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Antimicrobial Agents and Chemotherapy, August 2000, p. 2077-2080, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bactericidal Activity of Gentamicin against
Enterococcus faecalis In Vitro and In Vivo
Agnès
Lefort,1
Michel
Arthur,2
Louis
Garry,1
Claude
Carbon,1
Patrice
Courvalin,2 and
Bruno
Fantin1,*
Institut National de la Santé et de la
Recherche Médicale, EMI 9933, Hôpital Bichat-Claude
Bernard,1 and Unité des Agents
Antibactériens, Institut Pasteur,2 Paris,
France
Received 3 December 1999/Returned for modification 26 February
2000/Accepted 10 May 2000
 |
ABSTRACT |
The activity of gentamicin at various concentrations against two
strains of Enterococcus faecalis was investigated in vitro and in a rabbit model of aortic endocarditis. In vitro, gentamicin at
0.5 to 4 times the MIC failed to reduce the number of bacteria at
24 h. Rabbit or human serum dramatically increased gentamicin activity, leading to a 
3-log10 CFU/ml decrease in
bacterial counts when the drug concentration exceeded the MIC.
Susceptibility testing in the presence of serum was predictive of in
vivo activity, since gentamicin alone significantly reduced the number
of surviving bacteria in the vegetations if the peak-to-MIC ratio was
greater than 1. However, gentamicin selected resistant
mutants in rabbits. The intrinsic activity of gentamicin should be
taken into account in evaluation of combinations of gentamicin and cell
wall-active agents against enterococci.
| |
INTRODUCTION |
Enterococci are intrinsically
resistant to low levels of aminoglycosides due to inefficient active
transport across the cytoplasmic membrane (13). Thus,
aminoglycosides alone are considered inactive in the treatment of
enterococcal infections and are usually combined with inhibitors of
cell wall synthesis which may facilitate their uptake (16).
Therapeutic regimens with once-daily doses achieving high peak levels
of aminoglycosides in serum have been recommended since they provide
increased activity, in comparison to more traditional administration of
the drugs (1, 3, 4). Consistent with this notion, it was
shown that the bactericidal activity of aminoglycosides is
concentration dependent (6) and that high peak-to-MIC ratios could reduce the emergence of resistant mutants (5). We
conducted the present study to determine if the higher peak levels of
gentamicin in serum obtained with these new regimens affect the
antienterococcal activity of the drug.
| |
MATERIALS AND METHODS |
Bacterial strains and media.
Enterococcus faecalis
JH2-2 is susceptible to glycopeptides and 
-lactams and is
intrinsically resistant to low levels of aminoglycosides
(12). E. faecalis BM4281 is a JH2-2
transconjugant harboring a 250-kb chromosomal genetic element
conferring VanB-type resistance (21). BM4281 is
significantly less resistant to gentamicin (MIC = 5 µg/ml) than
JH2-2 (MIC = 16 µg/ml) (14).
In vitro susceptibility testing.
The MICs of gentamicin
(Unilabo, Levallois Perret, France) were determined by the method of
Steers et al. (23) with 105 CFU per spot on
brain heart infusion (BHI) agar (Difco Laboratories, Detroit,
Mich.) after 24 h of incubation. For time-kill curves, exponentially growing E. faecalis was diluted in glass
tubes containing 10 ml of BHI broth (pH 7.0; Difco) or 5 ml of
BHI broth and 5 ml of undiluted complement-intact rabbit serum (mixture
pH 7.4; Sigma Chemical Co., St. Louis, Mo.) or 5 ml of BHI broth and 5 ml of undiluted complement-intact human serum (mixture pH 7.4; Sigma)
to obtain 107 CFU/ml and was incubated with gentamicin at
various concentrations (0.5 to 4 times the MIC). Aliquots were taken
after 0, 3, 6, and 24 h of incubation and plated on BHI agar to
enumerate the surviving bacteria. In the experiment using human serum,
surviving bacteria were also enumerated at 48 h. A bactericidal
effect was defined as a 3-log10 CFU/ml difference between
the initial inoculum and the bacterial count after 24 h of incubation.
Experimental endocarditis.
Aortic endocarditis was induced
in female New Zealand White rabbits (2.2 to 2.5 kg) by insertion of a
polyethylene catheter through the right carotid artery into the left
ventricle (2). Twenty-four hours after catheter insertion,
the rabbits were inoculated by the ear vein with 108 CFU of
E. faecalis JH2-2 or BM4281 in 1 ml of 0.9% NaCl. The catheter was left in place throughout the experiment. Forty-eight hours
after inoculation, animals received gentamicin intramuscularly twice
daily for 5 days at 3 or 6 mg/kg of body weight per injection. Control
animals were sacrificed 48 h after inoculation (start of therapy)
or at the same time as treated animals (end of therapy).
For determination of peak and trough gentamicin levels in serum, blood
was sampled on day 5 of antimicrobial therapy 30 min and 12 h
after the last injection, respectively. Antibiotic concentrations were measured in serum by fluorescence polarization immunoassay (AxSYM System; Abbott Diagnostics, Rungis, France).
The animals were killed by an intravenous injection of pentobarbital.
At the time of sacrifice, the heart was removed and the chambers on the
left side were examined to confirm vegetative endocarditis. For each
rabbit, vegetations were excised, pooled, weighed, and homogenized in 1 ml of sterile distilled water. Vegetation homogenates were plated on
agar to count surviving bacteria and on agar containing gentamicin at
twice the MIC to enumerate mutants after 48 h of incubation. When
mutants were recovered on gentamicin-containing agar, 10 colonies per
rabbit were regrown on drug-free agar and then subcultured in
antibiotic-free broth before testing the MICs. The results were
expressed as log10 CFU per gram of vegetation.
Statistics.
Comparison of bacterial counts of the
vegetations of rabbits treated with various regimens was performed by
the Fisher test (22). A P value of <0.05 was
considered significant.
| |
RESULTS AND DISCUSSION |
In vitro activity of gentamicin.
A concentration-dependent
increase in bacterial killing of JH2-2 (MIC = 16 µg/ml) was
observed during the first 6 h of incubation with gentamicin (Fig.
1a). However, the number of bacteria
increased between 6 and 24 h for all gentamicin concentrations
tested. Regrowth of JH2-2 was previously shown to result from the
selection of mutants with moderate increases (two- to sixfold) in the
level of gentamicin resistance (14). Rabbit serum enhanced
the activity of gentamicin, leading to a bacteriostatic effect at 8 µg of gentamicin/ml (0.5 times the MIC) and a bactericidal effect at
24 h for gentamicin concentrations that were 20 µg/ml (1.2
times the MIC) (Fig. 1b). Similarly, killing of BM4281 (MIC = 5 µg/ml) was observed only in the presence of serum for gentamicin
concentrations that were 8 µg/ml (1.6 times the MIC) (Fig. 1b).
Thus, the supplementation of media with rabbit serum dramatically
increased the activity of gentamicin against JH2-2 and BM4281,
resulting in a bactericidal effect when the drug concentration exceeded
the MIC. Time-kill curves performed in the presence of human serum also
showed that adding serum increased the activity of gentamicin against
the two strains, with a 3-log CFU/ml decrease in bacterial titers when the concentration of gentamicin exceeded the MIC (Fig. 1c). However, bacterial regrowth was observed at 48 h for all
concentrations of gentamicin tested, suggesting the emergence of
gentamicin-resistant mutants (14).
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FIG. 1.
In vitro activity of gentamicin (Gent) against E. faecalis JH2-2 and BM4281 in BHI (a), 50% BHI and 50% rabbit
serum (b), or 50% BHI and 50% human serum (c). Gentamicin
concentrations are expressed in micrograms per milliliter.
|
|
Experimental endocarditis.
Treatment of rabbits with
gentamicin at 3 mg/kg twice a day (b.i.d.) led to peak levels in serum
of 7.0 ± 1.3 µg/ml (Table 1),
similar to those previously recommended with traditional therapeutic
regimens (18-20). Peak levels in serum obtained with a
dosage of 6 mg/kg b.i.d. (17.7 ± 1.5 µg/ml) were within the range achieved with current schedules of gentamicin administration (8; D. R. McNamara, A. N. Nafziger,
A. M. Menhinick, L. J. Cabelus, and J. S. Bertino,
Jr., Abstr. 39th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 1009, 1999).
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TABLE 1.
Gentamicin levels in serum and peak-to-MIC ratios in
rabbits with experimental endocarditis due to E. faecalis JH2-2 or BM4281
|
|
When bacterial titers in the vegetations of treated animals were
compared to those in controls left untreated for the same time period,
a statistically significant decrease was noted for both strains and
both gentamicin regimens (3 or 6 mg/kg b.i.d.). Compared to the initial
bacterial counts in the vegetations 48 h after inoculation of the
bacteria, which provides an estimate of the actual reduction of the
number of bacteria during therapy, the numbers of CFU were also
significantly decreased, except for the most resistant strain (JH2-2,
MIC = 16 µg/ml) and the low-dose gentamicin regimen (3 mg/kg
b.i.d.), which generated a peak-to-MIC ratio of <1 (Tables 1 and
2). None of the regimens prevented the
emergence of gentamicin-resistant mutants of JH2-2 or BM4281 (Table 2),
as previously reported for low doses of gentamicin in the same
experimental model (14) and as expected from in vitro
time-kill curves in the presence of human serum that showed bacterial
regrowth at 48 h, suggestive of the emergence of
gentamicin-resistant mutants (Fig. 1c). Analysis of the same set of
data in terms of reduction of the number of surviving bacteria as a
function of the peak-to-MIC ratio revealed a dose-dependent in vivo
killing of enterococci by gentamicin (Fig.
2).
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FIG. 2.
Activity of gentamicin in experimental endocarditis due
to E. faecalis. Reductions of bacterial counts in the
vegetations were plotted for different peak-to-MIC ratios of
gentamicin.
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|
A discrepancy was observed between the in vivo results and those
obtained in vitro in BHI medium. As shown previously, rabbit and human
serum enhanced the in vitro activity of gentamicin against E. faecalis (24, 26). Susceptibility testing performed in the presence of serum was predictive of in vivo activity in rabbits. The increase in the bactericidal activity of gentamicin in the presence
of serum may result from several factors. First, it may be due to
species-related factors, as suggested by the bactericidal activity of
vancomycin against enterococci in the presence of rat serum but not
rabbit or human serum (9). However, we noted in our study an
increase in the activity of gentamicin not only with rabbit serum but
also with human serum. Second, it was shown that the alkalinization of
nutrient broth and modifications in the concentrations of cations (such
as calcium or magnesium), the various components of complement, and
specific immunoglobulins of the serum may interact with antibiotics in
killing bacteria (7, 10, 25, 26). Finally, the increase in
bactericidal activity may be due to the interaction between gentamicin
and serum noncomplement cationic proteins, such as -lysin (9, 15, 26). Indeed, Traub et al. showed that the antagonization of
-lysin abolished the augmentation of gentamicin antienterococcal activity induced by the addition of rabbit, bovine, or human serum (26).
Conclusions.
It is well established for aminoglycosides that
the area under the concentration-time curve-to-MIC ratio and the
peak-to-MIC ratio are two pharmacokinetic parameters that are strongly
related and predictive of in vivo efficiency (6, 11, 17).
These parameters have not been evaluated for gentamicin alone in
enterococcal infections, since aminoglycosides are always used in
combinations. In this study, gentamicin was found to be significantly
active against E. faecalis in rabbit endocarditis, provided
that the peak exceeded the MIC (Table 2 and Fig. 2). Peak-to-MIC ratios greater than 1 are achievable in humans infected with low-level gentamicin-resistant enterococci and treated with currently recommended once- or twice-daily dosing regimens. For example, a single perfusion of 7 mg of gentamicin/kg was shown to provide peak levels in serum of
28.3 ± 6.5 µg/ml (McNamara et al., 39th ICAAC). However, these observations do not imply that gentamicin can be used in monotherapy in
the case of severe enterococcal infection, for two reasons. In clinical
practice, the peak-to-MIC ratio of gentamicin may not reliably exceed
1, depending on the dosage used and susceptibility of the strain. In
addition, gentamicin monotherapy selects gentamicin-resistant mutants
that may be responsible for therapeutic failure (14). However, our study indicates that the intrinsic activity of gentamicin should be taken into account in studying combinations of
aminoglycosides and agents active against the cell wall, such as
ampicillin or vancomycin. Besides increasing the activity of
aminoglycosides by facilitating their uptake into the cell
(16), combinations may also prevent the emergence of
gentamicin-resistant mutants (14). Conversely, the intrinsic
activity of gentamicin may be crucial for preventing the emergence of
resistance to cell wall-active agents, as shown for the emergence of
teicoplanin resistance in VanB-type glycopeptide-resistant enterococci
(2, 14). Finally, an evaluation of the synergistic effect of
combinations of cell wall-active agents and aminoglycosides against
enterococci should include a careful study of each drug separately.
| |
ACKNOWLEDGMENT |
A.L. was supported by the Fondation pour la Recherche
Médicale.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Service de
Médecine Interne, Hôpital Beaujon, 100, Boulevard du
Général Leclerc, 92118 Clichy Cedex, France. Phone: (33)
(1) 40 87 58 90. Fax: (33) (1) 40 87 54 95. E-mail:
bruno.fantin{at}bjn.ap-hop-paris.fr.
| |
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Antimicrobial Agents and Chemotherapy, August 2000, p. 2077-2080, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
