ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 257-260 Vol. 34, No.

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0066-4804/90/020257-04$02.00/0
Copyright © 1990, American Society for Microbiology

Efficacy of Ofloxacin in Experimental

Staphylococcus aureus Endocarditis
GLENN W. KAATZ,l* SUSAN M. SEO,1 STEVEN L. BARRIERE,2 LISA M. ALBRECHT,3t
AND MICHAEL J. RYBAK3
Department of Internal Medicine, Division of Infectious Diseases, Wayne State University School of Medicine,1 and
College of Pharmacy and Allied Health Professions, Wayne State University, and Department of Pharmacy
Services, Detroit Receiving HospitallUniversity Health Center,3 Detroit, Michigan 48201, and
Departments of Pharmaceutical Services, Pharmacology, and Medicine, Division of
Infectious Diseases, University of California, Los Angeles, Center for
the Health Sciences, Los Angeles, California 900242
Received 6 September 1989/Accepted 20 November 1989

The efficacy of ofloxacin was compared with that of vancomycin in the therapy of experimental Staphylo-
coccus aureus endocarditis. Rabbits infected with either a methicillin-susceptible (MSSA-1199) or a methicillin-
resistant (MRSA-494) test strain were treated with ofloxacin (20 mg/kg of body weight every 8 h) or vancomycin
(17.5 mg/kg of body weight every 6 h) for 4 days. The antimicrobial agents were found to be equally effective
in clearing bacteremia and in reducing bacterial counts in vegetations and in renal and splenic tissue of animals
infected with either test strain. The drugs were of equal efficacy in curing MRSA-494 endocarditis. No
resistance to ofloxacin emerged in either test strain during therapy. We conclude that in this model ofloxacin
is as efficacious as vancomycin and that, unlike for other fluoroquinolones we have evaluated, resistance to the
drug does not develop during therapy of this serious S. aureus infection.

Ofloxacin is a new fluoroquinolone antimicrobial agent of ofloxacin incorporated into Mueller-Hinton agar (Difco).
that possesses excellent in vitro activity against a broad Colonies were counted 48 h later.
range of bacteria, including methicillin-susceptible and Serum bactericidal titers (SBTs) were determined by using

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-resistant strains of Staphylococcus aureus (1, 10). Of the
currently available fluoroquinolones (both marketed and
investigational), only ciprofloxacin and pefloxacin show sim-
ilar in vitro potency against this pathogen (10). In addition to
its in vitro effectiveness, the pharmacokinetic profile of
ofloxacin is favorable and suggests that twice-daily dosing
should be adequate (9). Clearly, if ofloxacin maintains its
effectiveness against S. aureus in vivo, it may serve as an
alternative agent for the treatment of human infections
caused by this organism. We investigated this possibility by
comparing the therapeutic activities of parenterally admin-
istered ofloxacin and vancomycin by using the rabbit model
of S. aureus endocarditis. This model affords a severe test of
antimicrobial activity in a serious systemic infection.
MATERIALS AND METHODS
Organisms. The methicillin-susceptible and -resistant
strains of S. aureus used (MSSA-1199 and MRSA-494,
respectively) were bloodstream isolates from patients with
endocarditis.
In vitro studies. The MICs and MBCs of methicillin,
vancomycin, and ofloxacin for MSSA-1199 and MRSA-494
were determined by a microdilution method using Mueller-
Hinton broth (Difco Laboratories, Detroit, Mich.) supple-
mented with calcium and magnesium at concentrations of 50
and 25 mg/liter, respectively (4).
The frequencies at which test strains developed spontane-
ous mutational resistance to 2, 5, and 10 times their ofloxacin
MICs were determined by exposing exponential-growth-
phase organisms (-1010 CFU) to appropriate concentrations
*
Corresponding author.
t Present address: College of Pharmacy, University of Illinois at
Chicago, Chicago, IL 60680.
a microdilution method modified by the use of supplemented
Mueller-Hinton broth as the diluent, as the protein binding of
ofloxacin (25%) or vancomycin (50%) is insignificant (13).
Animal studies. Left-side endocarditis was established in
male New Zealand White rabbits (2 to 3 kg) as described
previously, using an intravenous bacterial inoculum of 106
CFU of either MSSA-1199 or MRSA-494 (5). Eighteen hours
later, all animals had 1 ml of blood withdrawn for culture.
Serial dilution and plating techniques were used to determine
CFU per milliliter of blood. Inclusion in the study required
that this blood culture be positive and that the catheter be
positioned properly across the aortic valve at autopsy.
Rabbits were then randomized to receive 4 days of either
ofloxacin (20 mg/kg of body weight intravenously every 8 h),
vancomycin (17.5 mg/kg of body weight intravenously every
6 h), or no treatment (controls). The dose administered was
adjusted for weight on a daily basis. Controls were sacrificed
at the time therapy was begun in animals receiving antimi-
crobial agents, and bacterial counts in vegetations and
tissues were determined (see below). The natural history of
endocarditis caused by both test strains was established by
determining the time between bacterial challenge and death
and by determining vegetation and tissue bacterial counts in
untreated animals allowed to succumb to the infectious
process.
Serum samples for the measurement of peak (obtained 1 h
postdose) and trough (just before a scheduled dose) antibi-
otic content and peak SBT were obtained from all animals at
the time of the first dose on day 2. Repeat blood cultures
were obtained before the first dose on day 3.
After 4 days of therapy, all MSSA-1199-infected animals
were sacrificed 8 to 10 h (for vancomycin) or 10 to 12 h (for
ofloxacin) after the final dose and autopsied in an aseptic
manner. For each animal a terminal blood culture was

257
258 KAATZ ET AL.
obtained, and vegetations and 500-mg (mean weight) sec-
tions of left kidney and spleen were removed for culture.
These specimens were weighed, suspended in 0.85% NaCl
(final volume, 1 ml), and homogenized. Quantitative bacte-
rial counts determined by serial dilution and plating tech-
niques were expressed as log1o CFU per gram (sensitivity
limit, 10 CFU per vegetation or tissue section; culture-
negative specimens were considered to contain 10 CFU for
numerical and statistical purposes). The effect of antibiotic
carry-over on cultured material was minimized by the vol-
ume of agar used in culture plates. This dilution effect was at
least 100-fold.
Because of the improved efficacy of both ofloxacin and
vancomycin in animals infected with MRSA-494 (see below),
randomly selected animals from each treatment group were
sacrificed after 4 days of therapy. These animals underwent
autopsy for the determination of vegetation and tissue bac-
terial counts as described above. Others (n = 10 for both
drugs) were monitored for 7 days posttherapy, with blood
cultures being obtained on days 3 and 7. These animals were
then sacrificed, and bacterial counts in vegetations and
tissues were determined. Those with sterile cultures from all
sites were considered cured of their infections.
Serum antibiotic content. Ofloxacin concentrations in se-
rum were determined by bioassay using an agar weil diffu-
sion method (11); Klebsiella pneumoniae 10031 was used as
the indicator organism. Vancomycin concentrations were
determined by fluorescence polarization immunoassay
(TDx; Abbott Diagnostics, Irving, Tex.) (12). Pooled normal
rabbit serum was used to prepare standards and dilute
unknowns as needed.
Resistance to ofloxacin. Isolates of MSSA-1199 and MRSA-
494 recovered from blood, vegetations, or tissues of animals
receiving ofloxacin were screened for the emergence of
resistance to the drug during therapy. This was done by
replica plating these organisms from their plates of original
growth onto tryptic soy agar (Difco) containing 5 or 10 times
the appropriate MIC of ofloxacin. Plates were examined for
growth 48 h later.
Statistical analysis. Comparisons of blood, vegetation, and
tissue bacterial counts were made by one-way analysis of
variance. Comparisons of frequencies of sterilization of
blood, vegetations, and renal and splenic tissue and of cure
of endocarditis were made by use of the Fisher exact test.
The Kruskal-Wallis H test was used for comparisons of
SBTs. A P value of <0.05 was considered significant.
RESULTS
In vitro studies. The MICs and MBCs of methicillin,
vancomycin, and ofloxacin for MSSA-1199 were 1.5 and 1.6,
0.5 and 0.5, and 0.4 and 0.8 p,g/ml, respectively. Correspond-
ing results for MRSA-494 were 21.8 and 59.5, 0.4 and 0.4,
and 0.4 and 0.8 ,ugIml, respectively. The frequency of
spontaneous mutational resistance to twice its ofloxacin
MIC was 3.71 x 10-9 for MSSA-1199; a frequency of
<1 x 10-10 was found for resistance to 5 and 10 times its
MIC. For MRSA-494, corresponding results were 4.79 x
lo-9, <1 x 10-10, and <1 x 10'1, respectively.
Animal studies. Death occurred in untreated rabbits in-
fected with either MRSA-494 or MSSA21199 within 2 days.
Vegetation and tissue bacterial counts in these animals were
0.5 to 1.0 loglo CFU per g higher than those found in controls
sacrificed at the initiation of antimicrobial therapy (data not
shown). These results establish the lethal and non-self-
curing nature of endocarditis caused by either test strain in
this animal model.
ANTIMICROB. AGENTS CHEMOTHER.
No differences were found in the intensity of pretreatment
bacteremia (log1o CFU per milliliter [mean ± standard
deviation]) for animals infected with MSSA-1199 and receiv-
ing vancomycin (3.83 + 0.83) or ofloxacin (3.75 + 1.13)
sacrificed after 4 days of therapy or for controls sacrificed 18
h after bacterial challenge (3.64 ± 0.72). The same was true
for animals infected with MRSA-494 and either treated with
vancomycin (3.14 ± 0.51) or ofloxacin (2.75 ± 0.76) or not
treated (controls; 3.10 ± 0.81).
Peak and trough concentrations of vancomycin in serum in
animals infected with MSSA-1199 were 30.4 ± 6.0 and 3.9 +
1.8 ,ug/ml, respectively (mean ± standard deviation). For
ofloxacin, the corresponding values were 8.3 ± 1.6 and 0.9 ±
0.4 ,ug/ml. The geometric mean peak SBT for animals
receiving vancomycin was 1:27, and for those receiving
ofloxacin it was 1:13. The mean peak SBT found in vanco-
mycin-treated animals was significantly higher than that in
animals treated with ofloxacin (P < 0.001).
For animals infected with MRSA-494, peak and trough
concentrations of vancomycin in serum were 29.7 ± 10.6 and
4.6 ± 3.4 ,ug/ml; for ofloxacin, corresponding results were
7.8 ± 1.4 and 0.9 ± 0.4 ,ug/ml. The geometric mean peak
SBTs were 1:15 (for vancomycin) and 1:9 (for ofloxacin). As
was true for animals infected with MSSA-1199, the mean
peak SBT found in animals treated with vancomycin was
significantly higher than that found in ofloxacin-treated ani-
mals (P < 0.001).
There were no differences in the frequencies of blood
culture sterilization after 2 or 4 days of therapy with vanco-
mycin or ofloxacin in animals infected with either test strain.
For vancomycin-treated animals with MSSA-1199 en-
docarditis, 80 and 100% had sterile cultures after 2 and 4
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days, respectively. For those treated with ofloxacin, 100 and
93% had sterile cultures at these times. For animals infected
with MRSA-494, blood cultures were sterile in 94% of
vancomycin-treated animals at both 2 and 4 days and in 88%
(2 days) and 100% (4 days) of ofloxacin-treated animals.
Quantitative bacterial counts in vegetations and tissues
are shown in Table 1. There were highly significant reduc-
tions in bacterial counts at each of these sites in rabbits
infected with either test strain and treated with vancomycin
or ofloxacin. In animals infected with the same test strain, no
differences were noted between vancomycin and ofloxacin in
these reductions or in the frequency of sterilization of
vegetations or tissues. Both drugs were more effective at
reducing bacterial counts in (and sterilizing) vegetations of
rabbits infected with MRSA-494 than of those infected with
MSSA-1199 (P < 0.001).
Cure of MRSA-494 endocarditis. Animals monitored for 7
days posttherapy had similar degrees of pretreatment bac-
teremia (vancomycin, 3.01 ± 0.73 [n = 10]; ofloxacin, 2.59 +
0.81 [n = 10]). Mean drug levels in serum and SBTs were
similar to those found in animals sacrificed after 4 days of
therapy. Both drugs produced a 100%o sterilization rate of
blood cultures during and after therapy.
Nine of ten vancomycin-treated animals and eight of ten
ofloxacin-treated animals had sterile vegetations and tissues
7 days posttherapy and were considered cured of their
infections. This difference was not significant.
Resistance to ofloxacin. No resistance to ofloxacin emerged
in either test strain during therapy.
DISCUSSION
Several fluoroquinolones, including ciprofloxacin, enoxa-
cin, fleroxacin, and pefloxacin, have been shown to compare
VOL. 34, 1990
TABLE 1. Vegetation and tissue bacterial counts
Infecting strain and No. of
treatment rabbits
MSSA-1199
Vancomycin 15
Ofloxacin 15
None (control) 11
MRSA-494
Vancomycin 16
Ofloxacin 16
None (control) 10
a Mean log1o CFU per gram ± standard deviation. Significant reductions in bacterial counts were noted for all animals infected with either test strain and
receiving antimicrobial therapy (P < 0.001 versus control).
b There were no significant differences between vancomycin and ofloxacin in the proportion of specimens sterilized in animals infected with the same test strain.
favorably with standard therapy in animals with experimen-
tal methicillin-susceptible or -resistant S. aureus infections
(3, 5-8, 14). On the basis of the results of our investigation,
the same appears to be true for ofloxacin. The drug cleared
bacteremia and reduced bacterial counts in vegetations and
tissues with an efficacy equal to that of vancomycin in
rabbits with either MSSA-1199 or MRSA-494 endocarditis.
In addition, ofloxacin was as effective as vancomycin in
curing MRSA-494 endocarditis. The greater efficacy of both
drugs in reducing bacterial counts in (and sterilizing) vege-
tations of rabbits infected with MRSA-494 likely was due to
the lower initial bacterial load in (>10-fold) and the smaller
size of (37% lower mean weight [69.5 versus 110.2 mg])
vegetations of those animals compared with vegetations of
animals infected with MSSA-1199.
Animals receiving vancomycin and infected with either
test strain had significantly higher peak SBTs than ofloxacin-
treated animals. Despite this difference, an improved micro-
biologic outcome was not realized for those receiving van-
comycin. We have found this to be true for ciprofloxacin and
fleroxacin in previous studies we have done with this animal
model (5-8). If these results can be corroborated in humans
with similar infections, attaining SBTs above 1:8 or 1:16
during fluoroquinolone therapy of S. aureus infections may
not be necessary. However, this conclusion is dependent on
the performance of the SBT procedure in a standardized
manner similar to that which we used.
We found a low frequency of in vitro spontaneous resis-
tance to ofloxacin in both of our test strains. Similar frequen-
cies have been found by others, supporting the contention
that spontaneous, and probably single-step, mutation to
ofloxacin resistance occurs rarely in S. aureus (1, 2). In our
study this low incidence was borne out in vivo, as no
resistance to the drug developed in either test strain during
therapy. In contrast, earlier studies we have done have
shown in vitro mutations of MSSA-1199 to twice its MIC of
ciprofloxacin or fleroxacin at frequencies which were 10- to
100-fold higher than those found in the current study with
ofloxacin. In addition, these earlier studies demonstrated
that a high proportion of rabbits infected with MSSA-1199
and treated with ciprofloxacin or fleroxacin yield isolates
that are resistant to the appropriate drug at 5 and 10 times the
MIC (6, 8; G. W. Kaatz, S. M. Seo, S. L. Barriere, L. M.
Albrecht, and M. J. Rybak, Abstr. Annu. Meet. Am. Soc.
Microbiol. 1989, A55, p. 10). We also have found that a
modest proportion of rabbits with MRSA-494 endocarditis
treated with fleroxacin will yield isolates resistant to more
than 5 times the MIC (7). These results suggest that the
development of resistance to ofloxacin during therapy of S.
OFLOXACIN IN EXPERIMENTAL S. AUREUS ENDOCARDITIS 259
Bacterial countsa (no. of culture-negative specimens') in:
Vegetation Kidney Spleen
7.08 ± 1.74 (0) 1.19 ± 0.05 (14) 1.27 ± 0.16 (13)
5.90 ± 1.81 (0) 1.23 ± 0.46 (14) 1.19 ± 0.12 (12)
10.27 ± 0.44 (0) 5.13 + 0.80 (0) 6.08 ± 0.40 (0)
2.48 ± 1.19 (14) 1.31 ± 0.54 (15) 1.64 ± 0.76 (14)
2.71 ± 1.05 (13) 1.24 ± 0.09 (16) 1.34 ± 0.12 (16)
8.98 ± 0.63 (0) 5.44 ± 1.79 (0) 5.24 ± 0.81 (0)
aureus infections may occur less commonly than with these
other fluoroquinolones. However, frequent evaluations of
MIC trends after the introduction of ofloxacin into clinical
practice would be in order. This is especially important in
light of several recent reports describing a high incidence of
ciprofloxacin resistance in S. aureus isolates (especially
methicillin-resistant strains) recovered from patients who
had received the drug, as well as from other patients and
hospital staff never treated with the drug who may have
acquired such strains nosocomially (H. M. Blumberg, D.
Rimland, and I. K. Wachsmuth, Program Abstr. 29th Inter-
sci. Conf. Antimicrob. Agents Chemother., abstr. no. 7,
1989; L. J. Strausbaugh, C. M. Jacobson, D. L. Sewell, and
T. T. Ward, 29th ICAAC, abstr. no. 1257, 1989).
In conclusion, we have established that ofloxacin is ther-
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apeutically equivalent to vancomycin in this model of a
serious methicillin-susceptible or -resistant S. aureus infec-
tion. If the low frequency at which in vitro and in vivo
resistance to ofloxacin develops in MSSA-1199 and MRSA-
494 can be corroborated with other strains of S. aureus, and
if the efficacy results with ofloxacin we found in this animal
model are supported by therapeutic trials in humans, this
drug may be the fluoroquinolone of choice for the treatment
of serious human infections caused by this organism.
ACKNOWLEDGMENTS
This study was supported by a grant from Ortho Pharmaceutical
Corporation, Raritan, N.J.
We thank Scott Eathorne, Kenneth Elman, Nancy Romatowski,
Ernest Siwik, and Frederic Smith for their technical assistance.
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