ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1993, p. 507-511 Vol. 37, No. 30066-4804/93/030507-05$02.00/0Copyright © 1993, American Society for Microbiology

Activity of Ampicillin-Sulbactam and Oxacillin inExperimental Endocarditis Caused by 3-Lactamase-

Hyperproducing Staphylococcus aureusANGELOS PEFANIS, CLAUDIE THAUVIN-ELIOPOULOS,* GEORGE M. ELIOPOULOS,

AND ROBERT C. MOELLERING, JR.Department ofMedicine, New England Deaconess Hospital, Boston, Massachusetts 02215,

and Harvard Medical School, Boston, Massachusetts 02115

Received 24 September 1992/Accepted 30 December 1992

Using a rat model of aortic valve infective endocarditis, we previously found that oxacillin was equallyeffective against an oxacillin-susceptible strain of Staphylococcus aureus and a--lactamase-hyperproducingborderline oxacillin-susceptible strain of S. aureus; also, ampicillin-sulbactam was less effective than oxacillinagainst both isolates and at low doses was less effective against the borderline-susceptible strain than against thefully oxacillin-susceptible strain (C. Thauvin-Eliopoulos, L. B. Rice, G. M. Ellopoulos, and R. C. Moellering,Jr., Antimicrob. Agents Chemother. 34:728-732, 1990). In the present study, we extended this work, usingalternative treatment schedules and additional bacterial strains. Extending treatment with low doses ofampicillin-sulbactam (500 and 250 mg/kg of body weight per day, respectively) to 6.5 days resulted inequalization of effectiveness against the previously studied strains BOSSA-1 and OSSA-1 (3.75 ± 1.61 loglo and4.71 ± 1.79 log1o CFU of residual viable bacteria per g, respectively). Against the borderline oxacillin-susceptible strain BOSSA-1, Increasing the sulbactam dosage from 500 to 2,000 m/kg/day while maintaininga fixed dose of ampicillin (1,000 mg/kg/day) by continuous infusion resulted in lower bacterial counts (4.93 ±1.84 log1o versus 3.65 ± 1.26 log1o CFU of residual viable bacteria per g, respectively), but this difference wasof only borderline significance; differences in efficacy between the low-dose and high-dose sulbactam regimenswere exaggerated when intermittent intravenous administration was used (6.19 + 1.90 log1, versus 3.37 + 1.41log1o CFU/g, respectively, P < 0.001). However, for any individual sulbactam dosage, the mode ofadministration(continuous versus intermittent infusion) did not affect the activity of the regimen. When additional strains wereused in the model, oxacillin and ampicillin-sulbactam (1,000 plus 2,000 mg/kday) were equally effective agalnstboth oxacillin-susceptible and borderline oxacillin-resistant strains of S. aureus. These results support thepredictions that oxacillin would be clinically effective in the treatment of infections caused by borderlineoxacillin-susceptible strains of S. aureus and that, except at very low doses, ampicillin-sulbactam would also beas effective against borderline-susceptible strains as against fully oxacillin-susceptible strains of S. aureus.

McDougal and Thornsberry (8) described isolates ofStaphylococcus aureus with reduced susceptibility to anti-staphylococcal penicillins based on production of largeamounts of ,B-lactamase. Inhibition of such isolates requiredmethicillin and oxacillin concentrations of 4 and 2 ,ug/ml,respectively, but full susceptibility could be restored bycombination of an antistaphylococcal penicillin with a ,B-lac-tamase inhibitor such as clavulanic acid or sulbactam. In aprevious study, we examined the in vivo activities of ampi-cillin-sulbactam and oxacillin in a rat model of experimentalendocarditis caused by such a borderline oxacillin-suscepti-ble S. aureus (BOSSA) strain in comparison with endocar-ditis caused by a fully oxacillin-susceptible S. aureus(OSSA) strain (12). We found that oxacillin was equallyeffective against BOSSA and OSSA strains, reducing bacte-rial densities in cardiac vegetations from more than 1010CFU/g to between io4 and io CFU/g. However, ampicillin-sulbactam was less effective than oxacillin against infectionscaused by either strain despite identical in vitro activities ofthe two antibiotic regimens against the OSSA strain (MIC =1 ,ug/ml, MBC = 2 ,ug/ml) and relatively similar activitiesagainst the BOSSA strain (MIC and MBC of oxacillin = 1and 32 ,ug/ml, MIC and MBC of ampicillin in conjunction

* Corresponding author.

with sulbactam = 4 and 32 ,ug/ml) in broth supplementedwith 50% rat serum.We speculated that these results may have been influenced

to a large degree by the striking ability of sulbactam toinduce 1-lactamase production in BOSSA strains and to amuch lower extent in OSSA strains, a phenomenon whichwe demonstrated in vitro (12). This hypothesis was sup-ported by the observation that addition of sulbactam tooxacillin tended to antagonize the in vivo activity of thelatter against both strains, although the differences observeddid not reach statistical significance.The present study extends our previous work by testing

the hypotheses that the ,B-lactamase-inducing activity ofsulbactam might be overcome with longer treatment periodsor with higher doses of the inhibitor. In addition, we exam-ined additional clinical isolates of both OSSA and 1-lacta-mase-hyperproducing BOSSA in the animal model to verifythe generality of our previous observations.

(This work was presented in part at the 32nd InterscienceConference on Antimicrobial Agents and Chemotherapy[9a].)

MATERIALS AND METHODS

Bacterial strains. The nine strains of S. aureus used in thisstudy were clinical isolates recovered in Boston, Mass.

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BOSSA-1 and OSSA-1 were the prototype P-lactamase-hyperproducing and fully oxacillin-susceptible strains, re-spectively, used in our previous study (12). For thesestrains, the MICs of oxacillin determined by broth microdi-lution testing in cation-supplemented Mueller-Hinton broth(BBL Microbiology Systems, Cockeysville, Md.) with 2%NaCl were previously found to be 1.0 and 0.25 ,g/ml,respectively (12). Susceptibilities were also determined withthe same medium supplemented with 50% pooled rat serumby using inocula of 5 x 105 to 1 x 106 CFU/ml, and the MICsfor both strains were 1.0 ,ug/ml (12). Five additional strainsof BOSSA and two additional strains of OSSA were alsoused in the study, as indicated below. All BOSSA strainswere examined for the presence of the mecA gene by S. Unalusing a previously reported polymerase chain reaction (PCR)method (14), and all were found to be negative. The ,-lac-tamase activities of cell lysates were determined by a spec-trophotometric method as described previously (9, 12), andall of the lysates behaved as hyperproducers of ,B-lactamase.

Antimicrobial agents. Ampicillin and sulbactam standardreference powders and the 2:1 ampicillin-sulbactam combi-nation for in vivo studies were generous gifts from the RoerigDivision, Pfizer, Inc., New York, N.Y. The combinationwas supplemented with additional sulbactam standard pow-der when the efficacy of high-dose sulbactam regimens wastested. Oxacillin susceptibility powder was a gift from Bris-tol-Myers Laboratories, Evansville, Ind. For in vivo studies,clinical oxacillin sodium (Bristol-Myers) was used.

Production of bacterial endocarditis. Bacterial endocarditiswas produced on the aortic valve of male Sprague-Dawleyrats as previously described (10). The previously describedtechnique was slightly modified by using a stainless steelinternal guide wire (stylets for blood pipettes; Becton Dick-inson, Franklin Lakes, N.J.) to achieve more reliable place-ment of the polyethylene catheter (PE 10 Intramedic tubing).Twenty minutes after placement of a polyethylene catheteracross the aortic valve, 106 CFU of the infecting strainsuspended in saline was injected into the catheter, whichwas then ligated and left in place for the duration of theexperiment. Establishment of bacterial endocarditis wasascertained at autopsy by evidence of macroscopic vegeta-tions and correct placement of the catheter across the valve.

Antimicrobial therapy. Treatment was begun 12 h afterbacterial challenge and was administered intravenously viaan indwelling central venous catheter inserted through thejugular vein into the superior vena cava (11). Delivery ofeither continuous or intermittent infusions was regulated bysyringe pumps (Orion Research, Inc., Cambridge and Har-vard Apparatus, South Natick, Mass.). Animals were ran-domly assigned to one of the following treatment groups.

(i) Prolongation of therapy. To determine whether exten-sion of therapy to 6.5 days would result in greater efficacythan previously noted with a 3.5-day regimen, rats infectedwith either BOSSA-1 or OSSA-1 received ampicillin (500mg/kg of body weight per day) plus sulbactam (250 mg/kg/day) by continuous intravenous infusion.

(ii) Continuous versus intermittent infusion. To determinewhether the mode of drug administration influenced theefficacy of the ampicillin-sulbactam combination against thehyperproducing strain (BOSSA-1), the more effective previ-ously studied regimen of ampicillin (1,000 mg/kg/day) plussulbactam (500 mg/kg/day) (12) was administered by eithercontinuous or intermittent intravenous infusions. For thelatter regimen, the total daily dose was administered in 16fractions with 15-min infusions beginning every 1.5 h. Thelatter regimen was chosen on the basis of pilot pharmaco-

kinetic studies to achieve serum drug concentrations equiv-alent to those achieved in humans after intermittent admin-istration of ampicillin. In this and subsequent experiments,treatment was administered for a total of 5.5 days forlogistical considerations.

(iii) Effect of increasing sulbactam dose. To determinewhether increasing the dose of sulbactam would overcomethe detrimental effects of I-lactamase production and im-prove the efficacy of ampicillin-sulbactam against BOSSA-1,two groups of rats were treated with ampicillin (1,000mg/kg/day) plus sulbactam (2,000 mg/kg/day), one by con-tinuous and the other by intermittent infusion, for 5.5 days.A third group was treated with oxacillin (600 mg/kg/day) bycontinuous infusion for comparison.

(iv) Studies with additional strains. To determine the gen-erality of previous observations, endocarditis was estab-lished with five additional BOSSA strains and two additionalOSSA strains. Four or five rats were infected with eachstrain and treated with either ampicillin (1,000 mg/kg/day)-sulbactam (2,000 mg/kg/day) or oxacillin (600 mg/kg/day) bycontinuous infusion for 5.5 days.With each of these regimens, a group of untreated rats was

included as controls to ensure reproducibility of the infectionwith the standardized inoculum.

Monitoring of therapy and outcome. Between days 3 and 5of therapy, serum antibiotic levels were determined once orsometimes twice by an agar well diffusion technique (1), withBacillus subtilis as the test organism for ampicillin andoxacillin and a P-lactamase-producing strain of Pasteurellahaemolytica as the test organism for sulbactam. All surviv-ing rats were sampled for serum antibiotic levels, but not allsamples yielded evaluable results because of occasionaltechnical difficulties with the microbiologic assay. At thebeginning of this study, animals were lightly anesthetizedwith ether for blood drawing, which required discontinuationof the intravenous infusion for several minutes before thesample was obtained. During the study, the use of ether wasproscribed by the institutional animal care committee, andthereafter animals were anesthetized with ketamine (Parke-Davis, Morris Plains, N.J.), which permitted completion ofblood sampling in less than 1 min after interruption of theinfusion.Animals surviving for the duration of antibiotic therapy

were sacrificed at least 2 h after discontinuation of antibiot-ics. In the study of prolonged antibiotic therapy, onlyanimals surviving 6.5 days were evaluated. In the remainderof the study, animals which were found dead during therapy,but which had received at least 3.5 days of treatment, wereincluded in the evaluation. Cardiac vegetations were asepti-cally removed, weighed, homogenized in saline, and dilutedserially in this solution for colony counts. Samples wereplated on both mannitol-salt agar plates and blood agarplates for colony counting. On the basis of the averagevegetation weight, the lower limit of detection by thismethod was approximately 2.3 loglo CFU/g of vegetation,and animals with vegetations yielding no growth by thistechnique were assigned a bacterial density of this magni-tude in calculations of mean values.

Statistical analysis. The chi-square test with Yates' correc-tion was used for evaluation of nominal data. Differences inbacterial densities within vegetations among treatmentgroups were assessed by the Kruskal-Wallis test with pair-wise comparisons; the Mann-Whitney test with the Bonfer-roni correction was used as appropriate for multiple compar-isons (5, 6).

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V-LACTAMASE-HYPERPRODUCING S. AUREUS IN VIVO 509

TABLE 1. Results of therapy of experimental S. aureus endocarditis with 6.5 days of ampicillin-sulbactam treatment(500 and 250 mg/kg/day, respectively) by continuous infusion

MIC (MBC) (,ug/ml) in: BacterialMean serum drug N f No. of sterile

Strain Regimen level + SD in CSMHB 2NaSMHB + survivors/no. vegetations/no. vegetationsxg/ml (no. of + 2% 2%NC faias of survivors or veogetationganimals) NaCl 50% rat of animals controls (mean /SD)]serum (en±S)

OSSA-1 Control 1/5 o/5b 10.19 ± 0.72Ampicillin 8.71 ± 5.56 (17) 16 (32) 8 (16)Ampicillin-sulbactam (2:1)C 7.53 ± 4.23 (14) 2 (4) 1 (2) 8/14 18d 4.71 ± 1.79

BOSSA-1 Control 3/6 0/6b 9.76 ± 0.92Ampicillin 10.19 ± 8.17 (13) 250 (>250) 125 (250)Ampicillin-sulbactam (2:1)c 7.26 + 4.15 (11) 4 (32) 4 (32) 10/15 4110d 3.75 ± 1.61

a CSMHB, cation-supplemented Mueller-Hinton broth.b Denominator is number of infected controls.c Drug level in serum, MIC, and MBC are those of ampicillin in the presence of sulbactam.d Denominator is number of survivors.

RESULTS

In vitro susceptibility studies. The susceptibilities ofBOSSA-1 and OSSA-1 to ampicillin-sulbactam are shown inTable 1, and the range of susceptibilities of the remainingstrains to these drugs and to oxacillin is given in Table 3.

Experimental endocarditis. (i) Effect of extending treatmentduration on efficacy of ampicillin-sulbactam. In our previousstudy, administration of low-dose ampicillin-sulbactam for a3.5-day treatment regimen was relatively ineffective in re-ducing bacterial densities within cardiac vegetations for bothBOSSA-1 (mean residual bacterial density standard devi-ation [SD], 9.0 + 1.6 log1o CFU/g) and OSSA-1 (7.1 + 2.0log1o CFU/g) (12). In the present study, we extended treat-ment with ampicillin-sulbactam (500 and 250 mg/kg/day,respectively) to 6.5 days. As shown in Table 1, serum druglevels were comparable to those previously observed (12).With 6.5 days of therapy, ampicillin-sulbactam was equallyeffective against BOSSA-1 and OSSA-1, with residual bac-terial densities within cardiac vegetations of 3.75 1.61log1o and 4.71 + 1.79 log1o CFU/g, respectively (P = 0.3)(Table 1). Differences in sterilization of vegetations with thetwo strains were not significant.

(ii) Effect of mode of administration on effectiveness ofampicillin-sulbactam. To determine whether the in vivoactivity of ampicillin-sulbactam against BOSSA-1 was de-pendent on the mode of administration, we compared treat-ment by continuous intravenous infusion with treatment bythe same daily dose given intermittently during the day. Thepeak and trough drug levels in serum drawn 5 min after theend of a 15-min intravenous infusion and just prior to thenext infusion, respectively, in animals receiving ampicillin

(1,000 mg/kg/day) and sulbactam (500 mg/kg/day) were asfollows (mean SD): ampicillin, 48.6 + 17.3 and 4.9 + 2.2jig/ml; sulbactam, 34.8 + 12.7 and 2.9 + 0.6 ,ug/ml. Inanimals treated by intermittent infusion, the final bacterialdensity within cardiac vegetations was 6.19 + 1.90 log1oCFU/g (13 rats). Administration of this dose of ampicillin-sulbactam for 5.5 days by continuous infusion resulted inresidual bacterial densities within vegetations of 4.9 ± 1.8log1o CFU/g (Table 2). This difference did not achievestatistical significance (P = 0.074).

(iii) Effect of increasing sulbactam dose on in vivo activity ofthe combination. This part of the study determined whetherincreasing the dose of sulbactam would enhance the efficacyof the combination against the ,B-lactamase-hyperproducingstrain BOSSA-1. Both the high-dose (2,000 mg/kg/day) andthe low-dose (500 mg/kg/day) sulbactam regimens (withampicillin at 1,000 mg/kg/day) were administered by contin-uous intravenous infusion for 5.5 days. The results areshown in Table 2. The residual bacterial densities withincardiac vegetations were reduced from 4.93 ± 1.84 log1o to3.65 ± 1.26 log1o CFU/g when the sulbactam dose wasincreased. Although direct comparison of these two regi-mens resulted in a P value of 0.027, this difference was notstatistically significant when the Bonferroni correction factorwas applied for additional comparisons. Thirty percent ofthe animals treated with the high-dose regimen had sterilecardiac vegetations, as compared with 4% of the animalstreated with the low-dose regimen; this difference was notstatistically significant. Results obtained with oxacillin ad-ministered by continuous infusion are also shown in Table 2for comparison.

TABLE 2. Results of 5.5-day continuous antibiotic infusion therapy of endocarditis due to S. aureus BOSSA-1

Bacterial densityRegimen Mean serum drug level No. of survivors/no. No. of sterile in vegetationsReglmen +~~~~SD (p.g/ml)a of animals vegOetati.onas/Sno. inveetationSD)SD(~~Lg/ml)' of aniinals of animals [logj0(CFU/g)(mean ±SD)]

Control 4/16 0/16 10.43 ± 0.84Oxacillin (600 mg/kg/day) 15.5 ± 7.11 13/14 4/14 4.57 ± 1.80Ampicillin (1,000 mg/kg/day) 27.7 ± 10.0, 22/25 1/25 4.93 ± 1.84

sulbactam (500 mg/kg/day) 31.0 ± 6.85Ampicillin (1,000 mg/kg/day) 29.7 t 13.1, 9/13 4/13 3.65 + 1.26

sulbactam (2,000 mg/kg/day) 41.6 + 14.1

a For ampicillin-sulbactam combinations, the first value is for ampicillin and the second is for sulbactam.

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TABLE 3. Results of continuous infusion therapy of experimental endocarditis due to additional strains of OSSA and BOSSA

No.ofterile Bacterial density in

Regimen Strains MIC range (MBC range) No. of survivors/ No. of sterile vegetationsReglmen Stralns ~~~~inFg/mla no. of animals vegetations/no. vegeatin+SD)in~Lg/mll n. of animals of animals [1ogj0(CFU/g)(mean ±SD)]

Controls OSSA 0/6 0/6 10.5 ± 0.23BOSSA 4/16 0/16 10.2 + 0.72

Oxacillin (600 mg/kg/day) OSSA <0.06-0.25 (32-64) 7/8 2/8 4.29 ± 1.78BOSSA 1-2 (2-250) 19/21 6/21 4.38 ± 1.65

Ampicillin (1,000 mg/kg/day) + OSSA .0.06-0.12 (16-64) 6/8 5/8 3.16 + 1.27sulbactam (2,000 mg/kg/day)

BOSSA 0.25-1 (1-250) 20/21 6/21 3.83 ± 1.43

a For ampicillin-sulbactam, the MIC and MBC are expressed as the concentration of ampicillin required to inhibit and kill, respectively, in the presence of 16jg of sulbactam per ml (8).

For any treatment group, excluding the small number ofanimals which died before completion of 5.5 days of therapydid not influence results. The mean bacterial densities cal-culated when these animals were excluded differed fromthose shown in Tables 2 and 3 by <0.35 log10 CFU/g.Likewise, statistical analyses of these values yielded resultsidentical to those obtained by including all animals receivingat least 3.5 days of treatment.

Administration by intermittent infusion was also examinedwith the higher-dose sulbactam regimen. In this group, peakand trough levels of ampicillin were 59.1 23.1 and 3.42.6 ,ug/ml and those of sulbactam were 94.0 + 4.2 and 7.90.3 p,g/ml, respectively. With the high-dose intermittentregimen, sterilization of vegetations (56%) and final bacterialdensities within vegetations (3.37 + 1.41 log1o CFU/g) indi-cated a benefit in comparison with the low-dose intermittentregimen (0% sterile, P = 0.004; bacterial density, 6.19 t 1.90log1o CFU/g, P < 0.001). Results observed with the high-dose intermittent regimen were equivalent to those seen withthe high-dose continuous regimen against BOSSA-1.

(iv) Studies with additional organisms. We evaluated theactivity of both oxacillin and ampicillin-sulbactam (high-dose regimen) against five additional BOSSA and two addi-tional OSSA strains to test the generality of our previousresults. Therapy was administered by continuous infusionfor 5.5 days, and the results are shown in Table 3. For bothdrug regimens, equivalent activities were found for BOSSAand OSSA strains, as determined by measuring bacterialdensities in vegetations. Identical rates of sterilization forthe two groups of strains were also noted with oxacillin (25to 29%), and although greater rates of sterilization withampicillin-sulbactam were seen with OSSA (63%) than withBOSSA (29%), these results were not statistically significant(P > 0.1).

DISCUSSION

Several factors have been implicated as potential mecha-nisms for borderline resistance to oxacillin among S. aureus

strains. Such strains may reflect true methicillin resistancewith synthesis of the low-affinity penicillin-binding proteinPBP 2a mediated by the mecA gene expressed heteroge-neously. Alternatively, resistance may result from a modifi-cation in the amount or affinity of normally migrating peni-cillin-binding proteins or may derive from increasedproduction of 3-lactamase (13). Even in the latter case,

however, additional factors which are not well understoodappear to be involved in borderline resistance to oxacillinbecause transformation of plasmids mediating production of

3-lactamase into different host strains does not necessarilyresult in borderline resistance to oxacillin despite hyperpro-duction of ,B-lactamase in the new host (2).The limited available experimental evidence suggests that

mecA-negative BOSSA strains are as susceptible to oxacillinin vivo as are fully oxacillin-susceptible strains of S. aureus(4, 7, 12). In the present study, using a rat endocarditismodel, we examined six clinical isolates of BOSSA whichwere negative for the mecA gene as determined by PCR,which produced large amounts of j-lactamase as measuredby relative rates of nitrocefin hydrolysis (12), and the oxacil-lin susceptibility of which was fully restored in the presenceof ,-lactamase inhibitors as originally described by McDou-gal and Thornsberry (8). Our results confirm the equivalentactivity of oxacillin in vivo against these isolates of BOSSAand fully oxacillin-susceptible strains of S. aureus (Table 3).

Previous work with BOSSA (oxacillin MIC = 2 ,ug/ml) inboth rat (3) and rabbit (7) experimental models of infectiveendocarditis has found amoxicillin-clavulanate equivalent tocloxacillin and ampicillin-sulbactam equivalent to oxacillin,respectively. Our own previous work in the rat model did notconfirm these results, with oxacillin proving more effectivethan ampicillin-sulbactam at two dosage regimens againstboth BOSSA and OSSA strains (12). We speculated that theability of sulbactam to induce 3-lactamase production inthese strains might have had a measurable negative impactduring the short-term, relatively low-dose therapy used, andwe cited as supporting evidence the observation that addi-tion of sulbactam to oxacillin appeared to slightly antagonizethe in vivo activity of the latter, albeit not to a statisticallysignificant degree, against both the BOSSA and OSSAstrains. In the present study, we demonstrated that evenwith the low-dose ampicillin-sulbactam regimen (500 and 250mg/kg/day, respectively), increasing the duration of therapyfrom 3.5 to 6.5 days resulted in equalization of activities ofthe drug combination against both the BOSSA-1 and OS-SA-1 strains used in the previous study (Table 1). Withhigher doses of ampicillin (1,000 mg/kg/day) combined withsulbactam (500 or 2,000 mg/kg/day), the in vivo activity ofthe combination regimen equalled that of oxacillin againstBOSSA-1 (Table 2) and against the additional strains ofBOSSA as well as OSSA studied (Table 3), bringing ourresults into concordance with those of the authors citedabove (3, 7).For two dosage regimens of sulbactam, comparison of

residual bacterial densities within cardiac vegetations fromanimals treated by continuous infusion with those in vege-tations from animals receiving treatment by frequent inter-mittent infusions indicated that the mode of antibiotic deliv-

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V-LACTAMASE-HYPERPRODUCING S. AUREUS IN VIVO 511

ery had no impact on the therapeutic outcome. With eithermode of administration, lower bacterial densities in vegeta-tions were observed in animals receiving the highest sulbac-tam regimen. When multiple comparisons were taken intoaccount, this difference retained statistical significance onlyfor animals treated with the intermittent infusion regimen.We conclude that oxacillin is likely to be equally effective

against BOSSA and OSSA strains, as is the combination ofampicillin with sulbactam at serum drug concentrations anddurations of therapy that were within a clinically relevantrange. When high enough concentrations of sulbactam wereachieved, oxacillin and ampicillin-sulbactam were equallyefficacious in the treatment of infections caused by bothBOSSA and OSSA strains. It is important to note, however,that administration of ampicillin-sulbactam in a 2:1 ratioresulted in disproportionately high levels of sulbactam in ratserum. This observation and the fact that the 1:2 ampicillinlsulbactam ratio used in our high-dose studies is not availablefor clinical use underscore the need for caution in extrapo-lating these results to the treatment of staphylococcal infec-tions in humans.

ACKNOWLEDGMENTS

This work was supported by a grant from the Roerig Division,Pfizer, Inc.Angelos Pefanis was supported by grants from the Greek Ministry

of Health and the Amphiarion Foundation of ChemotherapeuticStudies, Athens, Greece.

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