Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with ampicillin and broad-spectrum penicillins against defined beta-lactamase-producing aerobic gram-negative bacilli

The in vitro synergistic activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam, combined with ampicillin, ticarcillin, mezlocillin, azlocillin, piperacillin, and apalcillin, were determined against 34 strains of members of the Enterobacteriaceae family, Pseudomonas aeruginosa, Aeromonas hydrophila, and Haemophilus influenzae with characterized plasmid or chromosomal beta-lactamases or both. Strains were tested against fixed concentrations of beta-lactamase inhibitors (8 micrograms/ml) combined with doubling dilutions of beta-lactams. Synergy was defined as a fourfold or greater decrease in the MIC of the beta-lactam. Against Enterobacteriaceae producing Richmond and Sykes class III and V plasmid-mediated beta-lactamases, synergy was obtained against most strains with YTR 830- and clavulanate-beta-lactam combinations, with sulbactam being less effective. Against Enterobacteriaceae producing class I chromosomal beta-lactamases, combinations containing YTR 830 or sulbactam were more synergistic than combinations containing clavulanate. Against strains producing class V PSE enzymes, all three inhibitors were synergistic with piperacillin and apalcillin against strains producing PSE-1, -3, and -4 enzymes, while the PSE-2-producing strain was resistant to all inhibitors. YTR 830-beta-lactam combinations were also synergistic against strains producing the novel beta-lactamases OHIO-1, TLE-1, AER-1, and ROB-1. Overall, YTR 830 with piperacillin or apalcillin was the most effective combination.     

Comparative activity of beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with beta-lactams against beta-lactamase-producing anaerobes.

The in vitro activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with six beta-lactams against 88 beta-lactamase-producing anaerobes were determined. When combined with the beta-lactams, the three beta-lactamase inhibitors showed no synergy against the 10 Bacteroides fragilis homology group II strains. When the beta-lactams were combined with the inhibitors, their geometric mean MICs against the remaining 78 strains were reduced from 4.2 to 150.2 micrograms/ml to 0.2 to 12.9 micrograms/ml. The activity of the beta-lactams combined with the beta-lactamase inhibitors was significantly greater than that of the beta-lactams alone against all groups except B. fragilis homology group II, with 76 to 100% of the strains susceptible to ampicillin plus inhibitor and greater than or equal to 90% susceptible to the other combinations.     

Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate and sulbactam combined with extended-spectrum penicillins against ticarcillin-resistant Enterobacteriaceae and pseudomonads

The in-vitro synergistic activity of YTR 830, a new beta-lactamase inhibitor, combined with four extended-spectrum penicillins (ticarcillin, piperacillin, mezlocillin and apalcillin) against ticarcillin-resistant clinical isolates of Gram-negative enteric bacilli was compared with that of clavulanate and sulbactam. Synergy testing was performed with fixed concentrations of beta-lactamase inhibitors (8 mg/l) combined with doubling dilutions of beta-lactams in microdilution trays. Synergy was defined as a four-fold or greater decrease of beta-lactam MIC in the combination compared with the beta-lactam alone. For 79 ticarcillin-resistant Enterobacteriaceae, ticarcillin-YTR 830 and ticarcillin-clavulanate were synergistic against 90% of strains; for ticarcillin-sulbactam, 70% showed synergy. The synergistic activity of all three inhibitors was similar against strains resistant only to ticarcillin; for strains resistant to all four extended-spectrum penicillins, the activity of ticarcillin with YTR 830 and clavulanate was similar (synergy against 79% of strains) and superior to ticarcillin-sulbactam (synergy against 39% of strains). YTR 830 was more active than clavulanate against Serratia, Citrobacter, Proteus and Providencia spp. Piperacillin, mezlocillin and apalcillin susceptible strains, with MICs of 8-16 mg/l, showed synergy with inhibitors against 37-87% of strains. Amongst pseudomonads, no synergy was demonstrated against Pseudomonas aeruginosa; ticarcillin produced synergy with the inhibitors against Ps. maltophilia, while piperacillin-YTR 830 and apalcillin-YTR 830 were synergistic against Ps. cepacia. YTR 830 appears to have comparable in-vitro activity to that of clavulanate, and further development of this compound is warranted.     

Comparative in vitro activity of piperacillin combined with the beta-lactamase inhibitor tazobactam (YTR 830)

Combination with tazobactam substantially enhanced the activity of piperacillin against routine isolates of staphylococci, various Enterobacteriaceae, Acinetobacter anitratus, and Bacteroides fragilis. Tazobactam enhanced the activity of piperacillin more than fourfold against Pseudomonas aeruginosa spp. harboring eight of 12 plasmid-mediated beta-lactamases.     

Antibacterial characteristics of YTR 830, a sulfone beta-lactamase inhibitor, compared with those of clavulanic acid and sulbactam.

The antibacterial activity, binding to penicillin-binding proteins, and morphological changes effected by YTR 830, a sulfone beta-lactamase inhibitor, were studied in comparison with those of other beta-lactamase inhibitors. YTR 830 had very poor antibacterial activity, bound to PBP 2 of gram-negative organisms, and at the MIC caused rapid lysis of spheroplasts formed.     

In-vitro and in-vivo activities of sulbactam and YTR830H against Acinetobacter calcoaceticus

The antibacterial activities of sulbactam and YTR830H, both beta-lactamase inhibitors, were studied against Acinetobacter calcoaceticus, in vitro and in vivo. In-vitro susceptibility tests showed both to be very active against 80 clinical isolates of A. calcoaceticus. Against an intraperitioneal infection in mice, caused by A. calcoaceticus Ac54, the 50% effective doses of sulbactam and YTR830H were 1.62 and 16.7 mg/kg, respectively. Against A. calcoaceticus S31, a strain resistant to gentamicin, sulbactam and YTR830H were active both in vitro and in vivo.     

Synergy of amoxycillin combined with clavulanate and YTR 830 in experimental infections in mice

YTR 830, a new beta-lactamase inhibitor, is synergistic with amoxycillin in vitro against a number of beta-lactamase-producing organisms. The combination of amoxycillin-YTR 830 was compared to amoxycillin-clavulanate in the treatment of experimental Staphylococcus aureus, Citrobacter freundii and Proteus mirabilis infections in mice. Both combinations were synergistic with amoxycillin against all three test organisms. The amoxycillin-clavulanate combination was superior against S. aureus and C. freundii while amoxycillin-YTR 830 was more effective against P. mirabilis. The difference in efficacy between the two drug combinations appears to relate to the degree of protection afforded the animals by the beta-lactamase inhibitor alone. YTR 830 is a promising new agent and should undergo further investigation.     

Comparative in vitro and in vivo activities of piperacillin combined with the beta-lactamase inhibitors tazobactam, clavulanic acid, and sulbactam.

Tazobactam (YTR-830H), a novel beta-lactamase inhibitor, was compared with clavulanic acid and sulbactam for enhancement of the activity of piperacillin against beta-lactamase-producing, piperacillin-resistant clinical isolates. Piperacillin MICs were determined in media containing a fixed concentration of 2 or 4 micrograms of the inhibitors per ml. The higher concentration was generally more effective. Tazobactam was superior to sulbactam in enhancing the spectrum and potency of piperacillin. Although the calvulanic acid combination was more potent, tazobactam was effective for a similar spectrum of resistant gram-negative clinical isolates containing beta-lactamase. MICs were reduced to the susceptible range for Escherichia coli, Klebsiella pneumoniae, Proteus spp., Salmonella spp., and Shigella spp. Combinations with tazobactam and sulbactam, but not clavulanic acid, were effective against Morganella spp. Some antagonism of the activity of piperacillin was observed with clavulanic acid but not with tazobactam or sulbactam. The inhibitors were similarly effective with piperacillin against beta-lactamase-positive Staphylococcus spp. and the Bacteroides fragilis group. Piperacillin-tazobactam was more effective against a broader spectrum of gram-negative enteric bacteria than ticarcillin plus clavulanic acid was. Combinations with tazobactam or clavulanic acid had a broader spectrum of activity than combinations with sulbactam against bacteria that produce characterized plasmid-mediated enzymes of clinical significance. In particular, piperacillin with tazobactam or clavulanic acid, but not with sulbactam, inhibited TEM-1, TEM-2, and SHV-1 enzymes. In vitro activity was reflected in vivo. Tazobactam and clavulanic acid were superior to sulbactam in enhancing the therapeutic efficacy of piperacillin in mice infected with beta-lactamase-positive E. coli, K. pneumoniae, Proteus mirabilis, and Staphylococcus aureus. Only combinations with tazobactam and sulbactam were effective against the Morganella infection. Tazobactam has a good potential for enhancing the clinical efficacy of piperacillin.     

Comparative evaluation of a new beta-lactamase inhibitor, YTR 830, combined with different beta-lactam antibiotics against bacteria harboring known beta-lactamases.

YTR 830, a new beta-lactamase inhibitor, combined with amoxicillin or carbenicillin, showed a synergistic effect similar to that observed with clavulanic acid, and generally better than that with sulbactam, against strains harboring chromosome-encoded penicillinases and broad-spectrum beta-lactamases or plasmid-determined beta-lactamases. With ampicillin, YTR 830 showed the best synergistic activity of the inhibitors against Proteus morganii, Citrobacter freundii, and Enterobacter cloacae and their mutants with a derepressed chromosome-encoded cephalosporinase.     

In-vitro activity of the combinations of ampicillin with mecillinam or with beta-lactamase inhibitors against strains resistant to ampicillin

The in-vitro activity of ampicillin, of mecillinam and of combinations of ampicillin with mecillinam, clavulanic acid or 6 beta-bromopenicillanic acid has been studied against 126 Enterobacteriaceae resistant to ampicillin. The combination of ampicillin with mecillinam showed synergy or addition in 60% of the combinations tested. Synergy was seen especially when the strains were resistant to mecillinam, indifference when they were susceptible to mecillinam. The combination of ampicillin with mecillinam was more active than the combination with clavulanic acid against Escherichia coli, Klebsiella and Enterobacter, but not against Proteus, Morganella and Providencia. The combination of ampicillin with clavulanic acid was more active than the combination with 6 beta-bromopenicillanic acid in E. coli, Klebsiella and Enterobacter strains.     

Activity of ticarcillin/clavulanate and piperacillin/tazobactam (YTR 830; CL-298,741) against clinical isolates and against mutants derepressed for class I beta-lactamase

Piperacillin/tazobactam (YTR 830; CL-298,741) was tested against fresh and stock clinical isolates of Gram-negative bacilli, as well as against Gram-negative bacilli that had stably derepressed Class I beta-lactamases or that were hyperproductive of non-Class I beta-lactamases. Of 63 clinical isolates of the Enterobacteriaceae with ticarcillin (plus clavulanate) minimum inhibitory concentrations (MICs) of greater than or equal to 128 micrograms/ml, 16 had piperacillin/tazobactam MICs of less than or equal to 16/2 micrograms/ml. Of 48 clinical isolates of Pseudomonas spp. with ticarcillin (plus clavulanate) MICs of greater than or equal to 128 micrograms/ml, 35 had piperacillin/tazobactam MICs of less than or equal to 64/8 micrograms/ml. Tazobactam generally reduced piperacillin MICs by two- to greater than or equal to eightfold against stably derepressed mutants for Class I beta-lactamases.     

Activity of cefoperazone and two beta-lactamase inhibitors, sulbactam and clavulanic acid, against Bacteroides spp. correlated with beta-lactamase production.

A total of 102 isolates of Bacteroides spp. were studied for beta-lactamase production and susceptibility to cefoperazone alone or in combination with either of the beta-lactamase inhibitors sulbactam and clavulanic acid. The geometric mean minimal inhibitory concentration of cefoperazone alone was 31.5 micrograms/ml and when combined with 10 micrograms of sulbactam per ml or 2 micrograms of clavulanic acid per ml was reduced to 5.4 and 9.2 micrograms/ml, respectively. When bacterial suspensions were tested for beta-lactamase production with nitrocefin, 91 (89.2%) of these isolates produced the enzyme. The geometric mean minimal inhibitory concentrations of cefoperazone rose only slightly for isolates with low or intermediate enzyme activity but rose significantly for those with high activity. The addition of EDTA to cefoperazone significantly more frequently enhanced the activity of cefoperazone against beta-lactamase-negative as opposed to beta-lactamase-positive isolates. Furthermore, EDTA resulted in synergistic activity of the cefoperazone-sulbactam combination on beta-lactamase-positive isolates for which the combination had previously not shown a synergistic effect. This study demonstrates the relationship between beta-lactamase production and the resistance of Bacteroides spp. to cefoperazone and shows that inhibition of these enzymes can reverse this resistance.     

Studies to optimize the in vitro testing of piperacillin combined with tazobactam (YTR 830)

The combination of piperacillin and the beta-lactamase inhibitor tazobactam (formerly YTR 830) was studied to determine optimal disk concentrations and dilution testing conditions. In addition, the potency of the combination was compared to that of piperacillin alone. The spectrum of piperacillin was greatly expanded by the addition to tazobactam principally against beta-lactamase producing strains of Haemophilus influenzae, Escherichia coli, Morganella morganii, Proteus vulgaris, Providencia stuartii, Shigella spp., Neisseria gonorrhoeae, and Staphylococcus spp. Tazobactam was active alone against Branhamella catarrhalis (minimum inhibitory concentration [MIC] 50, less than or equal to 1 microgram/ml), gonococci (MIC 50, 0.5-4 micrograms/ml), and N. meningitidis (MIC 50, less than or equal to 1 microgram/ml). Studies with beta-lactamase-producing type strains showed tazobactam to have high affinity for plasmid-mediated enzymes (TEM-1 and 2, SHV-1, HMS-1, and some CARB or OXA types) and not chromosomal beta-lactamases. Piperacillin/tazobactam inhibited 93% of fluoro-quinolone resistant strains at less than or equal to 64/8 micrograms/ml but failed to suppress the growth of 15 strains producing stably depressed cephalosporinases. Comparisons of piperacillin/tazobactam results determined with 100/10-, 100/20-, and 100/30-micrograms disks established the 100/10-micrograms disk as most usable. Among five different MIC combinations the ratio of eight parts piperacillin to one part tazobactam or fixed concentration tests at greater than or equal to 4 micrograms tazobactam/ml were preferred, each producing very low occurrences (less than or equal to 1.6%) of false-resistance or -susceptibility when compared to disk test results. MICs determined by agar and broth microdilution methods were essentially the same. The recommended breakpoints for piperacillin/tazobactam MICs were identical to those now found in the NCCLS susceptibility testing standards with the following exceptions: (1) for tests with H. influenzae and Staphylococcus spp.--susceptible at greater than or equal to 21 mm (MIC less than or equal to 16/2 micrograms/ml) and resistant less than or equal to 20 mm (MIC less or equal to 32/4 micrograms/ml); and (2) all remaining nonspeudomonas isolates would be interpreted by the NCCLS piperacillin enteric bacilli susceptibility criteria. This newer beta-lactamase inhibitor combination appears to be worthy of further in vivo trials guided by these or similar tentative in vitro susceptibility testing parameters.     

In vitro activities of the beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam alone or in combination with beta-lactams against epidemiologically characterized multidrug-resistant Acinetobacter baumannii strains

Acinetobacter baumannii is an important nosocomial pathogen usually in the context of serious underlying disease. Multidrug resistance in these organisms is frequent. The beta-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam have intrinsic activity against Acinetobacter strains. To evaluate their potential therapeutic usefulness, we determined the in vitro activity of ampicillin, sulbactam, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, piperacillin, piperacillin-sulbactam, tazobactam, piperacillin-tazobactam, amoxicillin, clavulanic acid, amoxicillin-clavulanic acid, ticarcillin, and ticarcillin-clavulanic acid against multidrug-resistant A. baumannii. All isolates were epidemiologically characterized by RAPD [random(ly) amplified polymorphic DNA] analysis and/or pulsed-field gel electrophoresis and represented different strain types, including sporadic strains, as well as outbreak-related strains. The MICs were determined by agar dilution on Mueller-Hinton agar (using fixed concentrations, as well as fixed ratios for beta-lactamase inhibitors) and the E-test. The majority of E-test results were within two dilutions of those recorded by agar dilution, with the exception of piperacillin-tazobactam. Sulbactam was superior to clavulanic acid and tazobactam and may represent an alternative treatment option for infections due to multiresistant A. baumannii strains. beta-Lactamase inhibitors have intrinsic activity but do not enhance activity of beta-lactams against A. baumannii. Testing with the inhibitor added at a fixed concentration as recommended for piperacillin-tazobactam and ticarcillin-clavulanic acid by the National Committee for Clinical Laboratory Standards may falsely suggest high activity or gives uninterpretable results due to trailing. If combinations are used for testing, fixed ratios may give more useful results.     

In vitro efficacy of sulbactam combined with ampicillin against anaerobic bacteria.

An ampicillin-sulbactam combination was compared with ampicillin alone, chloramphenicol, clindamycin, and metronidazole against 272 strains of anaerobic bacteria. Chloramphenicol and ampicillin-sulbactam were the most effective, inhibiting 98 to 99% of strains tested at breakpoint (16 micrograms/ml). The combination of sulbactam and ampicillin was much more effective than ampicillin alone against Bacteroides fragilis strains but did not differ substantially from ampicillin alone against Fusobacterium spp., gram-positive rods, and gram-positive cocci.     

Comparative tolerability of ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole suspensions in children with otitis media.

The tolerabilities of ampicillin, amoxicillin, and trimethoprim-sulfamethoxazole (TMP-SMX) suspensions were evaluated in 263 children with otitis media. Because of watery stools, therapy was discontinued in 6 of 83 patients treated with ampicillin, in none of 89 patients treated with amoxicillin (P less than 0.01), and in 1 of 91 patients treated with TMP-SMX (P less than 0.03). Of the patients who completed the treatment courses, 13 recipients of ampicillin suffered loose or watery stools for 4 or more days, compared with 6 of the amoxicillin recipients (P less than 0.04) and 5 of the TMP-SMX recipients (P less than 0.02). Thus, ampicillin was clearly less well tolerated than either amoxicillin or TMP-SMX.     

Contribution of beta-lactamase and PBP amino acid substitutions to amoxicillin/clavulanate resistance in beta-lactamase-positive, amoxicillin/clavulanate-resistant Haemophilus influenzae

The roles of beta-lactamase and alterations in penicillin-binding protein in the development of amoxicillin and amoxicillin/clavulanate resistance in two beta-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains of Haemophilus influenzae were investigated. Seven beta-lactamase-negative, ampicillin-resistant (BLNAR) strains were also studied for comparison of their resistance mechanisms. All strains had been recovered from patients in Japan. The TEM type beta-lactamase of the two BLPACR strains had 100% homology with the amino acid sequences of published TEM-1 beta-lactamase, showing that amoxicillin/clavulanate resistance was not associated with mutations in this beta-lactamase. However, these strains, as well as the seven BLNAR strains, had multiple mutations in ftsI, which encodes penicillin binding protein 3 (PBP3). The transformation of H. influenzae Rd strain with amplified ftsI genes from two BLPACR and two BLNAR strains enabled the selection of amoxicillin/clavulanate-resistant transformants with the same mutations as their parent strains. We concluded that amoxicillin/clavulanate resistance in the two BLPACR strains was due to changes in PBP3. The possibility of the presence of an extended spectrum beta-lactamase was excluded in the BLPACR strains studied.     

Oxidant-scavenging activities of ampicillin and sulbactam and their effects on neutrophil functions.

Luminol-enhanced luminescence is a method used to measure formation of reactive oxygen intermediates important in the ability of neutrophils to kill microbes. Several studies have demonstrated that under some conditions of incubation, ampicillin can inhibit neutrophil-derived luminol-enhanced luminescence. We evaluated the mechanism(s) by which ampicillin inhibited the luminescent response of stimulated neutrophils. We also investigated sulbactam, a beta-lactamase inhibitor which has been given in combination with ampicillin and other beta-lactam antibiotics to increase their spectra, for possible similar effects. Both ampicillin and sulbactam attenuated luminol-enhanced luminescence by approximately 40%. Superoxide production was not prevented by added ampicillin, nor was superoxide scavenged by it. Myeloperoxidase reacts with H2O2 and Cl- to generate OCl-, which is believed to be the oxidizer of luminol that is primarily responsible for enhancement of neutrophil-derived luminescence. Hydroxyl radicals (HO.), which may also oxidize luminol, resulting in luminescence, can be formed from O2- and H2O2 via either myeloperoxidase-dependent (involving intermediate OCl-) or myeloperoxidase-independent (through a metal ion catalyst) reactions. Ampicillin scavenged H2O2 and OCl- and prevented 95% of Fenton reaction-generated HO. from reacting with 5,5-dimethyl-1-pyrroline-N-oxide. Sulbactam was found to scavenge OCl- and HO., but less avidly than ampicillin did. Neither ampicillin nor sulbactam inhibited myeloperoxidase activity. Sublethal concentrations of sulbactam had no significant effect on neutrophil killing of Staphylococcus aureus and Escherichia coli. Our results demonstrate a mechanism(s) by which ampicillin inhibits luminol-enhanced luminescence from stimulated neutrophils, namely, through scavenging of the oxidant(s) primarily responsible for the generation of luminescence.