coli, and (iii) release of labeled glucosamine-containing fragments from prelabeled cell wall of E. coli to assess the lysis capability of BK-218. The susceptibility of BK-218 to hydrolysis by 1-lactamases was also investigated. MATERIALS AND METHODS Antibiotics. BK-218 was synthesized at the Chemical Research Laboratory of BIOGAL Pharmaceutical Works, Debrecen, Hungary. The other compounds were obtained as follows: cefuroxime, Glaxo Research Ltd., Greenford, United Kingdom; cefoxitin, Merck Sharp & Dohme, Paris, France; and cephalexin and cefamandole, Eli Lilly & Co., Geneva, Switzerland. Solutions of antimicrobial agents were prepared on the day of use as directed by the manufacturers. *
Corresponding author. 349
350
SZAB6
ANTIMICROB. AGENTS CHEMOTHER.
ET AL.
C
2H20
o
CS NN N;CH2 I COONa CH3
FIG. 1. Chemical structure of BK-218.
chromatography on m-aminophenylboronic acid-agarose (hydrophobic spacer arm, type B) (9). Chromatofocusing was used to purify 1-lactamases of E. coli RTEM+ and E. cloacae P99. The sources of these strains are described in detail elsewhere (30, 32). Isolation of cytoplasmic membrane from Enterococcus faecalis ATCC 9790. Cytoplasmic membranes from E. faecalis were prepared as described by Coyette et al. (11) and were put in 1 mM phosphate buffer (pH 7) which contained 1 mM MgCl2. The protein content of membrane preparations was adjusted to 20 mg/ml. Isolation of cytoplasmic membrane from E. coli HB101 (3). Exponentially growing cells of E. coli were harvested by centrifugation at 6,000 x g for 20 min. The cells were suspended in 10 mM phosphate buffer (pH 7) which contained 0.14 M mercaptoethanol. After 3 min of ultrasonication (1.5 W; MSE, Ltd., London, United Kingdom), the intact cells were removed by centrifugation at 6,000 x g for 20 min. The supernatant was spun down at 36,000 x g for 30 min to isolate the membrane. The membrane was resuspended in 10 mM phosphate buffer (pH 7) and washed twice with the same buffer and in the same volume. Labeling of PBPs with radioactive penicillin. Membrane samples (containing about 200 ,ug of protein) were preincubated with different concentrations of cephalosporins for 10 min at 37°C and then incubated with 50 ,uM [3H]benzylpenicillin (specific activity, 773.3 GBq/mmol; Institute of Isotopes, Hungarian Academy of Sciences, Budapest) in a total volume of 21 p.l at 37°C for 10 min and subsequently supplemented with excess nonradioactive benzylpenicillin (BIOGAL Pharmaceutical Works) to a final concentration of 10 mM in a 5-pA volume. One microliter of 20% Sarkosyl-NL 97 was added to the membrane preparations of E. coli. Subsequently, they were incubated for 20 min at room temperature. The supernatant obtained at 40,000 x g for 30 min contained the inner-membrane fraction. Denaturing buffer (25 p.J) containing 10% (wt/vol) glycerol, 0.5 M Tris hydrochloride, 2% (wt/vol) sodium dodecyl sulfate (SDS), 0.01% (wt/vol) bromophenol blue, and 10% (vol/vol) 2mercaptoethanol was added. The suspension was boiled for 2 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis and fluorography. SDS-polyacrylamide gel electrophoresis was done as described by Laemmli and Favre (19) with acrylamide contents of 4 and 10% (wt/vol of each) in the stacking and running gels, respectively. After separation of the proteins at a constant 360 V, the gels were fixed in a solution containing 5% (vol/vol) methanol and 7.5% (vol/vol) acetic acid. Gels were placed in dimethyl sulfoxide to remove water, followed by 2 h of incubation in a 20% (wt/vol) solution of 2,5diphenyloxazole in dimethyl sulfoxide. Following rehydration, gels were dried and exposed to Medifort RP X-ray film for 7 to 10 days at -70°C (5). The band intensities were measured by densitometry (Kipp and Zonen densitometer
DD2). The half-saturation cephalosporin concentration (I50 value) for PBPs was calculated from the optical density of a cephalosporin-inhibited PBP band and from the optical density of the control band which was not treated with betalactam antibiotics. Incorporation of labeled glucosamine into cell wall of E. coli HB101. A 500-,ul sample of an exponentially growing culture (optical density, 0.5 at 550 nm) was pretreated with 3.8 x 10-5 M cephalosporin for 10 min (at 100 rpm and 37°C) in a rotatory shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.). After the addition of 9 kBq of N-acetyl-D-[1-3H]glucosamine (specific activity, 105 GBq/mmol; Amersham International plc, Amersham, United Kingdom), the culture was incubated for 15 min, the radioactivity was chased for 10 min with cold glucosamine (final concentration, 10 mM), and counts and disintegrations per minute retained in boiling SDS-precipitable material were measured (filtered on Millipore membrane; pore size, 0.45 ,um; Millipore Corp., Bedford, Mass.). The final concentration of SDS was 5%, and each precipitate was washed with 10 ml of the same SDS solution. The radioactivity was measured with a scintillation counter (Isocap/300; Nuclear-Chicago Corp., Des Plaines, Ill.). Release of cell wall fragments from E. coli HB101. NAcetyl-D-[1-3H]glucosamine (55.5 kBq) (specific activity, 105 GBq/mmol) was added to 3 ml of an exponentially growing culture of E. coli at 37°C (optical density, 0.5). After 90 min of incubation at 37°C and 200 rpm in a rotatory shaker (New Brunswick Scientific), the culture was chased with cold glucosamine. The cells were washed three times with fresh medium, centrifuged at 10,000 x g for 5 min, and suspended in a fresh culture medium containing 3.8 x 10-5 M antibiotic. The cultivation was continued under the same conditions as before. Samples were taken at different times, and the radioactivity of the supernatant (centrifugation at 10,000 x g for 5 min) was measured with the same scintillation counter as described above. Determination of Km, Vmax, and Vrel values. The kinetic experiments were done in a final volume of 3 ml of incubation mixture at 37°C in 0.05 M phosphate buffer, pH 7 (23, 25, 33). In the case of significant hydrolysis of some of the cephalosporins susceptible to certain 3-lactamases, evaluation of the kinetic parameters was made in the 0.5- to 5.0Km substrate concentration interval (33). Assessment of Ki values for enzymes. The inhibition studies were done under conditions similar to those described above. The enzyme samples were preincubated for 5 min, and the reaction was started with nitrocefin (Oxoid) as the substrate in each mixture (Km values were 56 p.M for P99 [33], 136 ,uM for TEM-1 [23], 15 p.M for the Cilote enzyme, 5 ,uM for PC1, and 0.1 ,uM for ,-lactamase I from B. cereus 569/H/9; unpublished data). The results were evaluated for two substrate concentrations (20 and 30 p,M) and for five inhibitor concentrations in the 0.5- to 5.0-Ki interval. 1BLactamase activity was assayed by measuring the A486 of nitrocefin in a cell of 1-cm pathlength, as described by O'Callaghan et al. (23). One unit is that amount of enzyme which is able to hydrolyze 1 ,umol of nitrocefin in 1 min at 37°C. Evaluation of the Ki values was made by the method of Dixon with a simple computer program.
351
IN VITRO INVESTIGATION OF BK-218, A NEW CEPHALOSPORIN
VOL. 34, 1990
TABLE 1. Comparative in vitro activity of BK-218, cefuroxime, cefamandole, cefoxitin, and cephalexin against gram-positive and gram-negative bacteria MIC (,ug/ml) Organism
Cefuroxime
Cefamandole
Cefoxitin
Cephalexin
s0.12 64 -O.12 0.5 -O.12 32 64 0.5 s0.12 s0.12
0.5 >128 1.0 4.0 1.0 >128 >128 0.25 128 2.0 s0.12 -O.12
2.0 >128 2.0 4.0 2.0 >128 32 1.0 0.5 0.25
2.0 >128 2.0 16.0 1.0 >128 >128 4.0 0.5 s0.12
0.25 s0.12 2.0 0.25 8.0 0.25 0.5
4.0 0.25 4.0 .0.12 4.0 4.0 2.0 2.0 4.0 >128 4.0 2.0 64 1.0 64 0.12 2.0 >128 >128 1.0 >128 >128
0.5 s0.12 1.0 0.12 4.0 0.5 0.5 0.25 1.0 >128 1.0 0.5 64 0.5 >128 0.12 1.0 64 >128 4.0 >128 >128
4.0 0.5 2.0 1.0 2.0 8.0 4.0 2.0 4.0 128 128 2.0 16 4.0 8.0 0.5 4.0 >128 >128 8.0 >128 128
8.0 4.0 8.0 4.0 8.0 8.0 16.0 4.0 8.0 >128 128 2.0 >128 32 >128 2.0 16.0 >128 >128 >128 >128 >128
BK-218
Gram-positive Staphylococcus aureus ATCC 25923 Staphylococcus aureus 4916 (methicillin resistant) Staphylococcus aureus 4916 (methicillin susceptible) Staphylococcus aureus NCTC 9789 (P-lactamase producing) Staphylococcus epidermidis OKI 110001 Enterococcus faecalis OKI 80171 Enterococcus faecalis ATCC 9790 Streptococcus pneumoniae OKI 84503 Streptococcus pyogenes OKI 80001 Micrococcus sp. strain OKI 117001 Gram-negative Escherichia coli HB101 Escherichia coli OKI 35034 Escherichia coli DCO Escherichia coli DC2 Yersinia enterocolitica OKI 98001 Shigellaflexneri OKI 20119 Shigella sonnei OKI 20045 Salmonella typhi OKI 10084 Enterobacter cloacae OKI 53001 Enterobacter cloacae P99 Citrobacterfreundii OKI 47001 Klebsiella pneumoniae ATCC 1200 Serratia marcescens OKI 56501 Proteus vulgaris OKI 60002 Morganella morganii OKI 63001 Providencia rettgeri OKI 65005 Proteus mirabilis OKI 61370 Bordetella bronchiseptica OKI 92003 Acinetobacter anitratus OKI 150001 Aeromonas hydrophila OKI 186001 Pseudomonas aeruginosa ATCC 27853 Xanthomonas maltophilia OKI 173006
.0.12 1.0 128 8.0 0.5 >128 2.0 >128 0.12 2.0 >128 >128 >128 >128 >128
BK-218 did not inhibit Bordetella bronchiseptica, Acine-
RESULTS Antibacterial activity. To assess the antibacterial spectrum of BK-218, we compared its activities against typical laboratory strains with those of cefuroxime, cefamandole, cefoxitin, and cephalexin (Table 1). BK-218 had excellent activity against gram-positive bacteria except methicillin-resistant S. aureus and E. faecalis. The MICs were lower than MICs of other beta-lactams. The MICs of BK-218 ranged from 0.12 to 8 ,ug/ml against most strains of the family Enterobacteriaceae, except cephalosporinase-producing E. cloacae, Serratia marcescens, and Morganella morganii strains.
tobacter anitratus, Aeromonas hydrophila, P. aeruginosa, or Xanthomonas maltophilia at 128 jig/ml. Affinity for PBPs of E. faecalis ATCC 9790 and E. coli HB101. The affinities for PBPs of BK-218, cefoxitin, and cephalexin were examined by measuring the competition of the investigated beta-lactam antibiotics with '4C-labeled penicillin G. Table 2 shows the results of the competition
quantitatively. BK-218, cefoxitin, and cephalexin had very high affinities for PBP 2 of E. faecalis (Table 2), but cefoxitin was a better inhibitor of PBP 1 than was BK-218. Cephalexin had mod-
TABLE 2. Iso values of BK-218, cefoxitin, and cephalexin for PBPs of E. faecalis ATCC 9790 and E. coli HB101 150' (mmol/ml) for PBP:
Organism and agent
E. faecalis BK-218
Cefoxitin Cephalexin E. coli BK-218
Cefoxitin Cephalexin a Data are means
+
1
2
3
4
5
6
0.3 ± 0.02 0.1 ± 0.02 24.7 + 3.50
0.3 ± 0.05 0.3 ± 0.04 0.6 ± 0.1
10.5 ± 2.0 50.7 ± 4.7 18.9 ± 1.5
12.2 + 1.9 >100 >100
ND ND ND
0.3 ± 0.04 20.6 ± 3.9 >100
0.3 ± 0.03 0.3 ± 0.03 >100
0.3 ± 0.03 >100 >100
0.63 ± 0.15 2.5 ± 0.1 15.0 ± 1.8
4.2 ± 0.6 6.0 ± 0.5 18.7 ± 3.2
standard deviations for three independent experiments. ND, Not determined.
5/6
3.5 ± 0.6 0.3 ± 0.04
>100
352
SZAB6
ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Comparison of Ki values of BK-218 with those of three laboratory-standard cephalosporins as measured with 3-lactamases of gram-negative and gram-positive origins
K; (,uM)
Source of enzyme
E. P. S. B. E.
l
25 Time [min]
40
FIG. 2. Release of labeled glucosamine from prelabeled cell wall of E. coli HB101.
erate affinity toward all the other PBPs of E. faecalis, including PBP 3. As shown in Table 2, BK-218 had higher or at least equal affinity for the lethal targets (PBP 1, PBP 2, and PBP 3) of E. coli (13) than cefoxitin and had significantly higher affinity than cephalexin. Incorporation of labeled glucosamine. An exponentially growing culture of E. coli HB101 was pretreated with 3.8 x 10' M cephalosporin. After 10 min of pretreatment, tritiated N-acetyl glucosamine (which incorporates predominantly into the cell wall; 12, 24) was added to the culture. Glucosamine incorporation was about 50% of the control (7,600 dpm) when BK-218 was added (3,600 dpm). Cefoxitin had less of an inhibitory effect (5,600 dpm) and cephalexin had essentially no effect (7,400 dpm) on the incorporation. Release of cell wall fragments. The cell wall of E. coli HB101 was labeled with [3H]glucosamine, after which the release of the radioactivity was measured under the influence of different cephalosporins. The control culture, which was not treated with any antibiotic, showed a low but continuous release of radioactivity because of the turnover of cell wall material (Fig. 2). The release of radioactivity was extensive in the culture treated with BK-218, while the effect of cefoxitin on release was moderate. Cephalexin did not cause significant release above that seen in untreated controls. P-Lactamase stability of BK-218. ,B-Lactamase stability of BK-218 was investigated with five beta-lactam-hydrolyzing enzymes. Two of the enzymes were of gram-positive-bacterial origin: S. aureus PC1 and B. cereus 569/H/9 P-lactamase I. The gram-negative strains and their enzymes were as follows: E. cloacae P99, P. aeruginosa Cilote CARB-3 type, and E. coli RTEM+ TEM-1. We found that only the P99 and Cilote enzymes were able to hydrolyze BK-218 to a significant extent (Vmax values, 130 and 100 ,umol/min, respectively), while BK-218 did not prove to be a substrate for the rest of the investigated enzymes (Vmax values, '10 nmol/min). To characterize these results, we correlated the data with the P-lactamase stabilities of cefuroxime, cefoxitin, and cephalexin. Of these
cloacae P99
aeruginosa Cilote aureus PC1 cereus 5691H/9 coli RTEM+
BK-218
Cefuroxime
80 160 7 40 670
1,200 5,000
35
290
3,500
Cefoxitin
Cephalexin
95 70 80 5,000 1,700
1,290 2,000
185 915
2,000
beta-lactams, only cephalexin was hydrolyzed by TEM-1 (120 ,umol/min), while its breakdown with the P99 enzyme was small (40 nmol/min). The compounds were not hydrolyzed by any other enzyme. Inhibition kinetic studies (Table 3) showed that BK-218 was the best inhibitor of PC1 and weakest against TEM-1. The standard cephalosporins were not as good inhibitors as BK-218 in most cases, except cefuroxime for P99. Cefoxitin and BK-218 inhibited P99 comparably, but BK-218 was less netive a%,tivv, npninqt araill3L Cilntf.
DISCUSSION BK-218 is a new cephalosporin antibiotic for oral use. It was very active against the 10 investigated gram-positive bacteria, except methicillin-resistant S. aureus and E. faecalis strains, and it showed good activity against numerous gram-negative laboratory strains, especially members of the Enterobacteriaceae. MICs of beta-lactam antibiotics alone do not elucidate characteristics such as binding to target enzymes, penetration of the outer membrane, effect of cell wall synthesis, and lysis. For example, the permeability barrier may prevent the beta-lactam from reaching the target enzyme even in the case of a compound which has high affinity for PBPs. The investigation of the latter by the use of isolated cytoplasmic membranes containing PBPs may help to clear up the problem. On the basis of these results, however, the rate of lysis and cell wall inhibition cannot be predicted, since several PBPs take part in the process. The inhibition of cell wall synthesis and the lysis of the cell wall can be measured by the use of labeled glucosamine which can incorporate into the peptidoglycan. In an investigation of the inhibition of PBPs of E. faecalis by BK-218, cefoxitin, and cephalexin, it was shown that BK-218 had lower affinity for PBP 1 than cefoxitin and higher affinity for PBP 3 than cefoxitin and cephalexin. PBP 3 is considered to be the major lethal target of E. faecalis (13). BK-218 was an excellent inhibitor of PBP 6, which is known to be a nonlethal target. As seen with carboxypeptidases of different strains, inactivation does not influence the MICs. BK-218 was very effective against numerous gram-negative strains. The effect of BK-218 on E. coli HB101 was investigated in detail. When the PBP-inhibiting effects of BK-218, cephalexin, and cefoxitin were compared, BK-218 proved to be the best inhibitor of the lethal targets of E. coli (28). The MIC (0.25 ,ug/ml) of BK-218 against E. coli HB101 is not significantly higher than the 150S for PBP 1 and PBP 2. This suggests that BK-218 may penetrate the outer membrane of E. coli well. The experiments on cell wall synthesis and lysis were done with concentrations of BK-218, cefoxitin, and cephal-
VOL. 34, 1990
IN VITRO INVESTIGATION OF BK-218, A NEW CEPHALOSPORIN
exin that were two magnitudes higher than the MIC of BK-218. This proved to be the optimal concentration (data obtained with other concentrations are not given here) for demonstrating the differences among the three investigated cephalosporins. This concentration promoted the quick and thus easily measurable inhibition of cell wall synthesis and lysis of labeled cell wall. The difference between the effects of BK-218 and cefoxitin on cell wall synthesis and lysis could not be predicted from the 150 values (Table 3) because it is not known how much the three lethal targets (PBP 1, PBP 2, and PBP 3) are involved quantitatively in these processes. We believe that the results given above on cell wall synthesis and lysis demonstrate more precisely the effect of beta-lactams on bacteria than do mere MICs and MBCs. BK-218 proved to be the best inhibitor of the S. aureus PC1 enzyme. Cefuroxime and cephalexin did not inhibit this enzyme significantly. Our results showed BK-218 to be a much better inhibitor of P-lactamase I from B. cereus 569/H/9 than the standard cephalosporins used in these experiments. Reference Ki data are available mainly for methicillin (14) and isoxazolyl penicillins (2) but not for cephalosporins, with the exception of cephalosporin C (1). Therefore, our values can be informative for research purposes. As to the Ki values of BK-218 compared with those of the standards for enzymes of gram-negative bacteria, one may observe a diverse picture. P99 was less inhibited by BK-218 than by cefuroxime and was inhibited to about the same extent as by cefoxitin and cephalexin. It is known that moxalactam is a good inhibitor of this enzyme (Ki, 62 nM; 8). The P. aeruginosa Cilote ,-lactamase was approximately equally inhibited by BK-218 and cefoxitin, but cefuroxime and cephalexin proved to be poor inhibitors. Comparative data from references show that cephaloridine (29) and cephalothin (3, 18) are not stable against P-lactamases of different P. aeruginosa strains and are also poor inhibitors. However, cefotaxime and ceftriaxone (31) are stable inhibitors for many enzymes of these strains. E. coli has not been extensively investigated, but the available Ki values are consistent with our findings (26). Ki values of cefuroxime (650 ,uM) and moxalactam (810 ,uM) (8) were found to be in the same range as that of BK-218. In sum, we regard BK-218 as a promising candidate for gram-negative-bacterial screening despite the fact that two investigated enzymes hydrolyzed it to some extent. LITERATURE CITED 1. Abraham, E. P., and G. G. F. Newton. 1956. Experiments on the degradation of cephalosporin C. Biochem. J. 62:658-665. 2. Abraham, E. P., and S. G. Waley. 1979. Beta-lactamases from Bacillus cereus, p. 311-338. In J. M. T. Hamilton-Miller and J. T. Smith (ed.), Beta-lactamases. Academic Press, Inc. (London), Ltd., London. 3. Berks, M., K. Redhead, and E. P. Abraham. 1982. Isolation and properties of an inducible and a constitutive beta-lactamase from Pseudomonas aeruginosa. J. Gen. Microbiol. 128:155-159. 4. Bill, N. J., and J. A. Washington II. 1977. Comparison of in vitro activity of cephalexin, cephadrine, and cefaclor. Antimicrob. Agents Chemother. 11:470-474. 5. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 6. Boyer, H. W., and D. Rouliand-Dunioix. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41:459-472. 7. Buck, R. E., and K. E. Price. 1977. Cefadroxil, a new broadspectrum cephalosporin. Antimicrob. Agents Chemother. 11: 324-330.
353
8. Cartwright, S. J., and S. G. Waley. 1983. Beta-lactamase inhibitors, p. 341-382. In Medical research news, vol. 3, no. 4. John Wiley & Sons, Inc., New York. 9. Cartwright, S. J., and S. G. Waley. 1984. Purification of lactamases by affinity chromatography on phenylboronic acid agarose. Biochem. J. 221:505-512. 10. Clarke, A. J., P. S. F. Mezes, and T. Viswanatha. 1980. A rapid procedure for the purification of Bacillus cereus 569/H lactamase. J. Appl. Biochem. 2:183-189. 11. Coyette, J., J.-M. Ghuysen, F. Binot, P. Adriaens, B. Meesschaert, and H. Venderhaeghe. 1977. Interaction between ,Blactam antibiotics and isolated membranes of Streptococcus faecalis ATCC 9790. Eur. J. Biochem. 75:231-239. 12. De Boer, W. R., F. J. Kruyssen, and J. T. M. Wouters. 1981. Cell wall turnover in batch and chemostat cultures of Bacillus subtilis. J. Bacteriol. 145:50-60. 13. Fontana, R., P. Canepari, G. Satta, and J. Coyette. 1980. Identification of the lethal target of benzylpenicillin in Streptococcus faecalis by in vivo penicillin binding studies. Nature (London) 287:70-72. 14. Kiener, P. A., and S. G. Waley. 1978. Reversible inhibitors of penicillinases. Biochem. J. 169:197-204. 15. Kiss, L., A. Tar, S. Gal, B. L. T6th-Martinez, and F. J. Hernadi. 1988. Modified general affinity adsorbent for large-scale purification of penicillinases. J. Chromatogr. 448:109-116. 16. Kitano, K., and A. Tomasz. 1979. Triggering of autolytic cell wall degradation in Escherichia coli by beta-lactam antibiotics. Antimicrob. Agents Chemother. 16:838-848. 17. Labia, R., M. Guionie, M. Barthelemy, and A. Philippon. 1981. Properties of three carbenicillin-hydrolysing 13-lactamases (CARB) from Pseudomonas aeruginosa: identification of a new enzyme. J. Antimicrob. Chemother. 7:49-56. 18. Labia, R., M. Guionie, J.-M. Masson, A. Philippon, and M. Barthelemy. 1977. Beta-lactamases produced by a Pseudomonas aeruginosa strain highly resistant to carbenicillin. Antimicrob. Agents Chemother. 11:785-790. 19. Laemmli, U. K., and M. Favre. 1973. Maturation of the head of bacteriophage T4. I. DNA packing events. J. Mol. Biol. 80: 575-599. 20. National Committee for Clinical Laboratory Standards. 1985. Approved standard M7-A. Standard methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. National Committee for Clinical Laboratory Standards, Villanova, Pa. 21. Neu, H. C., and K. P. Fu. 1978. Cefaclor: in vitro spectrum of activity and beta-lactamase stability. Antimicrob. Agents Che-
mother. 13:584-588. 22. Neu, H. C., and K. P. Fu. 1979. Cefatrizine activity compared with that of other cephalosporins. Antimicrob. Agents Chemother. 15:209-212. 23. O'Callaghan, C. H., A. Morris, S. Kirby, and A. H. Shingler. 1972. Novel method for detection of P-lactamases by using a chromogenic cephalosporin substrate. Antimicrob. Agents Chemother. 1:283-288. 24. Pooley, H. M. 1976. Layered distribution, according to age, within the cell wall of Bacillus subtilis. J. Bacteriol. 125: 1139-1147. 25. Samuni, A. 1975. A direct spectrophotometric assay and determination of Michaelis constants for the beta-lactamase reaction. Anal. Biochem. 63:17-26. 26. Sawai, T., M. Kanno, and K. Tsukamoto. 1982. Characterization of eight ,B-lactamases of gram-negative bacteria. J. Bacteriol. 152:567-571. 27. Simpson, I. N., S. J. Plested, and P. B. Harper. 1982. Investigation of the beta-lactamase stability of ceftazidime and eight other new cephalosporin antibiotics. J. Antimicrob. Chemother. 9:357-368. 28. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem. 72:341-352. 29. Sykes, R. B. 1982. The classification and terminology of enzymes that hydrolyze beta-lactam antibiotics. J. Infect. Dis.
145:762-765. 30. Tar, A., S. Gal, B. L. T6th-Martinez, F. J. Hernaidi, and L. Kiss.
354
SZABO ET AL.
1986. Use of chromatofocusing. for separation of P-lactamases. VII. Analytical and medium scale preparative chromatofocusing of the constitutive chromosomal cephalosporinase P99 from Enterobacter dloacae. J. Chromatogr. 368:427-432. 31. Then, R. L., and P. Angehrui. 1982. Trapping of nonhydrolyzable cephalosporins by cephalosporinases in Enterobacter cloacae and Pseudomonas aeruginosa as a possible resistance mechanism. Antimicrob. Agents Chemother. 21:711-721. 32. T6th-Martinez, B. L., S. Gil, and L. Kiss. 1983. Chromatofo-
ANTIMICROB. AGENTS CHEMOTHER.
cusing for separation of ,B-lactamases. I. Microscale separation of RTEM- and chromosomally mediated P-lactamases of Escherichia coli J6-2. J. Chromatogr. 262:373-378. 33. Waley, S. G. 1974. A spectrophotometric assay of beta-lactamase action on penicillins. Biochem. J. 139:789-790. 34. Zak, O., W. A. Vischer, C. Schenk, W. Tosch, W. Zimmerman, J. Regos, E. R. Suter, F. Kredolier, and J. Gelzer. 1976. CGP9000: a new orally active, broad-spectrum cephalosporin. J. Antibiot. 29:635455.
