Abstract
Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 β-lactamase. The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 μM. These values were 220- to 850-fold lower than those of tazobactam. Syn2190 showed in vitro synergy with ceftazidime and cefpirome. This synergy was dependent on the concentration of the inhibitor against group 1 β-lactamase-producing strains, such as Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, and Morganella morganii. However, against β-lactamase-derepressed mutants of P. aeruginosa, the MICs of ceftazidime plus Syn2190 were not affected by the amount of β-lactamase, and the values were the same for the parent strains. The MICs at which 50% of isolates are inhibited (MIC50s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria. Similarly, the MIC50s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates. The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo. Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant P. aeruginosa.
Expanded-spectrum cephalosporins show good therapeutic efficacies against various infectious diseases caused by gram-negative bacteria. However, due to long-term clinical usage, the problem of resistance of gram-negative bacteria to expanded-spectrum cephalosporins has occurred. This resistance is principally associated with the hyperproduction of chromosomally mediated cephalosporinases (16, 23), which are classified as group 1 β-lactamases (4). The increased levels of production of cephalosporinases among species of Enterobacter, Citrobacter, Serratia, Pseudomonas, and Acinetobacter may be induced by broad-spectrum β-lactams, and spontaneous mutation to a stably derepressed constitutive state may occur (1, 12). These cephalosporinases confer resistance to all cephalosporins (7, 22). The resistance mechanism of Pseudomonas aeruginosa is considered to be the combination of β-lactamase production and lower outer membrane (OM) permeability (10).
To overcome these clinical problems brought about by the increasing incidence of β-lactamase-producing organisms, three β-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam, have been developed. These β-lactamase inhibitors inactivate various types of group 2 β-lactamases, including extended- and broad-spectrum β-lactamases. However, their inhibitory activities against cephalosporinases are generally weak (2, 11). In our program to identify potent inhibitors of group 1 cephalosporinases, Syn2190 (Fig. 1) was found to possess potent inhibitory activity against cephalosporinases. This paper describes the in vitro and in vivo activities of Syn2190 in combination with various cephalosporins.
FIG. 1.
Chemical structure of Syn2190.
MATERIALS AND METHODS
Antibacterial agents.
Syn2190 and tazobactam were prepared by SynPhar Laboratories Inc. and Taiho Pharmaceutical Co., Ltd., respectively. Other antibiotics were obtained as commercial preparations.
Organisms.
Bacterial strains, listed in Tables 1 and 2, that produce the characterized β-lactamases were kindly provided by R. T. Testa (15) for Escherichia coli TEM-1 and TEM-2 and Klebsiella pneumoniae CTX-1, J. F. Acar for E. coli TEM-7 (8) and SHV-5 (9), J. D. Williams for K. pneumoniae 366L (19), and M. Galleni for Enterobacter cloacae P99 (13). Other bacterial strains were clinical isolates collected from 1988 to 1995 from several hospitals in Japan, and their biological properties were identified with the VITEK system (ASM III; Vitek System Inc.). All of the strains used were stored at −80°C as suspensions in 10% skim milk. β-Lactamase-derepressed mutants of P. aeruginosa were obtained as follows. The parent organism was incubated in Sensitivity Disk Agar (SDA-N; Nissui), which is a modified Mueller-Hinton medium adjusted with divalent cations containing 2 to 8× the MIC of ceftazidime, and the colony obtained after incubation for 48 h was used as a mutant. Alterations of penicillin-binding proteins (25), outer membrane proteins (20), and physiological characters were not observed for these mutants. The β-lactamases produced by the characterized β-lactamase-producing strains, listed in Table 2, were identified by their substrate-hydrolyzing profiles by using ampicillin and cephalothin.
TABLE 1.
β-Lactamase inhibitory activities of Syn2190 compared with those of tazobactam
| Enzyme classa | Organism | I50 (μM)
|
|
|---|---|---|---|
| Syn2190 | Tazobactam | ||
| Group 1 | P. aeruginosa 46012 | 0.010 | 2.264 |
| Group 1 | E. cloacae P99 | 0.006 | 4.995 |
| Group 1 | M. morganii | 0.002 | 0.433 |
| Group 1 | C. freundii | 0.002 | 1.931 |
| Group 2b (TEM-1) | E. coli TEM-1 | >20 | 0.007 |
| Group 2be (TEM-3) | K. pneumoniae CTX-1 | 7.894 | 0.008 |
| Group 2e | Proteus vulgaris | >20 | 0.002 |
The classification is based on that of Bush et al. (4).
TABLE 2.
MICs of ceftazidime alone or in combination with Syn2190 and tazobactam against β-lactamase-producing bacteria
| Organism | Enzyme classa | MIC (μg/ml)
|
||||
|---|---|---|---|---|---|---|
| Ceftazidime alone | Syn2190
|
Tazobactam
|
||||
| 1.0 μg/ml | 5.0 μg/ml | 1.0 μg/ml | 5.0 μg/ml | |||
| Staphylococcus aureus 54K | Group 2b | 12.5 | 12.5 | 12.5 | 12.5 | 6.25 |
| E. coli TEM-1 | Group 2b (TEM-1) | ≦0.20 | ≦0.20 | ≦0.20 | ≦0.20 | ≦0.20 |
| E. coli TEM-2 | Group 2b (TEM-2) | ≦0.20 | ≦0.20 | ≦0.20 | ≦0.20 | ≦0.20 |
| K. pneumoniae 336L | Group 2b (SHV-1) | 0.39 | 0.39 | 0.39 | 0.39 | ≦0.20 |
| E. coli TEM-7 | Group 2b (TEM-7) | 50 | 25 | 25 | 6.25 | 0.39 |
| K. pneumoniae CTX-1 | Group 2be (TEM-3) | 25 | 25 | 25 | 0.39 | 0.39 |
| E. coli SHV-5 | Group 2be (SHV-5) | 200 | 200 | 200 | ≦0.20 | ≦0.20 |
| Serratia marcescens CT98 | Group 1 | 6.25 | 6.25 | 6.25 | 6.25 | 6.25 |
| C. freundii CT76 | Group 1 | 50 | 12.5 | 6.25 | 25 | 12.5 |
| C. freundii 44032 | Group 1 | 200 | 25 | 3.13 | 200 | 100 |
| E. cloacae P99 | Group 1 | 50 | 12.5 | 0.78 | 25 | 12.5 |
| E. cloacae 40054 | Group 1 | 200 | 50 | 6.25 | 100 | 100 |
| E. aerogenes 41003 | Group 1 | 12.5 | 1.56 | 0.78 | 12.5 | 6.25 |
| M. morganii 36014 | Group 1 | 25 | 0.39 | ≦0.20 | 0.39 | ≦0.20 |
| P. aeruginosa CT144 | Group 1 | 50 | 6.25 | 1.56 | 50 | 25 |
| P. aeruginosa 46012 | Group 1 | 50 | 25 | 6.25 | 50 | 50 |
The classification is based on that of Bush et al. (4).
Assay of β-lactamase activity and β-lactamase inhibitory activity.
The enzymes were obtained as crude cell extracts prepared by ultrasonication. β-Lactamase activity was determined by the UV method (5). The amount of protein was measured by using the Bio-Rad Protein Assay. The concentration of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% (I50) was recorded as the residual activity of β-lactamase. Substrates (100 μM cephalothin for group 1 β-lactamases; 100 μM ampicillin or cefotaxime for group 2 β-lactamases) were added to the reaction mixture after preincubation of the enzyme with β-lactamase inhibitor for 5 min at 30°C. The reaction rate was measured by the UV method.
In vitro susceptibility tests.
A total of 106 cells of bacterial suspension per ml in broth cultures was spotted onto SDA-N plates with a twofold serial dilution of antibiotic either alone or in the presence of a β-lactamase inhibitor. A total of 1 or 5 μg of a β-lactamase inhibitor per ml was combined with antibiotic for the characterized β-lactamase-producing strains or was combined with antibiotic at a 1- to-1 ratio for the β-lactamase-derepressed mutant strains and clinical isolates. The MIC was defined as the lowest antibiotic concentration that prevented visible growth of bacteria after overnight incubation at 37°C.
Induction of β-lactamase.
The bacteria were incubated with each concentration of test compound for 2 h. The bacterial cells were washed twice with 50 mM phosphate buffer (pH 7.0) and were ultrasonicated. After centrifugation, the supernatant was used as the crude enzyme extract. β-Lactamase activity was determined with 100 μM cephalothin as the substrate by the UV method (5).
Therapeutic efficacy in mice systemic infection model.
A bacterial suspension (106 to 107 cells/ml) was mixed with an equal volume of 10% gastric mucin (Difco), and the mixture was inoculated intraperitoneally into mice. Male ddY mice weighing, on average, 21.5 g (age, 4 to weeks; Japan SLC Inc., Shizuoka, Japan) were used. The antibiotics, either alone or in the presence of a β-lactamase inhibitor at ratio of 1 to 1, were administered subcutaneously at 1 and 3 h after the inoculation. The 50% effective doses (ED50s) were calculated by the Probit method from the survival rate at 5 days after infection.
Therapeutic efficacy in murine urinary tract infection model.
A total of 0.1 ml of bacterial suspension (6.2 × 104 cells/mouse) was inoculated into the mouse urinary tract. Female ddY mice weighing, on average, 19.2 g (age, 4 weeks) were used. At 6 h postinoculation, the antibiotics, either alone or in the presence of a β-lactamase inhibitor at a ratio of 1 to 1, were administered twice daily for 2 days and once daily at 3 days after infection. At 5 days after infection, the kidneys were removed and were homogenized with 2 ml of saline, and the bacterial cells were counted serially by the pour plate method. Statistical analysis was performed by the Tukey method.
Plasma Syn2190 concentration.
Syn2190 was administered intravenously at a dose of 20 mg/kg of body weight. Blood was obtained from the inferior mesenteric vein of the mice with a heparinized syringe. Plasma Syn2190 concentrations were determined by high-pressure liquid chromatography. The half-life in plasma was calculated by the compartment method with the WinNonlic computer program.
RESULTS
β-Lactamase inhibitory activity.
The inhibitory activity of Syn2190 against β-lactamases was compared to that of tazobactam (Table 1). Tazobactam showed moderate inhibitory activity against group 1 β-lactamases produced by P. aeruginosa, E. cloacae, Morganella morganii, and Citrobacter freundii. The I50s of tazobactam were in the range of 0.433 to 4.995 μM. Syn2190 had stronger inhibitory activity against these enzymes. The I50s of Syn2190 were in the range of 0.002 to 0.010 μM, and these values were 220- to 850-fold lower than those of tazobactam. Against plasmid-mediated group 2b and 2be β-lactamases and chromosome-mediated group 2e β-lactamases, tazobactam showed stronger inhibition than Syn2190. The I50s of Syn2190 were 103- to 104-fold higher than those of tazobactam.
Synergy in combination with cephalosporin.
The synergistic activity of Syn2190 at 1.0 and 5.0 μg/ml with ceftazidime was tested against β-lactamase-producing bacteria (Table 2). Syn2190 plus ceftazidime showed synergy against group 1 β-lactamase-producing bacteria, but the synergy depended on the concentration of the inhibitor. When 1.0 μg of Syn2190 per ml was combined with ceftazidime, the MICs of ceftazidime were decreased 2- to 64-fold in comparison with those of ceftazidime alone against E. cloacae, Enterobacter aerogenes, M. morganii, C. freundii, and P. aeruginosa. However, with 5.0 μg of Syn2190 per ml, the MICs of ceftazidime were decreased 8- to 128-fold in comparison with those of ceftazidime alone. These synergistic activities of Syn2190 were 2- to 8-fold stronger at 1.0 μg/ml and 2- to 32-fold stronger at 5.0 μg/ml than those of tazobactam against all strains except M. morganii. In contrast, tazobactam showed stronger synergy than Syn2190 against group 2be β-lactamase-producing bacteria. The MICs of Syn2190 for all bacteria tested were ≥100 μg/ml (data not shown).
In vitro antibacterial activity of cephalosporin-Syn2190 against derepressed mutants.
Table 3 shows the antibacterial activities of ceftazidime-Syn2190 and cefpirome-Syn2190 at combination ratios of 1 to 1 against β-lactamase-derepressed mutants of P. aeruginosa. The MICs of ceftazidime-Syn2190 were 1.56 to 3.13 μg/ml for β-lactamase-derepressed mutants, and the reductions in the MICs were 16- to 32-fold in comparison with those of ceftazidime alone. These values were the same as the MICs of ceftazidime for the parent strains. The MICs of cefpirome-Syn2190 against derepressed mutants were 1.56 to 6.25 μg/ml, and the reductions in the MICs were 16- to 64-fold in comparison with those of cefpirome alone. These values were also the same or lower than the MICs of cefpirome for the parent strains.
TABLE 3.
MICs of ceftazidime and cefpirome in combination with Syn2190 against β-lactamase-derepressed mutants of P. aeruginosa
| Organism | Mutant | β-Lactamase activitya (mU/mg of protein) | MIC (μg/ml)
|
|||
|---|---|---|---|---|---|---|
| Ceftazidime
|
Cefpirome
|
|||||
| Alone | With Syn2190b | Alone | With Syn2190b | |||
| PAO1 | Parent | 6.59 | 1.56 | 1.56 | 3.13 | 3.13 |
| 4M-1 | 597 | 50 | 1.56 | 100 | 3.13 | |
| 8M-3 | 572 | 50 | 1.56 | 100 | 3.13 | |
| PAO4222 | Parent | 12.5 | 3.13 | 3.13 | 3.13 | 3.13 |
| 4M-3 | 253 | 100 | 3.13 | 100 | 6.25 | |
| 8M-1 | 229 | 100 | 3.13 | 100 | 6.25 | |
| PAO2354 | Parent | 10.6 | 1.56 | 1.56 | 6.25 | 3.13 |
| 2M-3 | 394 | 25 | 1.56 | 50 | 3.13 | |
| 4M-2 | 337 | 25 | 1.56 | 50 | 3.13 | |
| 46220 | Parent | 2.88 | 3.13 | 1.56 | 3.13 | 0.78 |
| DR-2 | 290 | 50 | 1.56 | 100 | 1.56 | |
β-Lactamase activity was assayed by the UV method with 100 μM cephalothin as the substrate.
The combination ratios of Syn2190 with ceftazidime or cefpirome were 1 to 1.
The antibacterial activities of cephalosporin-Syn2190 at a combination ratio of 1 to 1 against cephalosporin-resistant clinical isolates (MIC of cephalosporin, ≥12.5 μg/ml) are shown in Table 4. Syn2190 showed synergy with ceftazidime against all species tested. When the MIC at which 50% of isolates are inhibited (MIC50) of ceftazidime alone was 25 μg/ml for P. aeruginosa, that of ceftazidime-Syn2190 was reduced to 6.25 μg/ml. MIC50s of ceftazidime-Syn2190 were 12.5, 3.13, and 3.13 μg/ml and were reduced 8-, 8-, and 16-fold in comparison with those of ceftazidime for E. cloacae, E. aerogenes, and C. freundii, respectively. For S. marcescens, the MIC50 of ceftazidime-Syn2190 was twofold lower than that of ceftazidime. MIC90s of ceftazidime-Syn2190 were reduced one-eighth and one-half for P. aeruginosa and E. cloacae, respectively. The MIC50s of cefpirome-Syn2190 were 12.5, 3.13, and 25 μg/ml and were reduced one-eighth, one-eighth, and one-half in comparison with those of cefpirome alone for cefpirome-resistant P. aeruginosa, E. cloacae, and C. freundii, respectively. For S. marcescens, the MIC50 of cefpirome-Syn2190 was not reduced in comparison with that of cefpirome. The MIC90 of cefpirome-Syn2190 for E. cloacae was reduced one-fourth.
TABLE 4.
Antibacterial activities of a cephalosporin in combination with Syn2190 against cephalosporin-resistant clinical isolates
| Resistance and organism (no. of isolates) | Compoundb | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Ceftazidime resistanta | ||||
| P. aeruginosa (22) | Ceftazidime alone | 12.5–>100 | 25 | 100 |
| Ceftazidime-Syn2190 | 1.56–50 | 6.25 | 12.5 | |
| E. cloacae (42) | Ceftazidime alone | 12.5–>100 | 100 | >100 |
| Ceftazidime-Syn2190 | 0.78–>100 | 12.5 | 50 | |
| E. aerogenes (17) | Ceftazidime alone | 12.5–100 | 25 | 50 |
| Ceftazidime-Syn2190 | 1.56–50 | 3.13 | 50 | |
| C. freundii (47) | Ceftazidime alone | 12.5–>100 | 50 | >100 |
| Ceftazidime-Syn2190 | 0.39–>100 | 3.13 | 100 | |
| S. marcescens (31) | Ceftazidime alone | 12.5–>100 | 100 | >100 |
| Ceftazidime-Syn2190 | 1.56–>100 | 50 | >100 | |
| Cefpirome resistanta | ||||
| P. aeruginosa (25) | Cefpirome | 25–>100 | 100 | >100 |
| Cefpirome-Syn2190 | 3.13–>100 | 12.5 | 100 | |
| E. cloacae (26) | Cefpirome | 12.5–100 | 25 | 50 |
| Cefpirome-Syn2190 | 0.78–12.5 | 3.13 | 12.5 | |
| C. freundii (15) | Cefpirome | 12.5–>100 | 50 | >100 |
| Cefpirome-Syn2190 | 3.13–>100 | 25 | >100 | |
| S. marcescens (30) | Cefpirome | 12.5–>100 | 25 | >100 |
| Cefpirome-Syn2190 | 6.25–>100 | 25 | 100 | |
All isolates were resistant to ceftazidime or cefpirome (MICs, ≥12.5 μg/ml).
The combination ratios of Syn2190 with ceftazidime or cefpirome were 1 to 1.
Induction of β-lactamase.
For P. aeruginosa, treatment with 0.25 μg of imipenem per ml for 2 h induced a higher level of β-lactamase production: 2.16 U/mg of protein compared to a control value of 0.016 U/mg of protein (Fig. 2). Treatment with 10 μg of ceftazidime per ml induced a level of production of 0.166 U/mg of protein. This activity was almost 10-fold higher than that for the no-treatment control. On the other hand, Syn2190 and tazobactam did not induce β-lactamase production. In E. cloacae, treatment with 0.1 and 1.0 μg of imipenem per ml induced levels of β-lactamase production of 8.62 and 22.9 U/mg of protein, respectively, compared to a control value of 0.016 U/mg of protein. These activities were 200- and 470-fold higher in comparison with those for the no-treatment control. Treatment with 100 μg of ceftazidime per ml induced β-lactamase production that was almost 250-fold higher than that for the no-treatment control. However, Syn2190 and tazobactam did not induce β-lactamase production in P. aeruginosa, and the levels were only eight- and twofold higher, respectively, in comparison with the control value for E. cloacae, even with the highest concentrations tested.
FIG. 2.
Induction of β-lactamases of P. aeruginosa PAO1 and E. cloacae IFO13535 by Syn2190.
In vivo antibacterial activity of cephalosporin-Syn2190.
Table 5 shows the in vivo activities of Syn2190 combined with cephalosporins against murine systemic infections caused by group 1 β-lactamase-producing strains. Therapeutic efficacy in terms of the ED50s of ceftazidime-Syn2190 was compared with that of ceftazidime-tazobactam combined at a ratio of 1 to 1 against P. aeruginosa 94-46017 and P. aeruginosa 94-46209. The ED50s of ceftazidime-tazobactam of 190.2 and 54.9 mg/kg for these two strains, respectively, were similar to those of ceftazidime alone (175.8 and 85.1 mg/kg, respectively), whereas the ED50s of ceftazidime-Syn2190 of 37.2 and 31.2 mg/kg for these two strains, respectively, were five- and threefold superior to those of ceftazidime alone, respectively. For the combination of Syn2190 with ceftazidime, the ED50 was 95.8 mg/kg, although that of ceftazidime alone was >465 mg/kg against P. aeruginosa 46220 DR-2. Against E. cloacae, the ED50 of ceftazidime-Syn2190 was 387.4 mg/kg, although that of ceftazidime was >465 mg/kg. The ED50s of ceftazidime-Syn2190 against C. freundii and S. marcescens were decreased about one-fifth in comparison with those of ceftazidime alone. In combination with cefpirome, the ED50 of cefpirome-Syn2190 was 40.5 mg/kg against P. aeruginosa 46220 DR-2, although that of cefpirome alone was 234.9 mg/kg. Against E. cloacae, the ED50 of cefpirome-Syn2190 was 48.8 mg/kg, although that of cefpirome was 334.4 mg/kg. The ED50s of cefpirome-Syn2190 against C. freundii and S. marcescens were decreased about one-half in comparison with those of cefpirome.
TABLE 5.
Therapeutic efficacies of ceftazidime and cefpirome in combination with Syn2190 and tazobactam in a murine systemic infection model
| Organism (challenge dose [CFU/mouse]) | Compounda | MIC (μg/ml) | ED50 [mg/kg] (95% confidence limit) |
|---|---|---|---|
| P. aeruginosa 94-46017 (1.3 × 106) | Ceftazidime | 50 | 175 (150–195) |
| Ceftazidime-Syn2190 | 3.13 | 37.2 (34.9–40.5) | |
| Ceftazidime-tazobactam | 25 | 190 (159–230) | |
| P. aeruginosa 94-46209 (7.5 × 105) | Ceftazidime | 50 | 85.1 (76.3–95.8) |
| Ceftazidime-Syn2190 | 1.56 | 31.2 (29.3–32.6) | |
| Ceftazidime-tazobactam | 25 | 54.9 (52.1–58.1) | |
| P. aeruginosa 46220 DR-2 (1.6 × 106) | Ceftazidime | 50 | >465 |
| Ceftazidime-Syn2190 | 6.25 | 95.8 (56.7–162) | |
| Cefpirome | 100 | 234 (68.8–1330) | |
| Cefpirome-Syn2190 | 50 | 40.5 (23.7–65.6) | |
| E. cloacae 94-40017 (2.2 × 107) | Ceftazidime | >100 | >465 |
| Ceftazidime-Syn2190 | 3.13 | 387 (244–1120) | |
| Cefpirome | 12.5 | 334 (—b) | |
| Cefpirome-Syn2190 | 0.78 | 48.8 (19.5–104) | |
| C. freundii 94-44050 (7.3 × 105) | Ceftazidime | 100 | 69.8 (24.6–224) |
| Ceftazidime-Syn2190 | 1.56 | 14.4 (6.5–31.6) | |
| Cefpirome | 3.13 | 7.0 (—) | |
| Cefpirome-Syn2190 | 0.78 | 4.0 (0.8–9.0) | |
| S. marcescens 94-42004 (4.8 × 104) | Ceftazidime | 50 | 168 (83.2–437) |
| Ceftazidime-Syn2190 | 3.13 | 35.6 (10.7–87.4) | |
| Cefpirome | 12.5 | 47.9 (18.6–107) | |
| Cefpirome-Syn2190 | 6.25 | 31.2 (12.6–65.1) |
The combination ratios of Syn2190 and tazobactam with ceftazidime or cefpirome were 1 to 1.
—, not calculated.
The therapeutic efficacies of Syn2190 combined with a cephalosporin at a ratio of 1 to 1 against a urinary tract infection caused by P. aeruginosa 46220 DR-2 are shown in Fig. 3. In the control group, the viable cell counts in the kidneys gradually increased and reached a level of 107 cells/kidney after infection. In the groups administered ceftazidime and cefpirome alone twice daily for 3 days, the bacterial cell counts were constantly about 105 to 106 cells/kidney. On the other hand, the bacterial cell counts in the groups administered ceftazidime-Syn2190 and cefpirome-Syn2190 decreased every day and reached levels of 102 to 103 cells/kidney at 5 days after infection. Significant differences between the group that received a cephalosporin-Syn2190 and the group that received a cephalosporin alone were observed (P < 0.05) at almost all points.
FIG. 3.
Therapeutic efficacies of ceftazidime and cefpirome in combination with Syn2190 at a ratio of 1 to 1 in a murine urinary tract infection model with P. aeruginosa 46220 DR-2. The asterisks indicate a statistically significant difference between cephalosporin and cephalosporin-Syn2190 (P < 0.05, as determined by the Tukey method).
DISCUSSION
Three β-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam, have been developed and are marketed. These β-lactamase inhibitors have strong inhibitory activities against plasmid- and chromosome-mediated group 2 β-lactamases (2, 11), mainly penicillinases (4), and their clinical efficacies in combination with various penicillins such as ampicillin, ticarcillin, and piperacillin have been confirmed. On the other hand, the main factor related to resistance to cephalosporins was the high-level production of group 1 β-lactamases, mainly cephalosporinases (4). These β-lactamases are not satisfactorily inhibited by clavulanic acid, sulbactam, or even tazobactam. Syn2190 showed stronger inhibitory activity than tazobactam against group 1 β-lactamases from P. aeruginosa, E. cloacae, M. morganii, and C. freundii. Therefore, the combination of Syn2190 with a cephalosporin is considered effective and useful for the treatment of infections caused by cephalosporin-resistant bacteria.
In this study we found that the MICs of ceftazidime combined with Syn2190 were reduced to 1/2 to 1/128 in comparison with those of ceftazidime alone against group 1 β-lactamase-producing bacteria. This synergy was dependent on the concentration of the inhibitor. However, the synergy was not observed when ceftazidime-Syn2190 was tested against group 2 β-lactamase-producing strains. This finding may be of little importance, because most expanded-spectrum and “fourth-generation” cephalosporins are stable against this group of β-lactamases (3, 14).
The resistance of P. aeruginosa to cephalosporins is attributed to a number of mechanisms. Of note are the lower level of OM permeability and the increased level of production of β-lactamases (10). The limited OM permeability of P. aeruginosa is attributed to increases in cephalosporin MICs. To increase the rate of penetration across the OM as well as to increase stability against β-lactamases, we designed our inhibitor (Syn2190) to harbor a 1,5-dihydroxy-4-pyridone moiety at the 3-C position on the monobactam ring. Use of this moiety, which is able to use the tonB-dependent iron transport system, is believed to increase the rate of permeation across the OM (6, 17, 18, 24). We have demonstrated the ability of Syn2190 both to bind to free iron and to have a fast penetration rate (unpublished data). The inhibitory activity of Syn2190, combined with its improved penetration through the OM, might explain its ability to show synergy with ceftazidime against derepressed mutants of P. aeruginosa strains (Table 3).
The synergy of Syn2190 was also observed among clinical isolates of cephalosporin-resistant gram-negative bacteria (Table 4). The level of reduction was more prominent at the MIC50 rather than at the MIC90. A possible cause is the involvement of more than one mechanism of resistance, e.g., low-level affinity of binding of the cephalosporins combined with Syn2190 to the penicillin-binding proteins or the reversible nature of Syn2190.
Syn2190 combined with ceftazidime or cefpirome at a ratio of 1 to 1 was observed to have efficacy in vivo; in contrast, tazobactam showed little efficacy against systemic infections caused by P. aeruginosa. This efficacy occurred even though both ceftazidime (21) and cefpirome (26) possess longer half-lives in plasma than Syn2190. The half-lives of ceftazidime, cefpirome, and Syn2190 were 8.3, 11.4, and 4.5 min, respectively. The data presented above have therefore shown that the combination of Syn2190 plus a cephalosporin antibiotic, e.g., ceftazidime or cefpirome, may provide an effective treatment for drug-resistant infections caused by P. aeruginosa, E. cloacae, and M. morganii, among others.
Resistant gram-negative bacteria, such as P. aeruginosa and Enterobacter, Citrobacter, Morganella, and Serratia spp., that inducibly or constitutively produce large amounts of β-lactamase have recently been isolated in response to the frequent clinical use of expanded-spectrum cephalosporins, which are stable to β-lactamases (16, 23). The combination of a cephalosporin with the β-lactamase inhibitor Syn2190 is considered to be promising for the treatment of infections caused by these resistant strains and should be evaluated further. It is also expected to diminish the prevalence of resistant strains.
REFERENCES
- 1.Aronoff S C, Shlaes D M. Factors that influence the evolution of β-lactam resistance in β-lactamase-inducible strains of Enterobacter cloacae and Pseudomonas aeruginosa. J Infect Dis. 1987;155:936–941. doi: 10.1093/infdis/155.5.936. [DOI] [PubMed] [Google Scholar]
- 2.Aronoff S C, Jacobs M R, Johenning S, Yamabe S. Comparative activities of the β-lactamase inhibitors YTR830, sodium clavulanate, and sulbactam combined with amoxicillin or ampicillin. Antimicrob Agents Chemother. 1984;26:580–582. doi: 10.1128/aac.26.4.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bush K. Classification of β-lactamases: group 1, 2a, 2b, and 2b′. Antimicrob Agents Chemother. 1989;33:264–270. doi: 10.1128/aac.33.3.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Bush K, Jacoby G A, Medeiros A A. A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother. 1995;39:1211–1233. doi: 10.1128/aac.39.6.1211. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Bush K, Sykes R B. Methodology for the study of β-lactamases. Antimicrob Agents Chemother. 1986;30:6–10. doi: 10.1128/aac.30.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Choi K I, Cha J H, Pae A N, Cho Y S, Kho H Y, Chang M H, Kang H Y, Chung B Y. Studies on new catechol containing cephalosporins. III. Synthesis and structure-activity relationships of cephalosporins having a pyridone moiety at C-7 position. J Antibiot. 1997;50:279–282. [PubMed] [Google Scholar]
- 7.Doren G V, Pfaller M A, Jones R N The National Broad Spectrum β-Lactam Surveillance Group. Program and abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, D.C: American Society for Microbiology; 1997. In vitro surveillance study of the antimicrobial spectrum of newer cephalosporins and other β-lactam tested against non-enteric gram-negative bacilli—1997 result, abstr. E-95; p. 130. [Google Scholar]
- 8.Gutmann L, Kitzis M D, Billot-Klein D, Goldstein F, Tran Van Nhieu G, Lu T, Carlet J, Williamson R. Plasmid-mediated β-lactamase (TEM-7) involved in resistance to ceftazidime and aztreonam. Rev Infect Dis. 1988;10:860–866. doi: 10.1093/clinids/10.4.860. [DOI] [PubMed] [Google Scholar]
- 9.Gutmann L, Ferre B, Goldstein F W, Rizk N, Pint-Schuster E, Acar J F, Collatz E. SHV-5, a novel SHV-type β-lactamase that hydrolyzes broad-spectrum cephalosporins and monobactams. Antimicrob Agents Chemother. 1989;33:951–956. doi: 10.1128/aac.33.6.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Hancock R E W, Woodruff W A. Roles of porin and β-lactamase in β-lactam resistance of Pseudomonas aeruginosa. Rev Infect Dis. 1988;10:770–775. doi: 10.1093/clinids/10.4.770. [DOI] [PubMed] [Google Scholar]
- 11.Higashitani F, Hyodo A, Ishida N, Inoue M, Mitsuhashi S. Inhibition of β-lactamases by tazobactam and in-vitro antibacterial activity of tazobactam combined with piperacillin. J Antimicrob Chemother. 1990;25:567–574. doi: 10.1093/jac/25.4.567. [DOI] [PubMed] [Google Scholar]
- 12.Higashitani F, Nishida K, Hyodo A. Effects of tazobactam on the frequency of the emergence of resistant strains from Enterobacter cloacae, Citrobacter freundii and Proteus vulgaris. J Antibiot. 1995;48:1027–1033. doi: 10.7164/antibiotics.48.1027. [DOI] [PubMed] [Google Scholar]
- 13.Joris B, De Meester F, Galleni M, Reckinger G, Coyette J, Frere J M, Van Beeuman J. The beta-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6 beta-halogenopenicillinates. Biochem J. 1985;228:241–248. doi: 10.1042/bj2280241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kobayashi S, Arai S, Hayashi S, Fujimoto K. β-Lactamase stability of cefpirome (HR 810), a new cephalosporin with a broad antimicrobial spectrum. Antimicrob Agents Chemother. 1986;30:713–718. doi: 10.1128/aac.30.5.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Kuck H A, Jacobus N V, Petersen P J, Weiss W J, Testa R T. Comparative in vitro and in vivo activities of piperacillin combined with the β-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam. Antimicrob Agents Chemother. 1989;33:1964–1969. doi: 10.1128/aac.33.11.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Livermore D M. Clinical significance of beta-lactamase induction and stable derepression in gram-negative rods. Eur J Clin Microbiol. 1987;6:439–445. doi: 10.1007/BF02013107. [DOI] [PubMed] [Google Scholar]
- 17.Maejima T, Inoue M, Mitsuhashi S. In vitro antibacterial activity of KP-736, a new antibiotic. Antimicrob Agents Chemother. 1991;35:104–110. doi: 10.1128/aac.35.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Mochizuki H, Yamada H, Oikawa Y, Murakami K, Ishiguro J, Kosuzume H, Aizawa N, Mochida E. Bactericidal activity of M14659 enhanced in low-iron environments. Antimicrob Agents Chemother. 1988;32:1648–1654. doi: 10.1128/aac.32.11.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Moosdeen F, Makell J, Philpott-Howard J, Williams J D. Cefotetan activity against gram-negative aerobes and anaerobes. J Antimicrob Chemother. 1983;11(Suppl.):59–65. doi: 10.1093/jac/11.suppl_a.59. [DOI] [PubMed] [Google Scholar]
- 20.Nicas T I, Hancock R E W. Pseudomonas aeruginosa outer membrane permeability: isolation of a porin F-deficient mutant. J Bacteriol. 1983;153:281–285. doi: 10.1128/jb.153.1.281-285.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Okumura K, Tsuji H, Takeda K, Fukuda I, Nagaki T, Takano M, Higo K, Kinami J. Absorption, distribution metabolism and excretion of ceftazidime in mice, rats and rabbits. Chemotherapy (Tokyo) 1983;31(Suppl. 3):188–198. [Google Scholar]
- 22.Pfaller M A, Dosen G V, Jones R N The National Broad Spectrum β-Lactam Surveillance Group. Program and abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, D.C: American Society for Microbiology; 1997. Activity of broad-spectrum β-lactam antimicrobials versus Bush group 1 enzyme producing species, abstr. E-150; p. 141. [Google Scholar]
- 23.Schryvers A B, Ogunariwo J, Chamberland S, Godfrey A J, Rabin H R, Bryan L E. Mechanism of Pseudomonas aeruginosa persistence during treatment with broad-spectrum cephalosporins of lung infections in patients with cystic fibrosis. Antimicrob Agents Chemother. 1987;31:1438–1439. doi: 10.1128/aac.31.9.1438. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Silley P, Griffiths J W, Monsey D, Harris A M. Mode of action of GR69153, a novel catechol-substituted cephalosporin, and its interaction with the tonB-dependent iron transport system. Antimicrob Agents Chemother. 1990;34:1806–1808. doi: 10.1128/aac.34.9.1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Spratt R G. Distinct penicillin-binding proteins involved in the division, and shape of Escherichia coli. Proc Natl Acad Sci USA. 1975;72:2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Tabata S, Ogawa T, Inazu M, Hayashi S. Cefpirome sulfate: pharmacokinetics and mechanism of excretion in rats with experimentally induced renal and hepatic injuries. Chemotherapy (Tokyo) 1991;39(Suppl. 1):100–104. [Google Scholar]