ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1990, p. 728-732 Vol. 34, No.

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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 of sulbactam
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
of Staphylococcus aureus displaying borderline susceptibil-
ity to antistaphylococcal penicillins. These strains were
inhibited by methicillin and oxacillin at concentrations of 4
and 2 p,g/ml, respectively, and were characterized by the
production of large amounts of P-lactamase. Addition of a
f-lactamase inhibitor such as clavulanic acid or sulbactam
(This work was presented at the 28th Interscience Confer-
ence on Antimicrobial Agents and Chemotherapy [C. Thau-
vin-Eliopoulos, L. B. Rice, G. M. Eliopoulos, and R. C.
Moellering, Jr., Program Abstr. 28th Intersci. Conf. Antimi-
crob. Agents Chemother., abstr. no. 367, 1988].)

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restored activities of the penicillins to levels which inhibit
fully methicillin-susceptible S. aureus strains. Such strains
appear to be distinct from those demonstrating chromoso-
mally mediated intrinsic ,-lactamase resistance, which has
been associated with the presence of a low-affinity penicillin-
binding protein, termed PBP 2a or 2' (5). To differentiate
borderline susceptible strains from the latter group of intrin-
sically resistant organisms further, they have also been
called acquired-resistant strains (7, 8). Whether infections
caused by borderline susceptible staphylococcal isolates
would respond to ,B-lactams as well as infections caused by
fully susceptible strains, or whether these isolates should be
considered the same as intrinsically resistant strains for
clinical and therapeutic purposes, is unclear and remains to
be determined.
In this study, we established an aortic valve endocarditis
model in rats, using a fully oxacillin-susceptible strain of S.
aureus or a borderline susceptible clinical isolate of S.
aureus, to evaluate the role of P-lactamase production in the
therapeutic efficacies of oxacillin alone, oxacillin combined
with sulbactam, and ampicillin-sulbactam in combination.
* Corresponding author.
728
MATERIALS AND METHODS
Bacterial strains. The two strains of S. aureus used
throughout this study were clinical blood culture isolates
recovered in Boston, Mass. Susceptibilities were determined
by microdilution broth titration in cation-supplemented
Mueller-Hinton broth (CSMHB; BBL Microbiology Sys-
tems, Cockeysville, Md.) with 2% NaCl (9, 16). Strain 1 was
borderline oxacillin susceptible (BOSSA) with methicillin
and oxacillin MICs of 4 and 1 ,ug/ml, respectively. Addition
of sulbactam, 16 jig/ml (8), reduced MICs of these agents to
2 and 0.25 p.g/ml, respectively. Strain 2 was a fully oxacillin-
susceptible S. aureus (OSSA) strain with methicillin and
oxacillin MICs of 2 and 0.25 ,ug/ml, respectively. Sulbactam
alone inhibited both strains at 250 ,ug/ml. Antimicrobial
susceptibilities were also determined in CSMHB plus 2%
NaCl supplemented with 50% rat serum, using inocula of 5 x
105 to 1 x 106 CFU/ml. Inhibitory endpoints were read at 24
and 48 h of incubation at 35°C. At 48 h, 0.01-ml samples were
transferred to antibiotic-free plates for determination of
MBCs (11).
Antimicrobial agents. Ampicillin and sulbactam standard
reference powders and the 2:1 combination for in vivo
studies were generously provided by Roerig-Pfizer, Inc.,
New York, N.Y. Oxacillin was obtained from Bristol Lab-
VOL. 34, 1990
V-LACTAMASE-HYPERPRODUCING S. AUREUS
TABLE 1. Results of therapy of experimental S. aureus endocarditis
BOSSA
Regimen
Regimen ~~Drug
Du(g/ml levels
SD) MiC)
MC No. of survivors/
(q.g/ml)a no.ofanmas
no. of animals (%)
Controls 8/29 (28)
Oxacillin alone 10.9 ± 5.1 1 (32) 15/16 (94)
+ Sulbactam 6.2 ± 0.5 1 (8) 14/15 (93)
Ampicilin' 8.3 ± 2.6 125 (250) C 8 (16)
+ sulbactam (low dose) 5.9 ± 2.4 4 (32) 8/11 (73)
Ampicillind 16.8 ± 7.4 125 (250)
+ sulbactam (high dose) 9.5 ± 1.1 4 (32) 12/16 (75)
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
Sigma Chemical Co., St. Louis, Mo.
Evaluation of 1-lactamase activity. P-Lactamase activity of
cell lysates was determined by a spectrophotometric method
(10). Bacteria were grown in dextrose phosphate broth
(GIBCO Diagnostics, Madison, Wis.) to log phase at 35°C
and harvested when cell densities reached 109 CFU/ml. Cell
pellets were lysed by incubation with lysostaphin (50 ,ug/ml)
(Sigma) and Triton X-100 (0.05% final concentration) (Bio-
Rad Laboratories, Richmond, Calif.). For studies of ,B-
lactamase induction, cells were grown in the presence of
ampicillin or sulbactam at concentrations of 0.1 x the MIC of
each drug.
Production of bacterial endocarditis. Bacterial endocarditis
was established by the technique of Santoro and Levison
(12), with minor modifications as described previously (14,
15). Nonbacterial thrombotic endocarditis was created in
male Sprague-Dawley rats (180 to 250 g) by insertion of a
polyethylene catheter (PE 10 Intramedic tubing) across the
aortic valve via the right carotid artery. At 20 min after
catheterization, 106 CFU of the infecting strain in saline
were injected through the catheter, which was ligated and
left in place throughout the experiment. Establishment of
bacterial endocarditis was ascertained by positive blood
cultures drawn just before onset of therapy. Only animals
with both initial blood cultures positive for the infecting
strain and correct placement of the catheter across the aortic
valve (as established at autopsy) were included in the study.
With both strains, colony counts in cardiac vegetations
reached 8.0 to 8.5 log1o CFU/g by 12 h after infection.
Antimicrobial therapy. Treatment was started 12 h after
bacterial challenge and continued for 3.5 days. All antimi-
crobial agents were delivered by continuous intravenous
infusion via an indwelling central venous catheter inserted
through the external jugular vein into the superior vena cava,
as described previously (14). Antimicrobial agents were
carefully delivered with syringe pumps (Orion Research,
inc., Cambridge, Mass.). Animals were randomly assigned
to one of the following five groups: (i) oxacillin alone (600
mg/kg per day), (ii) oxacillin (600 mg/kg per day) plus
sulbactam (300 mg/kg per day), (iii) ampicillin (450 mg/kg per
day) plus sulbactam (225 mg/kg per day), (iv) ampicillin
(1,000 mg/kg per day) plus sulbactam (500 mg/kg per day),
and (v) an untreated control group.
Monitoring of therapy and outcome. On day 3 of therapy,
729
OSSA
Bacterial titer in MIC (MBC) No. of survivors/ Bacterial titer in
%) (log10vegetations
CFU/g + SD) (pgm)
(ml) no. animals (%) (log10vegetations
o offaias() CFtJ/g ± SD)
10.5 ± 0.5 3/28 (11) 10.4 + 0.1
4.8 ± 1.6 1 (2) 14/18 (78) 4.4 ± 1.7
6.1 ± 2.3 1 (4) 13/15 (87) 5.3 ± 1.6
9.0 ± 1.6 1 (2) 9/10 (90) 7.1 ± 2.0
8 (16)
6.6 ± 2.1 1 (2) 17/19 (89) 6.6 ± 1.9
antibiotic levels in serum were measured by using an agar
well diffusion technique (1), with Bacillus subtilis as the test
organism for ampicillin and oxacillin levels and with a
P-lactamase-producing strain of Pasteurella haemolytica as
the test organism for sulbactam levels. Animals surviving 3.5
days of treatment were sacrificed 2 h after discontinuation of
therapy. Cardiac vegetations were aseptically removed,
weighed, homogenized, and diluted in saline for bacterial
titer determination. Colony counts were performed on man-
nitol-salt agar plates with and without penicillinase to control
for potential antibiotic carry-over. The lower limit of detec-
tion by this method was 2.3 log1o CFU/g of vegetation.
Statistical evaluation. The chi-square test with Yates cor-
rection was used for evaluation of nominal data. Differences
in bacterial titers within vegetations among treatment groups
were assessed by analysis of variance followed by the
Bonferroni method for multiple comparisons (4).
RESULTS
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In vitro susceptilillity studies. Activities of antimicrobial
agents against the test strains determined in CSMHB plus
2% NaCl supplemented with 50% rat serum are shown in
Table 1. MICs of oxacillin against the two strains were
identical in this medium, and these were not affected in the
presence of sulbactam. The MIC of ampicillin-sulbactam
(2:1, expressed in terms of the ampicillin component) against
the BOSSA strain was fourfold higher than that against the
OSSA strain. In the absence of rat serum, MICs (micro-
grams per milliliter) against the BOSSA and OSSA strains in
CSMHB plus 2% NaCl were as follows: ampicillin-sulbac-
tam, 4 and 2; oxacillin, 1.0 and 0.25; oxacillin-sulbactam,
0.25 and 0.125. Strain 1 (BOSSA) demonstrated greater
tolerance to the bactericidal effects of oxacillin (MBC/MIC
ratio, 32) and ampicillin-sulbactam (MBC/MIC ratio, 8) than
did strain 2 (OSSA) (MBC/MIC ratios, 2 for both drugs).
P-Lactamase activity. As expected, P-lactamase activity of
cell lysates from strain 1 was substantially greater than that
of strain 2 (Fig. 1). Both ampicillin and sulbactam induced
production of P-lactamase, but in each case the resulting
hydrolytic activity was greater with BOSSA lysates. This
effect was particularly striking when BOSSA cells were
induced by incubation with sulbactam. The resulting lysate
achieved complete hydrolysis of substrate within 5 min.
Effects of preincubation with sulbactam or clavulanate on
730 THAUVIN-ELIOPOULOS ET AL.
100
C:
0
0
2- 75
-oU)
N
>% 50
0
L.
-C
C:
D
NIO 25 BO2SSA
~~~~amp-induced
0.0 1.0
FIG. 1. P-Lactamase activity of both infecting strains of S.
(Sb)-induced cells.
P-lactamase activity were also studied in five additional
BOSSA isolates. In four of these, exposure to sulbactam
induced P-lactamase activity, resulting in .90% nitrocefin
hydrolysis within 2 to 4 min, with the fifth strain hydrolyzing
this amount of drug by 60 min. In each case, preincubation
with clavulanic acid retarded nitrocefin hydrolysis by the
resulting cell lysate. Sulbactam induced P-lactamase activity
in two strains of OSSA which produced P-lactamase, but, as
expected, did not affect one penicillin-susceptible, non-
3-lactamase-producing isolate.
Experimental endocarditis. Results of therapy are shown in
Table 1. Antibiotic levels in serum achieved with both
oxacillin and ampicillin-sulbactam are clinically attainable in
humans and exceeded MICs against the test organisms.
Survival of animals in each treatment group exceeded that of
control animals (P 0.02), but did not differ among the
-
groups of treated animals. Oxacillin was equally effective
against BOSSA and OSSA infections, with residual titers
within vegetations of 4.8 ± 1.6 and 4.4 ± 1.7 log1o CFU/g,
respectively. Addition of sulbactam to oxacillin did not
enhance activity of the latter against either strain. Residual
bacterial titers were higher among animals treated with this
combination than among those treated with oxacillin alone,
but these differences were not statistically significant. Like-
wise, the high-dose ampicillin-sulbactam regimen was
equally effective against BOSSA and OSSA infections. This
regimen was less effective than oxacillin alone against both
strains (P = 0.01 for BOSSA and P = 0.002 for OSSA), but
ANTIMICROB. AGENTS CHEMOTHER.
2.0 3.0 4.0 5.0
Time (Hour)
aureus in uninduced cells, in ampicillin (amp)-induced cells, and in sulbactam
was comparable to the oxacillin-sulbactam regimen. Statis-
tically significant differences in response of animals infected
with BOSSA or OSSA strains were evident only among
those treated with low-dose ampicillin-sulbactam. With this
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regimen, significantly greater reductions in vegetation bac-
terial titers were observed with the OSSA than with the
BOSSA strain (P < 0.05).
DISCUSSION
Since the recognition that strains for which oxacillin MICs
are -1 ,ug/ml constitute a small but significant proportion of
recent isolates of S. aureus at some centers (7, 18), there has
been considerable concern about appropriate therapy of
infections due to such strains. Specifically, would a ,-lactam
alone prove successful or would such isolates more closely
resemble methicillin-resistant S. aureus and fail to respond
predictably? It has become amply clear, however, that
mechanisms of resistance to penicillinase-resistant penicil-
lins cannot always be accurately predicted solely from levels
of susceptibility. Strains of S. aureus for which oxacillin
MICs are between 1 and 2 ,ug/ml appear to represent a
heterogeneous group, some of which hyperproduce P-lacta-
mase, as defined by McDougal and Thormsberry (8), and
others of which may attribute oxacillin resistance to alter-
ations in penicillin-binding proteins (3, 13). In the present
study, we ascertained that the BOSSA strain studied was a
hyperproducer of ,-lactamase as compared with a clinical
isolate demonstrating full susceptibility to oxacillin.
VOL. 34, 1990
This study showed oxacillin to be equally effective against
the BOSSA and OSSA strains, with residual bacterial titers
of 4.4 to 4.8 log1o CFU/g. This observation is consistent with
results of other animal studies which have compared re-
sponse of fully susceptible and intermediately susceptible
strains of S. aureus to therapy with antistaphylococcal
penicillins. Cantoni et al. (2), using a rat endocarditis model,
found similar responses to cloxacillin of two strains, with
oxacillin MICs of 0.25 and 2 ,ug/ml. Chambers et al. (3),
using a rabbit model of endocarditis, noted identical re-
sponses to nafcillin of a fully susceptible strain (nafcillin
MIC, 0.5 ,ug/ml) and one characterized by a nafcillin MIC of
4 ,ug/ml. Penicillin-binding proteins of the latter strain were
examined and found to be similar to the pattern of methicil-
lin-susceptible S. aureus strains. However, in neither of
these studies was the level of ,-lactamase production in
intermediately resistant isolates formally tested. These data
are also consistent with results of a retrospective analysis of
clinical infections due to S. aureus strains with different
levels of resistance to oxacillin (7). Treatment with a peni-
cillinase-resistant penicillin was successful in 88.6% of in-
fections caused by fully susceptible strains and 7 of 10 (70%)
infections caused by acquired oxacillin-resistant isolates
(oxacillin resistant by disk diffusion; oxacillin MIC of c2
,ug/ml by broth dilution). These results are difficult to inter-
pret, however, in view of the fact that four of five (80%)
infections caused by oxacillin-resistant isolates (MIC, >8
jig/ml) also responded to treatment with a penicillinase-
resistant penicillin.
In attempting to reconcile the above results with the
fourfold difference in broth dilution oxacillin MICs and the
significant difference in ,-lactamase activity generated by
the two strains used in our study (Fig. 1), it is noteworthy
that susceptibility testing performed in the presence of 50%
rat serum yielded identical oxacillin MICs against the two
strains. Continuous administration of oxacillin (or repetitive
antibiotic dosing in the studies cited), resulting in drug
concentrations in serum 10-fold higher than the MIC, may
have partially negated any differences in P-lactamase pro-
duction apparent in the static environment of standard
susceptibility testing. While it is theoretically possible that
our results might also be explained by lower virulence of the
BOSSA strain, this seems highly unlikely in view of the fact
that bacterial titers in vegetations of untreated animals
inoculated with either strain were identical both 12 h after
infection (8.0 to 8.5 log1o CFU/g) and at sacrifice on day 4
(10.4 to 10.5 log1o CFU/g). Tolerance to the bactericidal
effects of oxacillin which we observed with strain 1 (MBC/
MIC ratio, 32) appears to be common among BOSSA
isolates (18), but is of uncertain relevance in vivo. Woods
and Yam (18) have reported that tolerance to oxacillin
among P-lactamase-hyperproducing strains of S. aureus
determined by MBC testing did not predict poor bactericidal
activity of the drug by time-kill techniques.
The combination of ampicillin plus sulbactam, adminis-
tered in the high-dose regimen, also demonstrated identical
efficacy against BOSSA and OSSA strains despite a 4-fold
difference in MICs and a 16-fold difference in MBCs against
the two isolates (Table 1). At the lower dose level, however,
the combination was significantly less effective against the
BOSSA strain. In the latter case, levels of ampicillin in
serum exceeded the MIC (of ampicillin in combination with
sulbactam) by only twofold. In addition, sulbactam levels
were likely to have been inadequate in view of the linear
relationship between sulbactam concentration and ampicillin
P-LACTAMASE-HYPERPRODUCING S. AUREUS 731
MBC (in combination) against penicillinase-producing S.
aureus (6).
In this study, oxacillin was more effective than ampicillin-
sulbactam (high dose) against both isolates tested. While it
might be possible to attribute this result to a superior
antibiotic level in serum/MIC ratio (10:1 for oxacillin, 4:1 for
ampicillin-sulbactam) for the BOSSA isolates, alternative
explanations would clearly have to be sought in the case of
the OSSA isolates (10:1 for oxacillin, 16:1 for ampicillin-
sulbactam). Examination of attained drug levels in serum in
relation to bactericidal endpoints yields similar conclusions.
Our results are also in contrast to those of other studies
which found ampicillin-sulbactam comparable to nafcillin
against methicillin-susceptible S. aureus (17) and amoxicil-
lin-clavulanate comparable to cloxacillin against both a fully
oxacillin-susceptible strain and one for which the oxacillin
MIC was 2 ,ug/ml (2).
We believe that these results may be explained by the
observation that sulbactam displayed a striking ability to
induce production of P-lactamase in each of the two strains
(Fig. 1). This phenomenon was probably reflected in the
diminished (although not statistically significant) efficacy of
oxacillin against both strains when the drug was combined
with sulbactam. This hypothesis provides an explanation for
the discordance of our results with those of the aforemen-
tioned studies with amoxicillin plus clavulanic acid (2),
which does not appear to be a potent inducer of the P-
lactamase (data not shown), and with ampicillin plus sulbac-
tam (17), which was dosed to achieve peak levels in serum
substantially higher than the steady-state mean levels in
serum achieved in the present study. Attainment of such
high drug concentrations in serum may be important because
the capacity of sulbactam to induce P-lactamase may well
reach a maximum level at modest drug concentrations, while
the P-lactamase-inhibitory activity of the agent appears to
increase proportionally with drug concentration (6).
We conclude that, against the representative strains stud-
ied here, both oxacillin and ampicillin-sulbactam (high dose)
were as effective against a P-lactamase-hyperproducing
BOSSA as against an OSSA strain. At the doses used,
ampicillin-sulbactam was less effective than oxacillin.
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Higher drug concentrations of ampicillin-sulbactam in serum
would probably have enhanced the efficacy of this combina-
tion. We have shown that sulbactam could induce produc-
tion of ,-lactamase in these strains. Combination of sulbac-
tam with oxacillin, while not affecting survival, appeared to
diminish efficacy in terms of final bacterial titers within
vegetations, although this effect did not reach statistical
significance.
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