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Antimicrobial Agents and Chemotherapy, January 2001, p. 243-251, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.243-251.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
In Vitro Activity of Trovafloxacin against Bacteroides
fragilis in Mixed Culture with either Escherichia
coli or a Vancomycin- Resistant Strain of Enterococcus
faecium Determined by an Anaerobic Time-Kill
Technique
Lorna E. T.
Stearne,*
Clarissa
Kooi,
Wil H. F.
Goessens,
Irma A. J. M.
Bakker-Woudenberg, and
Inge C.
Gyssens
Department of Medical Microbiology and
Infectious Diseases, Erasmus University Medical Center, Rotterdam,
The Netherlands
Received 26 June 2000/Returned for modification 26 August
2000/Accepted 25 October 2000
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ABSTRACT |
To determine the efficacy of trovafloxacin as a possible treatment
for intra-abdominal abscesses, we have developed an anaerobic time-kill
technique using different inocula to study the in vitro killing of
Bacteroides fragilis in pure culture or in mixed culture with either Escherichia coli or a vancomycin-resistant
strain of Enterococcus faecium (VREF). With inocula of
5 × 105 CFU/ml and trovafloxacin concentrations of
2 µg/ml, a maximum observed effect (Emax)
of 
6.1 (log10 CFU/ml) was attained with all pure and
mixed cultures within 24 h. With inocula of 108
CFU/ml, a similar Emax and a similar
concentration to produce 50% of Emax
(EC50) for B. fragilis were found in both
pure cultures and mixed cultures with E. coli. However, to
produce a similar killing of B. fragilis in the mixed
cultures with VREF, a 14-fold increase in the concentration of
trovafloxacin was required. A vancomycin-susceptible strain of E. faecium and a trovafloxacin-resistant strain of E. coli were also found to confer a similar "protective" effect
on B. fragilis against the activity of trovafloxacin. Using inocula of 109 CFU/ml, the activity of trovafloxacin was
retained for E. coli and B. fragilis and was
negligible against VREF. We conclude that this is a useful technique to
study the anaerobic killing of mixed cultures in vitro and may be of
value in predicting the killing of mixed infections in vivo. The
importance of using mixed cultures and not pure cultures is clearly
shown by the difference in the killing of B. fragilis in
the mixed cultures tested. Trovafloxacin will probably be ineffective
in the treatment of infections involving large numbers of enterococci.
However, due to its ability to retain activity against large cultures
of B. fragilis and E. coli, trovafloxacin could
be beneficial in the treatment of intra-abdominal abscesses.
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INTRODUCTION |
Several factors must be taken into
consideration when selecting an antibiotic for the treatment of
intra-abdominal abscesses. Firstly, the presence of mixed infections of
anaerobic and aerobic bacteria with different susceptibilities requires
the use of either dual therapy, e.g., a penicillin plus an
aminoglycoside, or broad-spectrum antibiotics, such as carbapenems or
cephalosporins (29). Secondly, the large numbers of
bacteria in a stationary phase of growth, as well as the pH and
anaerobic environment of the abscesses (4), could all
affect the activity of an antibiotic and consequently influence the
choice of treatment.
Bacteroides fragilis and Escherichia coli are the
most frequently isolated organisms from intra-abdominal abscesses
although other anaerobes and facultative bacteria, including
enterococcal strains, have been isolated in about 10 to 20% of cases
(10, 29). Recently, we have developed a subcutaneous
abscess model in the mouse by using a mixed infection of B. fragilis, E. coli, and autoclaved cecal contents (ACC)
(I. C. Gyssens, L. E. T. Stearne, S. L. C. E. Buijk, J. W. Mouton, I. A. J. M. Bakker-Woudenberg, and H. A. Verbrugh, Abstr. 38th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. A-37, 1998). Treatment of these
abscesses with high doses of imipenem or ceftizoxime was disappointing
despite the fact that both strains were susceptible to both
antibiotics, and, from kinetic studies, the concentrations of the drugs
were shown to be above MIC levels in serum and abscesses for the entire dosing interval. The inoculum effect shown with both these antibiotics (26; Gyssens et al., 38th ICAAC) as well as the reduced
activity of 
-lactams against stationary cultures (30,
32) could be a possible explanation for the reduced efficacy in vivo.
The quinolones have had a limited use in anaerobic mixed infections due
to their marginal activity or inactivity against anaerobes (27) or gram-positive strains (8).
Trovafloxacin is a broad-spectrum fluoroquinolone that is effective not
only against gram-negative organisms but also has an increased activity
against gram-positive and anaerobic strains (12). In one
study, trovafloxacin has been shown to inhibit 99.3% of isolates from
intra-abdominal infections (6). In addition, the activity
of trovafloxacin is virtually unaffected by changes in pH, inoculum
size, or anaerobic conditions (1, 8, 24) and is also
active against some cultures of nondividing cells (21).
These properties indicate that trovafloxacin could be a possible choice
of treatment for intra-abdominal abscesses while the long half-life
(3) and postantibiotic effect (25) would
facilitate its possible use in once-daily dosing regimens.
To date, the in vitro activity of trovafloxacin has been reported
in MIC and minimum bactericidal concentration (MBC) (12, 13) or time-kill (28, 31) studies using single
cultures. However, the killing of bacteria in pure culture may not
necessarily be identical to the killing in mixed culture (15,
22). We have developed a mixed culture anaerobic time-kill
technique for a comparative study of the in vitro activity of
trovafloxacin against B. fragilis in pure or mixed culture
with either E. coli or a vancomycin-resistant strain of
Enterococcus faecium (VREF). Since abscesses contain large
numbers of bacteria in a stationary phase of growth, we have used, in
addition to standard inocula (5 × 105 CFU/ml), large
numbers of actively growing (108 CFU/ml) or static
(109 CFU/ml) cultures to simulate in vivo conditions. In
addition, due to the elevated (protein) binding capacity reported for
trovafloxacin (3), we have investigated the effect of
intestinal contents (ACC) on the in vitro activity of trovafloxacin.
(This study was presented in part at the 39th Interscience Conference
on Antimicrobial Agents and Chemotherapy, abstr. 2333, p. 289, 1999.)
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MATERIALS AND METHODS |
Materials.
Wilkens Chalgren (WC) broth, WC agar, Eosin
Methylene Blue (EM) agar, Diagnostic Sensitivity Test (DST) agar, and
AnaeroGen anaerobic sachets and jars were all supplied by Unipath Ltd.
(Haarlem, The Netherlands). Columbia blood agar plates came from Becton Dickinson B.V. (Woerden, The Netherlands). Gentamicin was obtained from Centrafarm Services B.V. (Etten-Leur, The Netherlands) and vancomycin was from Eli Lilly Nederland B.V. (Nieuwegein, The Netherlands). Trovafloxacin (CP-99, 219-27, lot 25381-086-02) was
supplied by Pfizer Inc. (Groton, Conn.). The supplier of 5-ml polypropylene tubes was Greiner B.V. (Alphen a/d Rijn, The Netherlands).
Bacterial strains.
E. coli ATCC 25922, B. fragilis ATCC 23745, and E. faecium BM4147, a
vancomycin-resistant clinical isolate, were used throughout the
experiments. For comparative studies, B. fragilis ATCC
25285, E. faecium SH17, a vancomycin-susceptible clinical
isolate, and E. coli 29020, a trovafloxacin-resistant
clinical isolate, were also employed. Overnight cultures were obtained
by inoculating 30-ml volumes of WC broth with 0.1 ml of standardized
frozen bacterial suspensions and incubating aerobically (E. coli, VREF, and E. faecium) or anaerobically (B. fragilis) at 37°C for 18 h.
ACC.
ACC were obtained as previously described
(19). Briefly, the cecal contents were removed from
approximately 100 Swiss mice (Broekman Instituut B.V., Someren,
The Netherlands), diluted 1:4 with WC broth, homogenized, and filtered
twice through surgical gauze. The suspension was autoclaved in 5-ml
volumes at 121°C for 2 h and stored at 
80°C. Batches were
standardized by measuring the dry weight (Edwards Super Modulyo freeze
dryer) of a 1-ml sample of the autoclaved contents.
MIC and MBC determinations.
MICs were determined by the
standard broth microdilution method using inocula of 105 or
108 CFU/ml (23). All tests were performed in
duplicate and repeated on a separate day using freshly prepared
trovafloxacin mixtures and inocula. Microtiter plates were incubated at
37°C aerobically for 20 h (E. coli, VREF, and
E. faecium) or anaerobically for 48 h (B. fragilis). In experiments with the higher inocula (which were
visually turbid), the MIC was defined as the lowest concentration of
trovafloxacin that prevented a 0.1 increase in optical density at 600 nm (Biokinetics reader EL 340; Bio-tek Instruments) compared with that
of controls incubated at 4°C. MBCs were determined by plating
100-µl samples from all wells showing no bacterial growth after
incubation onto blood agar and then incubating at 37°C for 24 h
(E. coli, VREF, and E. faecium) or 48 h
(B. fragilis), and the MBC was defined as the lowest
concentration producing 99.9% killing of the initial inoculum.
Suspensions with the higher inocula (108 CFU/ml) were first
diluted 1:100 in phosphate-buffered saline (PBS) before determining the
MBCs. To determine the effect of ACC on the MBCs, tests were repeated
using inocula containing 4 mg (dry weight) of ACC/ml (final concentration).
Mixed-culture anaerobic time-kill technique.
Twofold-increasing trovafloxacin concentrations (0.015 to 64 µg/ml)
were used with final inocula of 5 × 105, 1 × 108, or 1 × 109 CFU/ml for each test
strain. Concentrated solutions of trovafloxacin were diluted in
prewarmed WC broth to yield two times the final required
antibiotic concentrations, from which four 2-ml volumes were placed in
polypropylene tubes with loose caps. Control samples contained 2 ml of
WC broth. Inocula were prepared by diluting 18-h cultures of B. fragilis ATCC 23745 and either E. coli ATCC 25922 or
VREF in prewarmed WC broth. Tubes containing broth with or without
antibiotic were carefully inoculated with 1 ml of E. coli
(or VREF) culture followed by 1 ml of B. fragilis culture. Cultures were placed in four separate anaerobic jars and incubated at
37°C on a shaker for 2, 4, 6, or 24 h. To prevent carryover of
any antibiotic, 1 ml of each test suspension was washed by centrifuging
at 13,000 × g for 2 min, removing 0.9 ml of the
supernatant and resuspending the pellet in 0.9 ml of PBS. The entire
procedure was repeated, and bacterial counts were performed on the
resulting suspensions by making duplicate serial 10-fold dilutions of
cultures in PBS and plating 20 µl of appropriate dilutions onto EM
agar (E. coli), blood agar (VREF), or WC agar containing 100 mg of gentamicin/liter (B. fragilis). Plates were incubated
at 37°C aerobically for 24 h (EM or blood agar) or anaerobically
for 48 h (WC agar). Experiments were also performed using pure
cultures of B. fragilis ATCC 23745. Counts were expressed as
log10 CFU/milliliter, and the lower threshold limit was 2.5 log10 CFU/ml. Experiments with the different inocula were
performed on different days. To determine daily variation, individual
experiments were repeated using a more limited number of
trovafloxacin concentrations.
For comparison, the technique was repeated with pure and mixed cultures
(108 CFU/ml) of other bacterial strains, namely B. fragilis ATCC 25285, E. faecium SH17, and E. coli 29020, using 2 µg of trovafloxacin/ml, and after 24 h
of incubation, bacterial counts were determined (when in mixed culture
with E. faecium, B. fragilis counts were determined on WC agar containing 6.25 mg of vancomycin/liter). The
experiment was performed three times on all pure and mixed cultures on
separate days by using freshly prepared trovafloxacin and inocula.
Control cultures without trovafloxacin were included with each
experiment, from which the reduction in bacterial counts of each
culture could be determined. In addition, the concentration of residual
trovafloxacin present in the supernatants of the enterococcal mixed
cultures after incubation was also measured using an agar diffusion
bioassay on DST agar with a Bacillus subtilis isolate as the
test organism. Standards were prepared in WC broth.
To determine the effect of intestinal contents on the activity of
trovafloxacin, the time-kill technique was performed using
concentrations of 0.25, 1, or 4 µg/ml and inocula of
E. coli ATCC
25922-
B. fragilis ATCC 23745 mixed cultures
(10
8 CFU/ml) with or without 4 mg (dry weight) of ACC/ml
(final concentration).
To exclude the possibility that the ACC could
affect the removal
of trovafloxacin during the washing procedure
outlined above by
"trapping" the antibiotic in the pellet, a
suspension containing
4 µg of trovafloxacin/ml and 4 mg of ACC/ml was
incubated at 37°C
for 24 h and centrifuged, and the pellet was
washed twice in PBS.
The trovafloxacin concentration in the pellet was
measured in
the bioassay previously described. Approximately 0.35 µg
of trovafloxacin/ml
was recovered from the pellet. However, in the
time-kill study,
this concentration would be further diluted 1:50 by
plating out
onto agar plates and would therefore have little influence
on
the outcome of the bacterial
counts.
The degree of ACC binding to trovafloxacin was assessed by incubating
0.5 to 16 µg of trovafloxacin/ml in WC broth with or
without 4 mg
(dry weight) of ACC/ml for 24 h at 37°C. After incubation,
the
trovafloxacin concentrations in the suspensions were determined
in the
bioassay. The binding capacity of ACC was determined by
measuring the
percent loss of activity of trovafloxacin at the
different
concentrations.
Statistical analysis.
The effect of trovafloxacin on the
bacterial counts of the different pure and mixed cultures
(E) was expressed as the difference, after 6 h of
incubation, between the log10 CFU/milliliter in the absence
and in the presence of trovafloxacin as described by the following
equation:
E = (Emax × Cs)/(EC50s + Cs),
where Emax is the maximum observed effect,
C is the concentration, EC50 is the
concentration at which 50% of the maximum effect is reached, and
s is a parameter for the steepness of the
concentration-effect relationship. EC50 and s
were calculated for each pure and mixed culture by using nonlinear
least-squares regression techniques (GraphPad Prism version 3.0 for
Windows; GraphPad Software, San Diego, Calif.). The 95% confidence
intervals were used to determine significant differences between cultures.
Comparison of the mean reduction in bacterial counts of the pure
cultures of
B. fragilis, VREF,
E. faecium, or
E. coli 29020
with the corresponding mixed cultures
was analyzed using the Tukey-Kramer
multiple comparison test. A
P value of <0.05 was considered
significant.
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RESULTS |
MIC and MBC determinations.
The MICs and MBCs of trovafloxacin
against B. fragilis ATCC 23745, B. fragilis ATCC
25285, E. coli ATCC 25922, VREF, and E. faecium
using different inocula are shown in Table
1. MICs and MBCs of trovafloxacin
increased for all strains when the inoculum was increased from
105 to 108 CFU/ml. However, the >64-fold
increase against VREF and E. faecium was much greater
than the 4- to 8-fold increase for E. coli and the two
B. fragilis strains. Trovafloxacin had MICs of
16 and >64 µg/ml for trovafloxacin-resistant E. coli 29020 inocula of 105 and 108 CFU/ml,
respectively. The bactericidal activity of trovafloxacin was decreased
in the presence of ACC as indicated by the 2- to 16-fold increase in
the MBCs of all strains.
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TABLE 1.
In vitro activity of trovafloxacin against B. fragilis, E. coli, VREF, and E. faecium using different
inocula with and without ACC
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Time-kill studies.
Fig. 1,
2, and 3
show the in vitro activity of trovafloxacin against B. fragilis ATCC 23745 in pure culture (Fig. 1) or in mixed culture
with either E. coli ATCC 25922 (Fig. 2) or VREF (Fig. 3)
using different inocula in the time-kill technique. Trovafloxacin produced a biphasic dose response whereby the bacterial killing was
concentration dependent up to an optimum bactericidal concentration that resulted in a maximum log10 CFU/milliliter reduction
in bacterial counts compared to counts of controls
(Emax). The technique was reproducible with the
mean difference in bacterial counts (log10 CFU/milliliter)
between duplicate experiments having a standard deviation (SD) of
<0.6. Trovafloxacin was effective in killing all standard inocula
(5 × 105 CFU/ml) within 24 h at concentrations
of 0.5 µg/ml (B. fragilis pure culture), 1 µg/ml
(B. fragilis-E. coli mixed culture), and 2 µg/ml (B. fragilis-VREF mixed culture), resulting in an
Emax of 6.1 for all strains. Similar
Emaxs were attained with B. fragilis pure cultures and B. fragilis-E. coli mixed cultures when
the inoculum was increased to 108 CFU/ml although a 2- to
4-fold increase in the trovafloxacin concentration (2 µg/ml) was
required compared to the concentration required for the standard
inocula. When the inoculum was further increased to 109
CFU/ml, trovafloxacin activity was retained against B. fragilis in pure culture. However, when in mixed culture with
E. coli, the killing of B. fragilis was reduced.
Trovafloxacin activity was retained against E. coli in the
same mixed culture.
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FIG. 1.
Time-kill studies of trovafloxacin against pure cultures
of B. fragilis ATCC 23745 using inocula of 5 × 105, 1 × 108, or 1 × 109 CFU/ml. All stated trovafloxacin
concentrations are in micrograms/milliliter.
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FIG. 2.
Time-kill studies of trovafloxacin against mixed
cultures of B. fragilis ATCC 23745 and E. coli
ATCC 25922 using inocula of 5 × 105, 1 × 108, or 1 × 109 CFU/ml. All stated
trovafloxacin concentrations are in micrograms/milliliter.
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FIG. 3.
Time-kill studies of trovafloxacin against mixed
cultures of B. fragilis ATCC 23745 and VREF BM4147 using
inocula of 5 × 105, 1 × 108, or
1 × 109 CFU/ml. All stated trovafloxacin
concentrations are in micrograms/milliliter.
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The presence of large and static inocula had a more pronounced effect
on the activity of trovafloxacin against mixed cultures
of
B. fragilis-VREF. To investigate these discrepancies further,
the
Emax and the EC
50 were calculated
from the bacterial counts
measured after 6 h of incubation with
trovafloxacin. Table
2 presents the
Emax and the EC
50 for
B. fragilis ATCC 23745 in pure
culture or in mixed culture with
E. coli ATCC 25922 or VREF. There
was little difference in
the
Emax for
B. fragilis between the
pure culture and the two mixed cultures with inocula of 5 × 10
5 and 1 × 10
8 CFU/ml. In addition,
using the same inocula, a similar EC
50 was
found for
B. fragilis in the pure cultures and the mixed cultures
with
E. coli. However, when in mixed culture with VREF,
there
was a 2-fold (inoculum, 5 × 10
5 CFU/ml) and a
14-fold (inoculum, 10
8 CFU/ml;
P < 0.05)
increase in the EC
50 for
B. fragilis compared
to
those of the pure cultures. With the higher inoculum, this
difference
in EC
50 was significantly different (
P < 0.05) from
those of both the pure culture and the mixed culture
with
E. coli.
Increasing the inoculum from 5 × 10
5 to 1 × 10
8 CFU/ml resulted in a
significant 12-fold increase in EC
50 for
B. fragilis in mixed culture with VREF. With inocula of
10
9 CFU/ml, the EC
50 for the mixed cultures
could not be accurately
determined due to inadequate killing of
B. fragilis. However,
compared to the other pure cultures, a
significant increase in
the EC
50 of
B. fragilis
in pure culture was detected.
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TABLE 2.
Activity of trovafloxacin against B. fragilis
ATCC 23745 in pure culture or in mixed culture with either E. coli ATCC 25922 or VREF BM4147 determined by using the time-kill
techniquea
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The size of the inoculum also had a significant effect on the killing
of VREF in the mixed culture with
B. fragilis. Increasing
the inoculum from 5 × 10
5 to 1 × 10
8 CFU/ml reduced the
Emax from
5.8 ± 1.1 (best-fit value ± standard
error) to 2.1 ± 0.24 log
10 CFU/ml and increased the EC
50 from
0.86 ± 1.03 to 4.63 ± 1.36 µg/ml, respectively (results
not shown).
In contrast, an
Emax of >5
log
10 CFU/ml for
E. coli was found
in all of the
mixed cultures with
B. fragilis (results not shown).
The
EC
50 for
E. coli in the 10
9-CFU/ml
mixed culture was 2.2 ± 2.2 µg/ml. (The EC
50 for
E. coli in the 5 × 10
5 and 1 × 10
8 CFU/ml cultures could not be accurately determined.)
These results suggested that VREF conferred a "protective" effect
on
B. fragilis against the activity of trovafloxacin. To
determine whether this phenomenon was also found with other strains,
experiments were repeated using inocula of 10
8 CFU/ml
of pure and mixed cultures of
B. fragilis ATCC 23745,
B. fragilis ATCC 25285, VREF,
E. faecium,
and trovafloxacin-resistant
strain
E. coli 29020. A
trovafloxacin concentration of 2 µg/ml
was used, and the bacterial
counts were determined after 24 h
(Table
3). (This concentration was chosen
because it was effective
in the killing of
B. fragilis in
both pure cultures and mixed
cultures with
E. coli.) The
reduction in bacterial counts of both
B. fragilis strains by
trovafloxacin was approximately 3 log
10 CFU/ml lower when
these strains were grown in mixed culture with
either VREF,
E. faecium, or
E. coli 29020 compared to that found
with
the pure cultures. This difference between the pure and mixed
cultures
was significant (
P < 0.01). There was no significant
difference in the reduction of
B. fragilis bacterial
counts between
the different mixed cultures. The killing of VREF,
E. faecium,
and
E. coli 29020 in all pure and
mixed cultures was negligible.
The residual concentrations of
trovafloxacin in the supernatants
of the enterococcal mixed cultures
after 24 h incubation at 37°C
were also measured in an agar diffusion
bioassay. A bacteria-free
control had a trovafloxacin concentration of
1.92 µg/ml compared
to an initial measured concentration of 2.2 µg/ml. The trovafloxacin
concentrations measured in all culture
supernatants ranged from
1.4 to 2.16 µg/ml (73 to 113% [median,
94%] of the concentration
found in the control).
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TABLE 3.
Reduction in bacterial counts of pure or mixed cultures
of B. fragilis, VREF, E. faecium, or E. coli strainsa
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The effect of ACC on the activity of trovafloxacin was also
investigated by using the time-kill technique. Figure
4 shows
the killing of
B. fragilis ATCC 23745 and
E. coli ATCC 25922 mixed
cultures by trovafloxacin with or without 4 mg of ACC/ml. The
intestinal contents had a detrimental influence on the killing
of both
strains by trovafloxacin at concentrations of 0.25 and
1 µg/ml.
However, this negative effect was minimal (
B. fragilis)
or
not found (
E. coli) with a trovafloxacin concentration of 4
µg/ml. This concentration dependent observation can be explained
by
the binding of ACC to trovafloxacin. At a concentration of
0.5 µg/ml,
there was a >90% loss of antibacterial activity of
trovafloxacin in
the presence of ACC. However, at 2 and 16 µg/ml,
the loss of activity
was reduced to 62 and 46%, respectively.
These results suggest that
there is a saturation point in the
binding of ACC to trovafloxacin.
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FIG. 4.
Effect of ACC on the in vitro activity of trovafloxacin
against mixed cultures of B. fragilis ATCC 23745 and
E. coli ATCC 25922 in the time-kill study. Trovafloxacin
concentrations (micrograms/milliliter) were inoculated with
108 CFU of either strain/ml with or without 4 mg (dry
weight) of ACC/ml.
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DISCUSSION |
The aim of the present study was to investigate the in vitro
activity of trovafloxacin, under anaerobic conditions, against mixed
cultures of B. fragilis-E. coli or B. fragilis-VREF to gain insight into the factors that could
influence its efficacy in the treatment of intra-abdominal abscesses.
Since abscesses contain large numbers of bacteria in a stationary phase
(Gyssens et al., 38th ICAAC), we studied the killing of large numbers
of actively growing (108 CFU/ml) and static
(109 CFU/ml) cultures to simulate conditions found in vivo.
The time-kill technique is a standard method used to follow the
kinetics of bacterial killing in vitro (9). Time-kill
studies under anaerobic conditions have been described by Spangler et al. (28); however, in contrast to their procedure, we have
developed an anaerobic time-kill technique that does not require an
anaerobic chamber. Although cultures were prepared under normal
laboratory conditions, an anaerobic environment was obtained within 30 min of incubation by using Anaerogen sachets (17) and, as
shown by control cultures in Fig. 1, 2, and 3, standard bacterial
growth curves were obtained throughout the 24-h incubation period. The rate and extent of bacterial killing by trovafloxacin was concentration dependent up to an optimum bactericidal concentration, with further concentration increases producing similar bactericidal activity. This
finding was reported also by Morrissey (21).
The MICs of trovafloxacin have been reported as being marginally
affected by an increase in inoculum size (1, 8, 24). We
have found a moderate effect of the inoculum on the killing of both
B. fragilis strains and E. coli as indicated by
the increase in MICs and MBCs when the inoculum was increased from
105 to 108 CFU/ml. However, these strains
remained below the susceptible breakpoint for trovafloxacin of 2
µg/ml (12). In contrast, a large inoculum effect was
found with both VREF and E. faecium in the present study and
is similar to that reported by Hayden et al. (14) on the
activity of levofloxacin against other clinically isolated enterococci.
In the present study, the EC50 was superior to the
Emax in differentiating between the activities
of trovafloxacin in the pure and mixed cultures. Previous studies also
found the EC50 to be a valuable parameter in the comparison
of the in vitro activities of different antibiotics against
Staphylococcus aureus strains (16). The killing
of B. fragilis ATCC 23745 in mixed culture with E. coli was similar to the killing of the anaerobe in pure culture by
the time-kill technique with inocula of 5 × 105 and
1 × 108 CFU/ml. However, when in mixed culture with
VREF, there was a 2-fold (inoculum, 5 × 105 CFU/ml)
and a 14-fold (inoculum, 108 CFU/ml) increase in the
trovafloxacin concentration required to obtain the same killing of
B. fragilis as in the pure cultures. The effect of the
inoculum on the killing of B. fragilis was also dependent on
the other bacterial strain present in the mixed culture. Increasing the
inoculum from 5 × 105 to 1 × 108
CFU/ml resulted in only a 2-fold increase in EC50 when the
anaerobe was in mixed culture with E. coli but a 12-fold
increase in the VREF mixed culture. This negative influence of VREF on
the activity of trovafloxacin was also found with a
vancomycin-susceptible strain of E. faecium in mixed culture
with different B. fragilis strains. Nagy and Földes
(22) have reported a similar protection of B. fragilis against the activity of metronidazole by
Enterococcus faecalis. The authors observed that the
antibiotic was inactivated by a cell extract of the enterococcus strain
but not by culture supernatants. However, the inactivation of
trovafloxacin by VREF or E. faecium was not found in the
present study since approximately 94% of trovafloxacin activity was
recovered from culture supernatants after a 24-h incubation compared to
the activity of a bacteria-free control. Another possible explanation
could be the production of an antagonistic substance by the
enterococcal strains (5, 15) that could interfere with the
activity of trovafloxacin. We have found, however, that culture
supernatants from VREF and E. faecium failed to confer
protection on B. fragilis ATCC 23745 (results not shown).
The emergence and selection of trovafloxacin-resistant mutants
(2, 5) with possible transfer of this resistance to
B. fragilis in the mixed cultures (7, 11, 18,
20) could also account for this protection although further
investigations would be required to confirm this hypothesis. It was
interesting to observe that this phenomenon was not restricted to
enterococcal mixed cultures but could be detected also in mixed
cultures with a trovafloxacin-resistant E. coli strain.
These results suggest that, since the killing of VREF, E. faecium, or E. coli 29020 in the mixed cultures was
minimal, the increased number of viable organisms was an important
factor in the reduced activity of trovafloxacin against B. fragilis. However, in mixed cultures with B. fragilis
and E. coli ATCC 25922 (inoculum, 109 CFU/ml),
the activity of trovafloxacin against the anaerobe was also reduced
despite the fact that there was an effective killing of the E. coli strain. However, the approximately 2 log10 CFU/ml larger number of viable E. coli cells remaining in these
mixed cultures compared to the 108 CFU/ml cultures may also
be a significant factor. The findings of the time-kill studies show,
therefore, that the activity of trovafloxacin against large numbers of
B. fragilis in mixed culture with different bacterial
strains could be compromised when the bacterial numbers of the
coexisting strain persist. Furthermore, the enterococcal strains, which
under standard laboratory tests would be regarded as trovafloxacin
susceptible, become totally resistant (inoculum, 109
CFU/ml) or require a significant increase in trovafloxacin
concentrations to produce an effective killing of these strains
(inoculum, 108 CFU/ml). For these reasons, trovafloxacin
would appear to have little use in the treatment of infections
involving large numbers of enterococci.
Previous studies have described different bactericidal mechanisms of
action of trovafloxacin (21). Mechanism A is the basic quinolone mechanism and requires bacteria to be undergoing
multiplication as well as protein or RNA synthesis while mechanism B
does not require these processes to be present. In the same study,
trovafloxacin was found to possess bactericidal mechanism B against
E. coli KL16 and mechanism A against E. faecalis
ATCC 19433. Similarly, by employing inocula that remained stable
throughout the 24-h incubation period, we have detected differences in
the killing by trovafloxacin against the different bacterial strains.
Bactericidal activity was retained against E. coli ATCC
25922 and B. fragilis (in pure culture), indicating that
active bacterial cell division was not a requirement for trovafloxacin
with these strains. On the other hand, active bacterial multiplication
appears to be of more importance for the bactericidal activity of
trovafloxacin against VREF, as demonstrated by the negligible killing
of the strain in the 109-CFU/ml cultures.
The protein binding capacity for trovafloxacin has been reported to be
70% in humans (3). ACC is composed of various
constituents, including proteins, fibrous material, and bacteria, and
since intra-abdominal abscesses comprise a certain amount of fecal
material, we wished to determine the binding capacity of this complex
mixture and investigate its effect on the activity of trovafloxacin. We have found that there is a saturation point in the binding of ACC to
trovafloxacin, which was confirmed by the in vitro activity studies. In
the MIC and MBC tests, a concentration of 1 µg/ml was required to
kill 99.9% of all strains, while in the time-kill studies, ACC had a
detrimental effect on the activity of trovafloxacin only when the
antibiotic concentrations were <4 µg/ml. These results suggest that,
providing that sufficiently high doses are employed, the presence of
fecal materials in intra-abdominal abscesses should have little effect
on the in vivo activity of trovafloxacin.
B. fragilis and E. coli are the most frequently
isolated organisms from intra-abdominal abscesses (4, 29)
and the effective in vitro killing of mixed cultures of these strains
outlined in the present experiments have been confirmed by us in vivo
using a mixed-infection subcutaneous abscess mouse model (I. C. Gyssens, L. E. T. Stearne, J. W. Mouton, W. H. Goessens, and H. A. Verbrugh, 39th Intersci. Conf. Antimicrob.
Agents Chemother., abstr. 800, 1999). Daily dosing of high
concentrations of trovafloxacin resulted in not only a significant
reduction in B. fragilis ATCC 23745 and E. coli
ATCC 25922 bacterial counts but also a significant reduction in abscess
weight and inflammation. In a similar mixed-infection mouse model with
B. fragilis ATCC 23745 and VREF BM4147, the killing of VREF
was ineffective while the killing of B. fragilis was
significantly lower than killing in the mouse model with E. coli. These results corroborate the in vitro results described here.
In conclusion, the time-kill technique is a useful procedure to study
the anaerobic killing of mixed cultures in vitro and may be of value in
predicting the killing of mixed bacteria in vivo. It is important that
the bacterial numbers and the mixed cultures studied in vitro reflect
those found in vivo when determining the efficacy of an antibiotic as a
treatment for mixed infections, as shown by the difference in the
killing of B. fragilis in the mixed cultures tested. Due to
its ability to retain activity against large cultures of B. fragilis and E. coli, trovafloxacin could be beneficial
in the treatment of intra-abdominal abscesses.
| |
ACKNOWLEDGMENTS |
This study was financially supported by Pfizer B. V.,
Capelle aan de IJssel, The Netherlands.
H. Mattie is gratefully acknowledged for his valuable comments on the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology and Infectious Diseases, Erasmus University
Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The
Netherlands. Phone: 31-01-4087665. Fax: 31-10-4089454. E-mail:
stearne{at}kmic.fgg.eur.nl.
| |
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Antimicrobial Agents and Chemotherapy, January 2001, p. 243-251, Vol. 45, No. 1
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.1.243-251.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
