Recommendations for Dilution Susceptibility Testing Concentrations of the Cefoperazone-Sulbactam Combination

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1987. p. 1725-1729 0095-1137/87/091725-05$02.00/O Copyright (O 1987, American Society for Microbiology Vol. 25, No. 9 In Vitro Antimicrobial Spectrum, Occurrence of Synergy, and Recommendations for Dilution Susceptibility Testing Concentrations of the Cefoperazone-Sulbactam Combination RONALD N. JONES,l* ARTHUR L. BARRY,1 RICHARD R. PACKER,' WILLIAM W. GREGORY,- AND CLYDE THORNSBERRY3 Tue Clinical Microbiology Institute, Tualatin, Oregon 970621; Pfizer Pha-rinaceuticals Inc., Newl, York, New York 100172; andi T/e Centzers foi Diseuise Control, Atlanta, Georgia 303333 Received 11 March 1987/Accepted 21 May 1987 Broth microdilution tests and antimicrobial interaction (synergy) studies using various combinations of cefoperazone and sulbactam were performed in an effort to determine the most appropriate in vitro dilution test system. The test results with cefoperazone and sulbactam were categorized as synergistic (complete or partial) for nearly 80% of the strains isolated from clinical trial patients. The results indicate that the cefoperazone-sulbactam fixed ratio (2:1) maximized the cefoperazone spectrum of activity and best approximated the parenteral formulation of the drug. The cefoperazone-sulbactam combination had a greater antimicrobial activity than did the other comparison beta-lactams, except for imipenem, tested against strains of the family Enterobacteriaceae. To be consistent with the National Committee for Clinical Laboratory Standards interpretive breakpoints for cefoperazone alone, the following MIC breakpoints should be applied to the combination (2:1 ratio): -<16/8,ug/ml, susceptible; 32/16,ug/ml, moderately susceptible; and -64/32,ug/ml, resistant. Beta-lactamase inhibitors, such as clavulanic acid, sulbactam, and YTR 830, have generally been used to expand the antimicrobial spectrum of beta-lactamase-labile beta-lactams (1-3, 5-9, 11, 16). Aminopenicillins and ticarcillin have received the greatest attention by virtue of studies of amoxicillin-clavulanate (Augmentin), ticarcillin-clavulanate (Timentin), and ampicillin-sulbactam (Unasyn) (1-3, 8). Penicillins are usually hydrolyzed by the more frequently encountered bacterial beta-lactamases such as the staphylococcal penicillinases and gram-negative bacterial plasmid-mediated enzymes, i.e., TEM, SHV-1, and OXA (15). Therefore, a significant spectrum enhancement could be achieved by combining penicillins with beta-lactamase inhibitors. The increased spectrum should also include remarkable activity against the Bacteroidesfragilis group and some other penicillin-resistant anaerobic bacteria (1, 3, 9). The in vitro recognition of these favorable combinations has focused on the testing of both drugs in dilution and disk diffusion systems. Combinations administered as oral agents have been tested in vitro in fixed ratios, attempting to simulate in vivo pharmacokinetic conditions (13, 15). In contrast, ticarcillin-clavulanic acid dilution tests use a single concentration of clavulanic acid (2.0,ug/ml) added to serial dilutions of ticarcillin. The kinetics of the drug at this concentration approximates the in vivo kinetics of the 100- or 200-mg intravenous inhibitor dose, and it is used in an attempt to recognize in vitro the favorable effects of the clavulanate (8, 13, 14). Cefoperazone, a widely used broad-spectrum cephalosporin, possesses acceptable beta-lactamase stability, especially against gram-positive and chromosome-mediated gramnegative enzymes (4). However, some more frequently isolated beta-lactamases found in members of the family Eniterobacteriaceae can efficiently destroy cefoperazone (5, 10, 12). Also, the cephalosporinases produced by the B. * Corresponding author. firagilis group hydrolyze this newer cephalosporin (1, 3, 5, 6). Sulbactam combined with cefoperazone prevents its destruction by some clinically prevalent beta-lactamases, especially those produced by Esciherichia coli and the anaerobes. However, because of the very wide spectrum of activity of cefoperazone, the increased benefit due to the sulbactam appears small (,<5%) when routinely isolated clinical strains are tested (9, 11). Therefore, in this investigation we concentrated our in vitro testing on those strains most often found to be resistant to cefoperazone and other newer cephalosporins. In addition, drug interaction studies with a fixed ratio and with single inhibitor concentrations were evaluated. The data were analyzed to determine the optimal method for testing the cefoperazone-sulbactam combination, correlating dilution test results to the parenteral formulation and pharmacokinetic properties of the drug. (This material was presented in part at the 25th Interscience Conference on Antimicrobial Agents and Chemotherapy [R. R. Packer, A. L. Barry, R. N. Jones, and W. W. Gregory, Program Abstr. 25th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 337, 1985].) MATERIALS AND METHODS Antimicrobial agents. Cefoperazone and sulbactam were obtained from Pfizer Inc., New York, N.Y. The other comparison beta-lactams were obtained from E. R. Squibb & Sons, Princeton, N.J. (aztreonam), Hoechst-Roussel Pharmaceuticals, Inc., Somerville, N.J. (cefotaxime), Glaxo, Inc., Research Triangle Park (ceftazidime), and Merck & Co., Inc., Rahway, N.J. (imipenem). Bacterial isolates. Recent isolates, typical of routine clinical strains, were collected from five geographically separate microbiology laboratories. This organism group was assembled to reflect the normal composition of clinically significant bacterial populations and included strains similar to those used in earlier studies of cefoperazone-sulbactam (10, 12). Additional strains included those resistant to cefoperazone 1725

1726 JONES ET AL. (MIC, -64 ptg/ml) and/or other newer cephalosporins (MIC,.32 kg/ml). In some instances, strains resistant to these drugs could not be identified among cited species. The 948 isolates are shown in Table 1. We also tested 333 gramnegative organisms isolated from patients treated with cefoperazone-sulbactam in previous clinical trials (data on file, Pfizer Inc.). This set of organisms included 126 anaerobes, 47 Pseudomonas aeruginosa isolates, 4 Pseudomonas spp. isolates, 19 Acinetobacter spp. isolates, 86 E. coli isolates, and 51 other isolates of Enterobacteriaceae. These latter organisms were tested for drug interaction (synergy) only. Antimicrobial susceptibility testing methods. MICs were determined by the broth microdilution method described in detail in previous reports (9-11) and by the National Committee for Clinical Laboratory Standards (13, 14). The inoculum contained ca. 5 x 105 CFU/ml. Mueller-Hinton broth was supplemented with 2 to 3% lysed horse blood or other appropriate reagents (14) for testing beta-hemolytic Streptococcus species, Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenza. Tests with strains of Neisseria gonorrhoeae were performed by the agar dilution procedure with Proteose Peptone no. 3 agar (Difco Laboratories, Detroit, Mich.) supplemented with 1% hemoglobin and 1% Kellogg defined supplement. Beta-lactamase tests were performed with nitrocefin reagent in microdilution trays. Cefoperazone-sulbactam MICs were determined in cationsupplemented Mueller-Hinton broth (Difco) for facultative strains and in Wilkins-Chalgren medium without agar for anaerobes. Fixed concentrations of sulbactam of 1.0, 2.0, or 4.0 ptg/ml were added to increasing twofold dilutions of cefoperazone (range, 0.015 to 512 ptg/ml), and the two drugs were also combined in a ratio of 2 parts cefoperazone to 1 part sulbactam. For the checkerboard drug interaction (synergy) technique, broth microdilution trays were prepared in which cefoperazone was combined with sulbactam. Methods and interaction definitions described previously and divalent cation-supplemented Mueller-Hinton broth or Wilkins- Chalgren broth were used (10). Each test strain was inoculated into the plastic trays at a final density of 5 x 105 CFU/ml (106 CFU/ml for anaerobes) and then incubated overnight (for aerobes) or for 48 h (for anaerobes), the MICs were read, and isobolograms were plotted. A total of 333 drug synergy studies were performed. Synergy was considered a questionable significance if either drug was highly active (MIC, 'z1,ug/ml) against a strain when tested singly. RESULTS The results of testing 948 organisms against cefoperazonesulbactam combinations and three comparison drugs are shown in Table 1. The addition of sulbactam to cefoperazone increased its in vitro spectrum of antimicrobial activity, principally among members of the Enterobacteriaceae and Bacteroides species. The presence of 2.0 ptg of sulbactam per ml converted 22 of 63 cefoperazone-resistant (MIC, -64 kg/ml) enteric bacilli to susceptible. Furthermore, by increasing the sulbactam concentration to 4 kg/ml, only two additional strains (a Serratia sp. and an Enterobacter aerogenes isolate) were converted to susceptible. Among the members of the Enterobacteriaceae, the species that usually produce type I cephalosporinases were rendered more cefoperazone susceptible, whereas Serratia, Salmonella, and Klebsiella strains were less favorably affected. Sulbactam at any concentration or test ratio did not significantly expand J. CLIN. MICROBIOL. the already excellent cefoperazone spectrum against Pseudomonas species, Staphylococcus species, streptococci, Branhamella catarrhalis, H. influenza, or Neisseria species. Direct inhibitory antimicrobial activity of sulbactam against the Acinetobacter spp. isolates was also observed (MIC for 90% of isolates tested, 2.0 ptg/ml; data not shown). The 205 Bacteroides strains were tested by the broth microdilution method. Of 71 cefoperazone-resistant Bacteroides strains, 56 (79%) and 62 (87%) were susceptible to cefoperazone when combined with 2.0 and 4.0 tg of sulbactam per ml, respectively. When tested at a 2:1 ratio (cefoperazone-sulbactam), all but two strains of B. fragilis required for inhibition MICs of <32/16 tg/ml. The cefoperazone-sulbactam MICs for 50 and 90% of the Bacteroides strains were 4.0/2.0 and 16/8.0 fig/ml, respectively. The comparison broad-spectrum beta-lactams also failed to inhibit many of these strains. As many as 72% of Citrobacter freundii and 45% of Enterobacter cloacae strains were resistant to ceftazidime. Against the Salmonella spp. and Serratia spp., aztreonam, cefotaxime, and ceftazidime were all superior to cefoperazone with or without sulbactam. Cefoperazone was most effective against the gram-positive species, and the activity was not affected by the addition of sulbactam. Aztreonam was not active against gram-positive cocci. Both cefotaxime and ceftazidime failed to inhibit some coagulase-negative staphylococcal and enterococcal strains that were inhibited by cefoperazone. Imipenem was also tested against the organisms listed in Table 1 (data not shown). The members of the Enterobacteriaceae, S. aureus, Streptococcus spp., and enterococci were all susceptible to imipenem, but for P. aeruginosa and other Pseudomonas spp., imipenem resistance rates were 3 and 15%, respectively. The in vitro antimicrobial spectrum of cefoperazone with and without sulbactam and those of four other drugs are shown in Table 2. Cefoperazone-sulbactam inhibited 87% of the highly resistant members of the Enterobacteriaceae at the susceptible breakpoint concentration for cefoperazone (<16 1tg/ml) and 91% at the moderately susceptible concentration of c32,xg/ml. This spectrum was equal or superior to those of all other beta-lactams tested except imipenem. Cefoperazone-sulbactam was comparable or slightly superior to other comparison drugs against the Pseudomonas spp. and Acinetobacter spp. The coverage of gram-negative anaerobes by cefoperazone was improved from 65 to >99% by adding sulbactam in a 2:1 ratio. The proportions of anaerobes for which cefoperazone MICs were s32 Ftg/ml in combination with 2.0 and 4.0 tg of sulbactam per ml were 95 and 97%, respectively. The aerobic strains were further analyzed for the incidence of at least fourfold reductions in cefoperazone MICs brought about by the addition of sulbactam. A fixed inhibitor concentration of 2 or 4,ug/ml was more active. A total of 173 strains showed a significant reduction of the cefoperazone MIC (fourfold lower) associated with any concentration or ratio of sulbactam. Nearly all of these isolates were already highly susceptible to cefoperazone alone (MIC, s1,ug/ml). The log2 dilution change in the aerobic-organism cefoperazone MICs with 4,ug of sulbactam per ml was -3.4. The cefoperazone dilution change at a 2:1 ratio was -2.1. Statistics for the gram-negative anaerobic bacteria were similar, with a mean -2.3 log2 dilution change in the cefoperazone MIC at the 2:1 ratio. The value for the fixed 4,ig/ml sulbactam concentration test was -2.5 dilutions. Synergy testing was performed on 333 additional strains isolated from 242 patients participating in the cefoperazone-

VOL. 25, 1987 CEFOPERAZONE-SULBACTAM DILUTION TESTS 1727 TABLE 1. Percentage of 948 tested strains resistant to cefoperazone, cefoperazone-sulbactam combinations. aztreonam, cefotaxime, and ceftazidime % Resistant' Organism Cefoperazone with sulbactam at: (no. of strains tested) Aztreonam Cefotaxime Ceftazidime 0 ug/ml 2,ug/ml" 4 pg/ml 2:1 ratio Citrobacter diversus (10) 0 0 0 0 0 0 0 Citrobacterffreundii (18) 56 39 39 6 56 22 72 Enterobacter aerogenes (41) 17 8 5 3 22 5 37 Enterobacter agglomerans (10) 10 0 0 0 0 10 10 Enterobacter cloacae (47) 41 32 32 28 32 38 45 Escherichia coli (34) 9 3 3 0 3 3 3 Klebsiella spp. (30) 20 13 13 13 10 0 0 Morganella morganii (10) 0 0 0 0 0 0 0 Proteus mirabilis (24) 0 0 0 0 0 0 0 Proteus vulgaris (10) 10 0 0 0 0 10 0 Providencia rettgeri (10) 10 10 10 0 0 0 0 Providencia stuarfii (19) 0 0 0 0 0 0 0 Salmnonella spp. (9) 22 22 22 il 0 0 0 Serratia spp. (43) 30 19 16 16 0 2 0 Shigella spp. (8) 0 0 0 0 0 0 0 Acinetobacter anitratus (17) 29 6 6 0 30 6 6 Pseudoinonas aeruginosa (68) 6 6 6 4 15 28 4 Pseudomonas spp. (40)' 15 15 15 10 40 30 10 Branhamnella (catarrhalis (12) 0 0 0 0 0 0 0 Haemopohilus influenzae (40)d 0 0 0 0 0 0 0 Neisseria meningitidis (20) 0 0 0 0 0 0 O Neisseria gonorrhoeae (52)' 0 0 0 0 0 0 0 Staphylococcus aureus (58) 0 0 0 0 100 0 0 Staphylococcus spp., coagulase negative (28) 4 4 4 4 100 7 29 Streptococcus agalactiae (20) 0 0 0 0 100 0 0 Streptococcus pneumoniae (20) 0 0 0 0 100 0 0 Streptococcus pyogenes (20) 0 0 0 0 5 0 0 Enterococcus faecalis (25) 4 4 4 4 100 88 100 Bacteroides fragilis (78) 44 13 8 3 NDf ND ND Bacteroides distasonis (10) 20 20 20 0 ND ND ND Bacteroides ovatus (31) 55 6 0 0 ND ND ND Bacteroides thetaiotaomicron (21) 57 0 0 0 ND ND ND Bacteroides vulgatus (23) 13 0 0 0 ND ND ND Bacteroides bivius (19) 0 0 0 0 ND ND ND Bacteroides spp. (23) 13 4 4 0 ND ND ND a Resistance defined by National Committee for Clinical Laboratory Standards (13, 14) interpretive criteria: cefotaxime and cefoperazone. -64 pig/ml: ceftazidime and aztreonam,.32,tg/mlr imipenem, -16,tg/ml. Resistance for the cefoperazone-sulbactam combinations were based on the cefoperazone breakpoints. b Fixed concentrations of 1 ktg of sulbactam per ml with cefoperazone produced essentially identical MIC results. Includes P. acido'orans (3 strains), P. cepacia (3 strains), P.fluorescens (16 strains). P. maltophilia (4 strains), P. plutida (5 strains), and P. stutzeri (9 strains). d Includes 20 beta-lactamase-producing isolates. e Includes 25 beta-lactamase-producing isolates. f ND, Not determined. sulbactam clinical trials (Table 3). Of the 137 members of the Enterobacteriaceae tested, 74 could not be evaluated because the cefoperazone MIC for the isolate was already lower than the dilution schedule used, i.e., '1.0 Ftg/ml. At such a low level, synergy would be of questionable clinical relevance. Of the 63 evaluatable strains, there was a synergistic interaction between cefoperazone and sulbactam with 47. All of the remaining strains showed partial synergism. Among the anaerobic bacteria, cefoperazone and sulbactam were synergistic for 88% of the 120 evaluatable strains. Beta-lactamase was detected in only 2 of the 7 anaerobic strains showing drug indifference. The Pseudomonas spp. isolates were generally indifferent (59%) to the combination, with only three strong beta-lactamase-producing isolates showing complete synergy (P. acidovorans, P. aeruginosa, and P. maltophilia). Eighteen other beta-lactamaseproducing pseudomonads showed a partially synergistic interaction between cefoperazone and sulbactam. Synergy tests with the Acinetobacter spp. essentially showed direct inhibition by sulbactam, although drug interaction analyses yielded 11% synergy, 50% partial synergy, and 39% additive interactions. DISCUSSION The broad spectrum of cefoperazone antimicrobial activity could be expanded by the presence of sulbactam at various concentrations (1, 3, 5, 7, 9, 11, 16). Furthermore, previous investigators have concluded that when routine fresh clinical isolates are tested, the addition of sulbactam at concentrations greater than 2.0 or 4.0 fig/ml rarely enhances the spectrum of cefoperazone (9, 11). This observation was

1728 JONES ET AL. J. CI-IN. MICROBIOL. TABLE 2. Spectra of activity of six beta-lactams and combinations against three major groups of bacteria Bacterial group (no. of strains tested) and Cumulative 7Sf inhibited at MIC (1tg/ml): antimierobial agent <0.06 0.12 0.25 0.5 1 2 4 8 16 32 Enterobac ieriac-eae (323) Cefoperazone 14 24 36 49 59 64 69 71 76 81(' Cefoperazone-sulbactam (2:1) 10 25 39 51 61 67 74 80 87 91 Aztreonam 57 69 73 75 77 78 80 83 88 96 Cefotaxime 47 57 67 70 73 74 76 77 82 91 Ceftazidime 29 53 67 72 77 79 80 82 84 88 Imipenem 6 30 49 65 80 95 98 100 NT" Pseudomwnas and A inetobac(te- spp. (125) Cefoperazone 2 5 7 13 45 57 77 88 Cefoperazone-sulbactam (2:1) 2 6 13 25 61 72 87 94 Aztreonam 1 2 6 13 48 61 74 88 Cefotaxime 2 2 6 10 16 23 52 73 Ceftazidime 3 9 il 42 74 88 93 93 95 Imipenem 6 22 33 72 83 91 93 94 NT Gram-negative anaerobes (205) Cefoperazone 6 9 il 20 43 65 Cefoperazone-sulbactam (2:1) 13 24 41 66 95 99 ' Boldface values represent the highest moderatcly susceptible or intermediate MICs by National Committee for Clinical Laboratory Standards interpretive criteria for aerobes (13. 14). h NT, Not tested. comparable to that reported for ticarcillin-clavulanate by Fuchs et al. (8). However, the clinical infusions of cefoperazone-sulbactam have been established as a 2:1 ratio (United States), in contrast to the single 100- or 200-mg clavulanic acid dose delivered with 3 g of ticarcillin. This elevated sulbactam dose produces significantly higher concentrations in tissue and serum. In this study, we evaluated the in vitro activity of cefoperazone-sulbactam at a ratio of 2:1 against a population of organisms with unusually high cephalosporin resistance. The 2:1 ratio clearly increased the spectrum of cefoperazone compared with that at a fixed concentration of 1.0, 2.0, or 4.0 Fig of sulbactam per ml. Use of a 2:1 ratio added several cephalosporinase-producing strains of C. jfieundii, E. aerogenies, and E. cloa(ae to the spectrum of cefoperazone. On the other hand, little influence on the activity of cefoperazone was found with a 2:1 ratio in tests with Serrauia inarcescens isolates. This observation may be related to the high affinity of cefoperazone for the S. inarcescens betalactamase (12). The greatest benefit of this combination, however. seemed to be achieved against the more clinically prevalent plasmid-mediated beta-lactamases (15) and favorable interactions were apparent with al tested sulbactam concentrations. The 2:1 ratio better simulates drug formulations and pharmacokinetics and allows recognition of the expanded spectrum of the combination against members of the Enterobacteria(ceae and some nonenteric gram-negative organisms resistant to other newer beta-lactams (Table 1). As observed in earlier studies, cefoperazone MICs in the susceptible range (-16 ig/ml) were synergistically reduced by the presence of sulbactam (9, 11). This was especially relevant for beta-lactamase-producing H. influenzae (16), the penicillinase-positive staphylococci, Bac reroides spp., and a number of isolates of Enterohacteriaceae. A few more anaerobic gram-negative bacilli were also susceptible to cefoperazone when tested with sulbactam at the 2:1 ratio compared with fixed concentrations. Of the strains tested, 83% were synergistically inhibited by the combination. Our results confirm the high in vitro cefoperazone-sulbactam susceptibility rates (96.6%) reported by Appelbaum et al. (1). Although we found 92.7% of Bacteroides strains to be susceptible to cefoperazone when it was combined with 2.0 1tg of sulbactam per ml, as suggested by Appelbaum et al., >99% were susceptible to cefoperazone at the 2:1 ratio. This in vitro spectrum of activity was slightly better than that of cefoxitin (94.3% susceptible [1]). Barry et al. (3) have reported that cefoperazone-sulbactam was highly effective against 63 strains of the B. firaigilis group. In their studies, however, fixed sulbactam concentrations of 4.0 and 8.0 fig/ml produced results identical to those with a 2:1 ratio. Approximately 97%c of their B. fi-agilis isolates were inhibited by cefoperazone-sulbactam concen- TABLE 3. Interaction between cefoperazone and sulbactam against 333 bacterial strains isolated from patients participating in cefoperazone-sulbactam clinical trials No. of strains with interaction category: Organism Synergistic synergistic category (no. of strains tested) Synergistic Complete Partilal Additive Indifferent Not evaluatable positive for beta-laictamase' Acinetoba(ter spp. (19) 2 9 7 O 1 91 Bacteroides spp. (126) 70 35 8 7 6 91 E.clierichiIia coli (86) 27 12 0 O 47 92 Other Enteroba(-teria(eeae (51) 20 4 0 0 27 96 Pseudom0onas spp. (51) 3 18 0 30 0 100 Beta-lactamase positive by the nitrocefin broth test in microdilution trays.

VOL. 25* 1987 trations of <16/8 1tg/ml. The B. firagilis cefoperazone MICs were decreased by approximately four to eightfold when sulbactam was added. Similar findings were reported by Crosby and Gump (5). Sulbactam and some other betalactamase inhibitors have only modest antimicrobial activity against anaerobic bacteria, with MICs usually in the range of 16 to 64 p.g/ml (1, 3, 5). Therefore, the fixed ratio of inhibitor and cefoperazone should have greater antimicrobial effects in the clinical setting and in diagnostic tests than the 2 ptg/ml single concentration advocated for ticarcillin-clavulanic acid (8, 13, 14). In conclusion, we recommend that the cefoperazonesulbactam antimicrobial combination be tested in vitro at a 2:1 ratio by standardized dilution methods (13, 14). Consideration should be given to reassessing the validity of proposed 75/15,ug cefoperazone-sulbactam disk tests (15). A higher sulbactam disk content, with a ratio closer to 2:1, may be required to recognize the cefoperazone-resistant strains that could respond (MICs, <16,ug/ml) in dilution tests with a 2:1 ratio (Barry et al., in press). Results of in vitro dilution testing of cefoperazone-sulbactam (2:1 ratio) indicate that it has the widest spectrum of antimicrobial activity when compared directly with other newer cephalosporins, penicillins, aminoglycosides, and clavulanic acid combinations (12). Only imipenem inhibited more bacterial strains. LITERATURE CITED 1. Appelbaum, P. C., M. R. Jacobs, S. K. Spangler, and S. Yamabe. 1986. Comparative activity of 1-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with,-lactams against,3-lactamase-producing anaerobes. Antimicrob. Agents Chemother. 30:789-791. 2. Aronoff, S. C., M. R. Jacobs, S. Johenning, and S. Yamabe. 1984. 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