Efficacy of Oxacillin and Ampicillin-Sulbactam Combination in Experimental Endocarditis Caused by ,B-Lactamase

Home , Oxacillin

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 728-732 Vol. 34, No. 5 0066-4804/90/050728-05$02.00/0 Copyright X) 1990, American Society for Microbiology Efficacy of Oxacillin and Ampicillin-Sulbactam Combination in Experimental Endocarditis Caused by ,B-Lactamase- Hyperproducing Staphylococcus aureus CLAUDIE THAUVIN-ELIOPOULOS,* LOUIS B. RICE, GEORGE M. ELIOPOULOS, AND ROBERT C. MOELLERING, JR. Department of Medicine, New England Deaconess Hospital,* and Harvard Medical School, Boston, Massachusetts 02215 Received 13 July 1989/Accepted 31 January 1990

Optimal therapy of infections caused by borderline oxacillin-susceptible, 3-lactamase-hyperproducing Staphylococcus aureus has not been established. We used a rat model of aortic valve endocarditis to examine efficacies of antibiotic regimens against a borderline oxacillin-susceptible strain as compared with a fully susceptible S. aureus strain. Animals were treated with oxacillin alone or in combination with sulbactam or with ampicillin-sulbactam combinations at two dose levels. Infections caused by the borderline susceptible and fully susceptible strains responded equally wel to oxacillin alone, with residual bacterial titers in vegetations falling to 4.8 ± 1.6 and 4.4 ± 1.7 (mean ± standard deviation) log,0 CFU/g, respectively. Addition ofsulbactam to oxacillin (1:2) did not enhance the efficacy of oxacillin against either strain in the animal model. A high-dose regimen of ampicillin-sulbactam (2:1) yielding mean (+ standard deviation) levels in serum of 16.8 ± 7.4 and 9.5 ± 1.1 ,ug/ml, respectively, proved equally effective against both strains (bacterial titers, 6.6 log1o CFU/g). However, at lower doses (8.3 + 2.6 and 5.9 ± 2.4 ,ug/ml), the combination showed greater efficacy against the fully susceptible strain, with residual titers of 7.1 + 2.0 versus 9.0 + 1.6 log1o CFU/g (P < 0.05). In vitro studies revealed that the ,B-lactamase inhibitor sulbactam was also a potent inducer of staphylococcal P-lactamase at clinically relevant concentrations. Based on this short-term in vivo therapy study, oxacillin would be predicted to be clinically effective in the therapy of infections caused by borderline oxacillin-susceptible strains of S. aureus, while the combination of ampicillin with sulbactam appears to be inferior to oxacillin alone against such infections.

In 1986, McDougal and Thornsberry (8) described isolates (This work was presented at the 28th Interscience Confer- of Staphylococcus aureus displaying borderline susceptibil- ence on Antimicrobial Agents and Chemotherapy [C. Thau- ity to antistaphylococcal penicillins. These strains were vin-Eliopoulos, L. B. Rice, G. M. Eliopoulos, and R. C. inhibited by methicillin and oxacillin at concentrations of 4 Moellering, Jr., Program Abstr. 28th Intersci. Conf. Antimi- and 2 p,g/ml, respectively, and were characterized by the crob. Agents Chemother., abstr. no. 367, 1988].) production of large amounts of P-lactamase. Addition of a f-lactamase inhibitor such as clavulanic acid or sulbactam MATERIALS AND METHODS restored activities of the penicillins to levels which inhibit fully methicillin-susceptible S. aureus strains. Such strains Bacterial strains. The two strains of S. aureus used appear to be distinct from those demonstrating chromoso- throughout this study were clinical blood culture isolates mally mediated intrinsic ,-lactamase resistance, which has recovered in Boston, Mass. Susceptibilities were determined been associated with the presence of a low-affinity penicillin- by microdilution broth titration in cation-supplemented binding protein, termed PBP 2a or 2' (5). To differentiate Mueller-Hinton broth (CSMHB; BBL Microbiology Sys- borderline susceptible strains from the latter group of intrin- tems, Cockeysville, Md.) with 2% NaCl (9, 16). Strain 1 was sically resistant organisms further, they have also been borderline oxacillin susceptible (BOSSA) with methicillin called acquired-resistant strains (7, 8). Whether infections and oxacillin MICs of 4 and 1 ,ug/ml, respectively. Addition caused by borderline susceptible staphylococcal isolates of sulbactam, 16 jig/ml (8), reduced MICs of these agents to would respond to ,B-lactams as well as infections caused by 2 and 0.25 p.g/ml, respectively. Strain 2 was a fully oxacillin- fully susceptible strains, or whether these isolates should be susceptible S. aureus (OSSA) strain with methicillin and considered the same as intrinsically resistant strains for oxacillin MICs of 2 and 0.25 ,ug/ml, respectively. Sulbactam clinical and therapeutic purposes, is unclear and remains to alone inhibited both strains at 250 ,ug/ml. Antimicrobial be determined. susceptibilities were also determined in CSMHB plus 2% In this study, we established an aortic valve endocarditis NaCl supplemented with 50% rat serum, using inocula of 5 x model in rats, using a fully oxacillin-susceptible strain of S. 105 to 1 x 106 CFU/ml. Inhibitory endpoints were read at 24 aureus or a borderline susceptible clinical isolate of S. and 48 h of incubation at 35°C. At 48 h, 0.01-ml samples were aureus, to evaluate the role of P-lactamase production in the transferred to antibiotic-free plates for determination of therapeutic efficacies of oxacillin alone, oxacillin combined MBCs (11). with sulbactam, and ampicillin-sulbactam in combination. Antimicrobial agents. Ampicillin and sulbactam standard reference powders and the 2:1 combination for in vivo studies were generously provided by Roerig-Pfizer, Inc., * Corresponding author. New York, N.Y. Oxacillin was obtained from Bristol Lab- 728 VOL.V-LACTAMASE-HYPERPRODUCING34, 1990 S. AUREUS 729

TABLE 1. Results of therapy of experimental S. aureus endocarditis BOSSA OSSA levels RegimenRegimen Du(g/ml~~Drug SD) MiC)MC No. of survivors/ Bacterial titer in MIC (MBC) No. of survivors/ Bacterial titer in no.ofanmasno. of animals (%)%) vegetations (pgm)(ml) no.o offaias()animals (%) vegetations (q.g/ml)a (log10 CFU/g + SD) (log10 CFtJ/g ± SD) Controls 8/29 (28) 10.5 ± 0.5 3/28 (11) 10.4 + 0.1 Oxacillin alone 10.9 ± 5.1 1 (32) 15/16 (94) 4.8 ± 1.6 1 (2) 14/18 (78) 4.4 ± 1.7 + Sulbactam 6.2 ± 0.5 1 (8) 14/15 (93) 6.1 ± 2.3 1 (4) 13/15 (87) 5.3 ± 1.6 Ampicilin' 8.3 ± 2.6 125 (250) C 8 (16) + sulbactam (low dose) 5.9 ± 2.4 4 (32) 8/11 (73) 9.0 ± 1.6 1 (2) 9/10 (90) 7.1 ± 2.0 Ampicillind 16.8 ± 7.4 125 (250) 8 (16) + sulbactam (high dose) 9.5 ± 1.1 4 (32) 12/16 (75) 6.6 ± 2.1 1 (2) 17/19 (89) 6.6 ± 1.9 a In CSMHB plus 2% NaCl and 50%o rat serum. MICs were determined at 24 h, and MBCs were determined at 48 h of incubation. b 450 mg of ampicillin per kg; 225 mg of sulbactam per kg per day. c -, Ampicillin alone was not used for treatment. d 1,000 mg of ampicillin per kg; 500 mg of sulbactam per kg per day. oratories, Evansville, Ind. Methicillin was obtained from antibiotic levels in serum were measured by using an agar Sigma Chemical Co., St. Louis, Mo. well diffusion technique (1), with Bacillus subtilis as the test Evaluation of 1-lactamase activity. P-Lactamase activity of organism for ampicillin and oxacillin levels and with a cell lysates was determined by a spectrophotometric method P-lactamase-producing strain of Pasteurella haemolytica as (10). Bacteria were grown in dextrose phosphate broth the test organism for sulbactam levels. Animals surviving 3.5 (GIBCO Diagnostics, Madison, Wis.) to log phase at 35°C days of treatment were sacrificed 2 h after discontinuation of and harvested when cell densities reached 109 CFU/ml. Cell therapy. Cardiac vegetations were aseptically removed, pellets were lysed by incubation with lysostaphin (50 ,ug/ml) weighed, homogenized, and diluted in saline for bacterial (Sigma) and Triton X-100 (0.05% final concentration) (Bio- titer determination. Colony counts were performed on man- Rad Laboratories, Richmond, Calif.). For studies of ,B- nitol-salt agar plates with and without penicillinase to control lactamase induction, cells were grown in the presence of for potential antibiotic carry-over. The lower limit of detec- ampicillin or sulbactam at concentrations of0.1 x the MIC of tion by this method was 2.3 log1o CFU/g of vegetation. each drug. Statistical evaluation. The chi-square test with Yates cor- Production of bacterial endocarditis. Bacterial endocarditis rection was used for evaluation of nominal data. Differences was established by the technique of Santoro and Levison in bacterial titers within vegetations among treatment groups (12), with minor modifications as described previously (14, were assessed by analysis of variance followed by the 15). Nonbacterial thrombotic endocarditis was created in Bonferroni method for multiple comparisons (4). male Sprague-Dawley rats (180 to 250 g) by insertion of a polyethylene catheter (PE 10 Intramedic tubing) across the RESULTS aortic valve via the right carotid artery. At 20 min after catheterization, 106 CFU of the infecting strain in saline In vitro susceptilillity studies. Activities of antimicrobial were injected through the catheter, which was ligated and agents against the test strains determined in CSMHB plus left in place throughout the experiment. Establishment of 2% NaCl supplemented with 50% rat serum are shown in bacterial endocarditis was ascertained by positive blood Table 1. MICs of oxacillin against the two strains were cultures drawn just before onset of therapy. Only animals identical in this medium, and these were not affected in the with both initial blood cultures positive for the infecting presence of sulbactam. The MIC of ampicillin-sulbactam strain and correct placement of the catheter across the aortic (2:1, expressed in terms of the ampicillin component) against valve (as established at autopsy) were included in the study. the BOSSA strain was fourfold higher than that against the With both strains, colony counts in cardiac vegetations OSSA strain. In the absence of rat serum, MICs (micro- reached 8.0 to 8.5 log1o CFU/g by 12 h after infection. grams per milliliter) against the BOSSA and OSSA strains in Antimicrobial therapy. Treatment was started 12 h after CSMHB plus 2% NaCl were as follows: ampicillin-sulbac- bacterial challenge and continued for 3.5 days. All antimi- tam, 4 and 2; oxacillin, 1.0 and 0.25; oxacillin-sulbactam, crobial agents were delivered by continuous intravenous 0.25 and 0.125. Strain 1 (BOSSA) demonstrated greater infusion via an indwelling central venous catheter inserted tolerance to the bactericidal effects of oxacillin (MBC/MIC through the externaljugular vein into the superior vena cava, ratio, 32) and ampicillin-sulbactam (MBC/MIC ratio, 8) than as described previously (14). Antimicrobial agents were did strain 2 (OSSA) (MBC/MIC ratios, 2 for both drugs). carefully delivered with syringe pumps (Orion Research, P-Lactamase activity. As expected, P-lactamase activity of inc., Cambridge, Mass.). Animals were randomly assigned cell lysates from strain 1 was substantially greater than that to one of the following five groups: (i) oxacillin alone (600 of strain 2 (Fig. 1). Both ampicillin and sulbactam induced mg/kg per day), (ii) oxacillin (600 mg/kg per day) plus production of P-lactamase, but in each case the resulting sulbactam (300 mg/kg per day), (iii) ampicillin (450 mg/kg per hydrolytic activity was greater with BOSSA lysates. This day) plus sulbactam (225 mg/kg per day), (iv) ampicillin effect was particularly striking when BOSSA cells were (1,000 mg/kg per day) plus sulbactam (500 mg/kg per day), induced by incubation with sulbactam. The resulting lysate and (v) an untreated control group. achieved complete hydrolysis of substrate within 5 min. Monitoring of therapy and outcome. On day 3 of therapy, Effects of preincubation with sulbactam or clavulanate on 730 THAUVIN-ELIOPOULOS ET AL. ANTIMICROB. AGENTS CHEMOTHER.

100

C: 0 0 2- 75 -o U) N >% 50 0 L. -C C: D NIO 25 ~~~~amp-inducedBO2SSA

0.0 1.0 2.0 3.0 4.0 5.0

Time (Hour) FIG. 1. P-Lactamase activity of both infecting strains of S. aureus in uninduced cells, in ampicillin (amp)-induced cells, and in sulbactam (Sb)-induced cells.

P-lactamase activity were also studied in five additional was comparable to the oxacillin-sulbactam regimen. Statis- BOSSA isolates. In four of these, exposure to sulbactam tically significant differences in response of animals infected induced P-lactamase activity, resulting in .90% nitrocefin with BOSSA or OSSA strains were evident only among hydrolysis within 2 to 4 min, with the fifth strain hydrolyzing those treated with low-dose ampicillin-sulbactam. With this this amount of drug by 60 min. In each case, preincubation regimen, significantly greater reductions in vegetation bac- with clavulanic acid retarded nitrocefin hydrolysis by the terial titers were observed with the OSSA than with the resulting cell lysate. Sulbactam induced P-lactamase activity BOSSA strain (P < 0.05). in two strains of OSSA which produced P-lactamase, but, as expected, did not affect one penicillin-susceptible, non- DISCUSSION 3-lactamase-producing isolate. Since the recognition that strains for which oxacillin MICs Experimental endocarditis. Results oftherapy are shown in are -1 ,ug/ml constitute a small but significant proportion of Table 1. Antibiotic levels in serum achieved with both recent isolates of S. aureus at some centers (7, 18), there has oxacillin and ampicillin-sulbactam are clinically attainable in been considerable concern about appropriate therapy of humans and exceeded MICs against the test organisms. infections due to such strains. Specifically, would a ,-lactam Survival of animals in each treatment group exceeded that of alone prove successful or would such isolates more closely control animals (P - 0.02), but did not differ among the resemble methicillin-resistant S. aureus and fail to respond groups of treated animals. Oxacillin was equally effective predictably? It has become amply clear, however, that against BOSSA and OSSA infections, with residual titers mechanisms of resistance to penicillinase-resistant penicil- within vegetations of 4.8 ± 1.6 and 4.4 ± 1.7 log1o CFU/g, lins cannot always be accurately predicted solely from levels respectively. Addition of sulbactam to oxacillin did not of susceptibility. Strains of S. aureus for which oxacillin enhance activity of the latter against either strain. Residual MICs are between 1 and 2 ,ug/ml appear to represent a bacterial titers were higher among animals treated with this heterogeneous group, some of which hyperproduce P-lacta- combination than among those treated with oxacillin alone, mase, as defined by McDougal and Thormsberry (8), and but these differences were not statistically significant. Like- others of which may attribute oxacillin resistance to alter- wise, the high-dose ampicillin-sulbactam regimen was ations in penicillin-binding proteins (3, 13). In the present equally effective against BOSSA and OSSA infections. This study, we ascertained that the BOSSA strain studied was a regimen was less effective than oxacillin alone against both hyperproducer of ,-lactamase as compared with a clinical strains (P = 0.01 for BOSSA and P = 0.002 for OSSA), but isolate demonstrating full susceptibility to oxacillin. VOL. 34, 1990 P-LACTAMASE-HYPERPRODUCING S. AUREUS 731

This study showed oxacillin to be equally effective against MBC (in combination) against penicillinase-producing S. the BOSSA and OSSA strains, with residual bacterial titers aureus (6). of 4.4 to 4.8 log1o CFU/g. This observation is consistent with In this study, oxacillin was more effective than ampicillin- results of other animal studies which have compared re- sulbactam (high dose) against both isolates tested. While it sponse of fully susceptible and intermediately susceptible might be possible to attribute this result to a superior strains of S. aureus to therapy with antistaphylococcal antibiotic level in serum/MIC ratio (10:1 for oxacillin, 4:1 for penicillins. Cantoni et al. (2), using a rat endocarditis model, ampicillin-sulbactam) for the BOSSA isolates, alternative found similar responses to cloxacillin of two strains, with explanations would clearly have to be sought in the case of oxacillin MICs of 0.25 and 2 ,ug/ml. Chambers et al. (3), the OSSA isolates (10:1 for oxacillin, 16:1 for ampicillin- using a rabbit model of endocarditis, noted identical re- sulbactam). Examination of attained drug levels in serum in sponses to nafcillin of a fully susceptible strain (nafcillin relation to bactericidal endpoints yields similar conclusions. MIC, 0.5 ,ug/ml) and one characterized by a nafcillin MIC of Our results are also in contrast to those of other studies 4 ,ug/ml. Penicillin-binding proteins of the latter strain were which found ampicillin-sulbactam comparable to nafcillin examined and found to be similar to the pattern of methicil- against methicillin-susceptible S. aureus (17) and amoxicillin-susceptible S. aureus strains. However, in neither of lin-clavulanate comparable to cloxacillin against both a fully these studies was the level of ,-lactamase production in oxacillin-susceptible strain and one for which the oxacillin intermediately resistant isolates formally tested. These data MIC was 2 ,ug/ml (2). are also consistent with results of a retrospective analysis of We believe that these results may be explained by the different observation that sulbactam displayed a striking ability to clinical infections due to S. aureus strains with induce production of P-lactamase in each of the two strains levels of resistance to oxacillin (7). Treatment with a peni- (Fig. 1). This phenomenon was probably reflected in the cillinase-resistant penicillin was successful in 88.6% of in- diminished (although not statistically significant) efficacy of fections caused by fully susceptible strains and 7 of 10 (70%) oxacillin against both strains when the drug was combined infections caused by acquired oxacillin-resistant isolates with sulbactam. This hypothesis provides an explanation for (oxacillin resistant by disk diffusion; oxacillin MIC of c2 the discordance of our results with those of the aforemen- ,ug/ml by broth dilution). These results are difficult to inter- tioned studies with amoxicillin plus clavulanic acid (2), pret, however, in view of the fact that four of five (80%) which does not appear to be a potent inducer of the P- infections caused by oxacillin-resistant isolates (MIC, >8 lactamase (data not shown), and with ampicillin plus sulbac- jig/ml) also responded to treatment with a penicillinase- tam (17), which was dosed to achieve peak levels in serum resistant penicillin. substantially higher than the steady-state mean levels in In attempting to reconcile the above results with the serum achieved in the present study. Attainment of such fourfold difference in broth dilution oxacillin MICs and the high drug concentrations in serum may be important because significant difference in ,-lactamase activity generated by the capacity of sulbactam to induce P-lactamase may well the two strains used in our study (Fig. 1), it is noteworthy reach a maximum level at modest drug concentrations, while that susceptibility testing performed in the presence of 50% the P-lactamase-inhibitory activity of the agent appears to rat serum yielded identical oxacillin MICs against the two increase proportionally with drug concentration (6). strains. Continuous administration of oxacillin (or repetitive We conclude that, against the representative strains stud- antibiotic dosing in the studies cited), resulting in drug ied here, both oxacillin and ampicillin-sulbactam (high dose) concentrations in serum 10-fold higher than the MIC, may were as effective against a P-lactamase-hyperproducing have partially negated any differences in P-lactamase pro- BOSSA as against an OSSA strain. At the doses used, duction apparent in the static environment of standard ampicillin-sulbactam was less effective than oxacillin. susceptibility testing. While it is theoretically possible that Higher drug concentrations of ampicillin-sulbactam in serum our results might also be explained by lower virulence of the would probably have enhanced the efficacy of this combina- BOSSA strain, this seems highly unlikely in view of the fact tion. We have shown that sulbactam could induce produc- that bacterial titers in vegetations of untreated animals tion of ,-lactamase in these strains. Combination of sulbac- inoculated with either strain were identical both 12 h after tam with oxacillin, while not affecting survival, appeared to infection (8.0 to 8.5 log1o CFU/g) and at sacrifice on day 4 diminish efficacy in terms of final bacterial titers within (10.4 to 10.5 log1o CFU/g). Tolerance to the bactericidal vegetations, although this effect did not reach statistical effects of oxacillin which we observed with strain 1 (MBC/ significance. MIC ratio, 32) appears to be common among BOSSA isolates (18), but is of uncertain relevance in vivo. Woods LITERATURE CITED and Yam tolerance to oxacillin 1. Anhalt, J. P. 1985. Assays for antimicrobial agents in body (18) have reported that fluids, p. 1009-1014. In E. H. Lennette, A. Balows, W. J. among P-lactamase-hyperproducing strains of S. aureus Hausler, Jr., and H. J. 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