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Antimicrobial Agents and Chemotherapy, July 1999, p. 1747-1753, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Combinations of Vancomycin and 
-Lactams Are
Synergistic against Staphylococci with Reduced Susceptibilities
to Vancomycin
Michael W.
Climo,1,2,*
Roberto L.
Patron,1 and
Gordon L.
Archer1,3
Departments of
Medicine1 and
Microbiology/Immunology,3 Medical
College of Virginia Campus of Virginia Commonwealth University, and the
Hunter Holmes McGuire Veterans Affairs Medical
Center,2 Richmond, Virginia
Received 7 December 1998/Returned for modification 26 February
1999/Accepted 6 April 1999
 |
ABSTRACT |
Evidence of synergism between combinations of vancomycin and
-lactam antibiotics against 59 isolates of methicillin-resistant staphylococci (Staphylococcus aureus, Staphylococcus
epidermidis, and Staphylococcus haemolyticus) for
which vancomycin MICs ranged from 1 to 16 µg/ml were tested by broth
microdilution checkerboard, disk diffusion, agar dilution, and
time-kill antimicrobial susceptibility tests. The combination of
vancomycin and oxacillin demonstrated synergy by all test methods
against 30 of 59 isolates; no antagonism was seen. Synergy with
vancomycin was also found by modified disk diffusion testing for
ceftriaxone, ceftazidime, cefpodoxime, and amoxicillin-clavulanate but
not for aztreonam. Evidence of synergy correlated directly with
vancomycin MICs. The efficacy of vancomycin given alone and in
combination with nafcillin was tested in the rabbit model of
experimental endocarditis caused by three clinical isolates of
glycopeptide-intermediate-susceptible S. aureus (GISA) (isolates HIP5827, HIP5836, and MU50). Two of the GISA isolates (isolates MU50 and HIP5836) were extremely virulent in this model, with
27 of 42 (64%) animals dying during the 3-day trial. Therapy with
either vancomycin or nafcillin given as a single agent was ineffective
for animals infected with HIP5827 or MU50. However, the combination of
vancomycin and nafcillin resulted in a mean reduction of 4.52 log10 CFU/g of aortic valvular vegetations per g compared
to the reduction for controls for animals infected with HIP5827 and a
reduction of 4.15 log10 CFU/g for animals infected with
MU50. Renal abscesses caused by HIP5827 were sterilized significantly better with the combination of vancomycin and nafcillin than by either
treatment alone. We conclude that the combination of vancomycin and
-lactams with antistaphylococcal activity is an effective regimen
for the treatment of infections with clinical strains of staphylococci
which demonstrate reduced susceptibility to glycopeptides.
| |
INTRODUCTION |
Glycopeptides have been used
successfully for the treatment of serious methicillin-resistant
Staphylococcus aureus (MRSA) infections for the past 30 years. Clinical strains of S. aureus that demonstrate
reduced susceptibility to glycopeptides have recently been described
from geographically diverse sources (4, 5, 14, 22, 24, 33,
34). For these strains, known collectively as
glycopeptide-intermediate-susceptible S. aureus (GISA),
vancomycin MICs range from 8 to 16 µg/ml. Although the vancomycin
MICs for these isolates remain below achievable levels in serum,
clinical infections have responded poorly to the glycopeptides vancomycin and teicoplanin (32). Most patients have required a combination of therapeutic approaches for successful treatment, including surgery, prolonged vancomycin therapy, or the use of additional antistaphylococcal agents.
Although the first clinical isolate of GISA was described in 1997 (14), laboratory-derived strains of S. aureus
which demonstrate reduced susceptibility to glycopeptides have been
under investigation since the early 1990s (9, 17, 18, 26, 28,
29). These laboratory-derived strains are easily produced by step
passage in the presence of increasing concentrations of glycopeptides. In one report of a laboratory-derived vancomycin-resistant isolate of
S. aureus, Sieradzki and Tomasz (28) noted that
this highly vancomycin resistant isolate became extremely sensitive to
-lactam antibiotics in comparison to its vancomycin-sensitive,
methicillin-resistant parent. He termed this phenomenon "the seesaw
effect" and attributed it to the alterations in cell wall composition
required for vancomycin resistance. The previous report suggested that
the alterations required for the optimal expression of vancomycin
resistance affected the expression of methicillin resistance as well.
Investigators have also noted that combinations of glycopeptides and
-lactams can demonstrate additive or synergistic activity against
MRSA isolates in in vitro tests, since they act as inhibitors at
different stages of cell wall synthesis (2, 23, 30). These
data, in combination with the observations of Sieradzki and Tomasz
(28), raised the possibility that GISA strains may also
respond synergistically to combinations of vancomycin and -lactams.
In this report, we characterize the in vitro susceptibilities of
staphylococcal isolates with a broad range of vancomycin MICs to
combinations of vancomycin and -lactams by the use of broth
microdilution checkerboard testing and the disk diffusion, agar
dilution, and time-kill methods. We then evaluated the effectiveness of
combination vancomycin-nafcillin treatment of experimental aortic valve
endocarditis caused by clinical GISA isolates in rabbits.
| |
MATERIALS AND METHODS |
Bacterial strains.
Fifty-nine methicillin-resistant
staphylococcal isolates were examined. Forty-one methicillin-resistant
staphylococcal strains (21 MRSA, 10 methicillin-resistant
Staphylococcus epidermidis [MRSE], and 10 methicillin-resistant Staphylococcus haemolyticus [MRSH]
strains) were taken from a collection of geographically diverse
clinical strains maintained at the Medical College of Virginia Campus
of Virginia Commonwealth University as described previously
(7). Ten MRSE isolates collected between August 1998 and
October 1998 were recovered from patients with infections of central
venous catheters. Three MRSA isolates with reduced susceptibility to
vancomycin (vancomycin MIC, 8 µg/ml; isolates HIP5827 [Michigan],
HIP5836 [New Jersey] and MU50 [Japan]) and vancomycin-susceptible
isolate MU3 (vancomycin MIC, 2 µg/ml) were kindly provided by Fred
Tenover of the Centers for Disease Control and Prevention
(34). Four staphylococcal strains with higher levels of
vancomycin resistance were produced by step passage as described
previously (8, 18). MRSA 27619VR (vancomycin MIC, 8 µg/ml)
is an isogenic derivative of the vancomycin-susceptible parent S. aureus 27619 (7, 8). MRSA 5827HR (vancomycin MIC, 16 µg/ml) is an isogenic derivative of HIP5827. Finally, two MRSH isolates with higher levels of vancomycin resistance were isolated following successive overnight passages of S. haemolyticus
27280 in increasing concentrations of vancomycin. They were designated 27280-2 (vancomycin MIC, 8 µg/ml) and 27280-3 (vancomycin MIC, 16 µg/ml), respectively.
Antimicrobial susceptibility testing.
MICs were determined
by the broth microdilution method in cation-adjusted Mueller-Hinton
broth (Becton Dickinson, Cockeysville, Md.) according to the standards
of the National Committee for Clinical Laboratory Standards
(19). The MIC was the lowest concentration of antibiotic
that yielded no visible growth after incubation at 37°C for 24 h.
Checkerboard synergy testing was performed by the microdilution method
in microtiter trays with cation-adjusted Mueller-Hinton broth.
Combinations of vancomycin and oxacillin were tested at concentrations
of 0.25 to 16 and 0.125 to 128 µg/ml, respectively. Microtiter plates
were incubated at 37°C and were read at 24 and 48 h. The
fractional inhibitory concentration (FIC) index was calculated by
adding the FICs (MIC of drug A in combination with drug B/MIC of drug A
alone) of vancomycin and oxacillin. An FIC index of 
0.5 was defined
as synergy, an FIC index of >0.5 to 4.0 was defined as additive or
indifferent, and an FIC index of >4.0 was defined as antagonistic.
Checkerboard test results represented the average of duplicate tests.
Time-kill assays were performed in 20 ml of cation-adjusted
Mueller-Hinton broth inoculated with the test organisms to a final concentration of from 5 × 106 to 5 × 107 CFU/ml. Vancomycin was tested at the MIC, 2× the MIC,
and 4× the MIC. Oxacillin was used at a concentration of 6 µg/ml.
Bacterial counts were taken at 0, 1, 4, and 24 h by plating 0.1-ml
aliquots and serially diluting each onto Mueller-Hinton agar. Synergy
between vancomycin and oxacillin was defined as a reduction in the
initial inoculum of >2 log10 CFU/ml (99.9%) at 24 h.
Synergy between vancomycin and oxacillin was also determined by a
modified disk diffusion method. Clinical isolates of GISA
(HP5827,
HP5836, and MU50) underwent Kirby-Bauer disk diffusion
testing with
commercially prepared brain heart infusion agar (BHIA)
containing 6 µg of vancomycin (Remel, Lenexa, Kans.) per ml, and
antimicrobial
disks (BBL Sensi-Disc; Becton Dickinson) containing
oxacillin (1 µg),
amoxicillin-clavulanate (30 µg), ceftriaxone
(30 µg), aztreonam (30 µg), and cefpodoxime (10 µg) according
to the guidelines of the
National Committee for Clinical Laboratory
Standards (
20).
Synergy was defined as an enhancement in the
zone of inhibition
surrounding the antibiotic disk on vancomycin
agar compared to the zone
size around disks placed on BHIA containing
no
antibiotic.
Population analysis profiles were generated from overnight cultures of
staphylococcal strains grown in Mueller-Hinton broth.
Overnight
cultures were diluted until the turbidity matched that
of a 0.5 McFarland standard and serial dilutions were then plated
on
Mueller-Hinton agar plates containing vancomycin ranging in
concentrations from 1 to 16 µg/ml. In tests for synergy, oxacillin
at
a fixed concentration of 6 µg/ml was also added to
vancomycin-containing
agar. The plates were incubated for 48 h,
and the numbers of colonies
were counted and plotted
graphically.
Experimental infection.
The rabbit model of aortic valve
endocarditis, which has been described previously (7, 8,
21), was used to evaluate the antibiotic treatment regimens.
Seventy-two hours after transcarotid placement of a polyethylene
catheter across the aortic valve, rabbits were injected intravenously
through the marginal ear vein with 1 ml of an overnight culture
containing 107 CFU of the test organism per ml. The test
organisms included strains HIP5827, HIP5836, and MU50. Blood samples
for culture were obtained 24 h later, and the rabbits were
randomly assigned to one of the following treatment groups: vancomycin
(Abbott Laboratories, Chicago, Ill.) given at 30 mg/kg of body weight
intravenously every 12 h, nafcillin (Bristol-Meyer Squibb,
Princeton, N.J.) given at 200 mg/kg intramuscularly every 8 h, and
vancomycin given at 30 mg/kg intravenously every 12 h plus
nafcillin given at 200 mg/kg intramuscularly every 8 h, or no
treatment (control group). The surviving animals were killed with
intravenous pentobarbital after a total of 3 days of antibiotic
treatment. Rabbits with negative blood cultures at 24 h were
excluded from subsequent analysis. To reduce the possibility of
antibiotic carryover, rabbits were not killed until at least 18 h
after administration of the last dose. The heart and kidneys were
removed aseptically from each rabbit. Aortic valve vegetations were
removed from each rabbit's heart and weighed, and serial dilutions of
vegetation homogenates were made. The kidneys were examined, and areas
of abscess or infarct were removed, weighed, homogenized in saline, and
serially diluted. Tissue homogenates were also plated onto
Mueller-Hinton agar containing increasing concentrations of vancomycin
in order to generate population analysis profiles. Cultures were read
after 48 h. Titers of bacteria were expressed as log10
CFU per gram of vegetation or kidney tissue. Sterile vegetation and
kidney cultures contained 2 and 1 log10 CFU/g,
respectively (the limit of detection).
Inclusion criteria.
For the final analysis, data for animals
that fulfilled the following criteria were included: (i) positive blood
culture at 24 h, (ii) survival for at least 24 h of
antibiotic treatment, (iii) proper placement of the catheter across the
aortic valve at necropsy with macroscopic evidence of aortic valve
endocarditis (visible vegetations), and (iv) aortic valve vegetation
and kidney tissue yielding pure cultures of the test organism.
Statistical analysis.
The mean numbers of bacteria per gram
of vegetation and kidney tissue in all treatment groups were compared
by analysis of variance. Sterile aortic valve and kidney cultures were
entered as 2 and 1 log10 CFU/g, respectively (the limit of
detection). The Student-Newman-Keuls test was used to adjust for
multiple comparisons. For analysis of the sterilization of tissue
cultures, we used Fisher's exact test. A P value of <0.05
was considered statistically significant for all tests.
| |
RESULTS |
Vancomycin--lactam synergy against methicillin-resistant
staphylococcal strains.
A total of 59 methicillin-resistant
staphylococci underwent antimicrobial susceptibility testing and
testing for synergy between vancomycin and oxacillin by the broth
microdilution checkerboard method. We included coagulase-negative
staphylococcal species in our examination because the glycopeptide MICs
for these isolates are increased compared to those for S. aureus. The results of these tests are presented in Table
1. Vancomycin-oxacillin combinations demonstrated synergy against all three clinical isolates of GISA as
well as 4 of 10 MRSH and 18 of 20 MRSE isolates. Synergy was seen
against all four step-passage isolates with higher levels of vancomycin
resistance (isolates 27619VR, 5827HR, 27280-2, and 27280-3). No
evidence of synergy was seen against 22 MRSA isolates for which
vancomycin MICs were 2 µg/ml. No antagonism was noted against any
of the isolates tested.
Among the staphylococcal isolates, the synergy of the combination of
vancomycin and oxacillin was directly associated with
the level of
vancomycin resistance. The combination was more likely
to exhibit
synergism against isolates of staphylococci with higher
levels of
vancomycin resistance. The combination consistently
demonstrated
synergy against those isolates for which vancomycin
MICs were
4
µg/ml. This is shown in Fig.
1, which
plots the FIC
index for isolates as a function of the vancomycin MIC.
The FIC
index for isolates is inversely correlated with the vancomycin
MIC, indicating that higher levels of vancomycin resistance are
associated with increasing synergy between the combination of
vancomycin and
-lactams. This observation was also confirmed
in the
examination of the step-passage isolates: 27619VR (vancomycin
MIC, 8 µg/ml), 27280-2 (vancomycin MIC, 8 µg/ml), and 27280-3
(vancomycin
MIC, 16 µg/ml). In all three of these isolates the
development of
vancomycin resistance was associated with the development
of synergism
of the drug combination (FIC indices, 0.19190, 0.25195,
and 0.1255 respectively), whereas synergism was not exhibited
for either of the
parent strains (strains 27619 and 27280; vancomycin
MICs, 1 and 2 µg/ml, respectively; FIC indices, 0.6875 and 0.75,
respectively).
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FIG. 1.
Results of checkerboard testing of combinations of
vancomycin and oxacillin against staphylococcal isolates with various
degrees of vancomycin susceptibility. The FIC index is plotted against
the MIC for each isolate of S. haemolyticus ( ), S. epidermidis ( ), and S. aureus ( ). Solid circles,
S. haemolyticus isolates 27280, 27280-2, and 27280-3 (vancomycin MICs, 2, 8, and 16 µg/ml, respectively); solid squares,
S. aureus isolates 27619 and 27619VR (vancomycin MICs, 1 and
8 µg/ml, respectively). The dashed line represents a FIC index of
0.5. Vancomycin and oxacillin demonstrated synergy against isolates
whose data fall below this line. The solid line represents the best-fit
line, with a corresponding correlation factor of 0.563.
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The synergistic activity of vancomycin in combination with oxacillin
against GISA isolates was confirmed in time-kill experiments,
the
results of which are presented in Fig.
2.
Vancomycin at concentrations
up to 4 × the MIC showed no evidence
of bactericidal activity.
However, a bactericidal effect was seen with
the combination of
vancomycin (8 µg/ml) and oxacillin (6 µg/ml) at
24 h against two
GISA isolates (isolates HIP5827 and HIP5836),
with reductions
in bacterial counts of 2.44 and 3.06 log
10
CFU/ml, respectively.
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FIG. 2.
Time-kill curves for GISA HIP5827 and HIP5836. Results
for the following groups are represented; controls (squares),
vancomycin at 8 µg/ml (circles), oxacillin at 6 µg/ml (diamonds),
and vancomycin at 8 µg/ml in combination with oxacillin at 6 µg/ml
(triangles).
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Synergism against GISA isolates could also be demonstrated by a
modified disk diffusion method. For GISA isolates inoculated
onto a
BHIA plate containing vancomycin (6 µg/ml), an enhanced
zone of
inhibition surrounding the oxacillin (1 µg) disk was seen
compared to
that of disks on agar containing no antibiotic (no
zone), indicating
synergistic activity between the vancomycin
contained within the agar
and the oxacillin diffusion disk (Fig.
3). Enhanced zones of inhibition could be
demonstrated with a
number of
-lactams including ceftriaxone,
ceftazidime, cepodoxime,
and amoxicillin-clavulanate, but could not be
demonstrated with
aztreonam. The largest zones of inhibition were seen
with amoxicillin-clavulanate.
Similar synergism could be demonstrated
by double-disk potentiation
methods with oxacillin (1 µg) and
vancomycin (30 µg) diffusion
disks placed 15 to 20 mm apart (data not
shown). However, the
enhanced zones of inhibition surrounding the
vancomycin disks
were subtle and more difficult to interpret.
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FIG. 3.
Modified Kirby-Bauer disk diffusion testing of a GISA
isolate. HIP5827 was inoculated onto BHIA plates containing vancomycin
at 6 µg/ml (A) and BHIA with no antibiotics (B), followed by the
placement of antimicrobial disks. OX, oxacillin; CRO, ceftriaxone; and
AMC, amoxicillin-clavulanic acid.
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We were also interested in seeing whether synergism was related to
suppression of subpopulations with a lower level of vancomycin
resistance. To test this hypothesis, we performed population analysis
with an MRSH isolate for which the vancomycin MIC was 16 µg/ml
(isolate 27280-3). This isolate was produced following two overnight
passages in increasing concentrations of vancomycin. Population
analysis profiles confirmed the heterotypic nature of resistance
among
isolates with reduced vancomycin susceptibility compared
to the nature
of resistance among more vancomycin-susceptible
isolates. Population
analysis profiles were completed on vancomycin-containing
agar as well
as vancomycin-containing agar with a fixed concentration
of oxacillin
(6 µg/ml). These results are presented in Fig.
4 and demonstrate that synergy between
vancomycin and oxacillin
occurred, with suppression of the
subpopulation with the highest
level of vancomycin resistance.
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FIG. 4.
Population analysis profiles of S. haemolyticus 27280-3. Overnight cultures were plated on media
containing vancomycin ( ) and vancomycin and oxacillin at 6 µg/ml
().
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Endocarditis.
Results of treatment of endocarditis caused by
three clinical strains of GISA (strains HIP5827, HIP5836, and MU50)
following a 3-day course of vancomycin (30 mg/kg twice a day) are
presented in Table 2. Vancomycin
treatment was ineffective against all three strains, with a
<1-log10 CFU/g reduction in mean aortic valve vegetation
counts compared to the counts for untreated controls. There was also
high rate of mortality among both vancomycin-treated animals (5 of 12;
42%) and untreated control animals (12 of 12; 100%) infected with
HP5836 and MU50, establishing the virulence of these strains. In
contrast, none of the rabbits infected with HIP5827 and treated with
vancomycin and only 42% of control animals died during the 4 day
trial.
In order to determine whether selection of resistant subpopulations
within vegetation material was a possible cause of the
failure of
vancomycin treatment, we determined the population
analysis profiles of
cultured vegetation material on vancomycin-containing
agar. The
profiles of all three clinical GISA isolates are presented
in Fig.
5. In the population analysis profiles,
vegetation material
was serially diluted on agar plates containing
increasing concentrations
of vancomycin. The number of bacteria
collected from untreated
control animals was compared to the number of
bacteria cultured
from animals treated with vancomycin. The profiles of
the vegetation
material from control animals were nearly identical to
those seen
for the material from animals treated with vancomycin. No
evidence
of an increase in the proportion of cultured bacteria
resistant
to higher levels of vancomycin was detected. Thus, the
selection
of a highly vancomycin resistant subpopulation following
vancomycin
treatment did not occur.
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FIG. 5.
Population analysis profiles of bacteria isolated from
aortic valve vegetation material following 3 days of vancomycin
treatment. Rabbits infected with MU50, HIP5827, and HIP5836 were
treated with no antibiotics (solid symbols) or vancomycin at 30 mg/kg
intravenously twice a day for 3 days (open symbols).
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We next sought to confirm the in vitro data that indicated synergy
between vancomycin and
-lactam antibiotics in the endcarditis
model.
Following the establishment of endocarditis with GISA HIP5827
and MU50,
the animals were treated with either vancomycin, nafcillin,
or the
combination of the two antibiotics for 3 days. The results
of these
studies are presented in Table
2.
In the treatment of endocarditis due to HIP5827, a modest reduction in
the aortic valve vegetation counts was seen when either
vancomycin or
nafcillin was given as a single agent:
0.64 and
1.51
log
10 CFU/g, respectively. The combination of vancomycin
and nafcillin was significantly better than either drug given
alone,
with a reduction in mean log aortic valve vegetation counts
of
4.53
log
10 CFU/g compared to the counts for untreated controls.
Combination therapy also resulted in sterilization of kidney abscess
tissue from 89% of animals, whereas sterilization of kidney abscesses
occurred for only 12.5% of animals treated with either antibiotic
alone.
The results seen for the treatment of clinical isolate GISA MU50
paralleled those seen for the treatment of HIP5827. Mean
log aortic
valve counts were reduced by 4.06 log
10 CFU/g compared
to
the counts for untreated controls in rabbits treated with the
combination. Again, therapy with either vancomycin or nafcillin
as a
single agent was ineffective, with a <1.0-log
10 CFU/g
reduction
in mean aortic valve vegetation counts compared to the counts
for untreated controls. The rate of kidney abscess sterilization
was
significantly higher among rabbits treated with the combination
(57%)
than among animals treated with either vancomycin (33%)
or nafcillin
(14%).
| |
DISCUSSION |
The emergence of decreasing levels of vancomycin susceptibility
among clinical isolates of staphylococci has recently raised fears that
effective antimicrobial treatment options for these isolates may soon
be severely limited. Clinical data have suggested that patients
infected with these isolates respond poorly to vancomycin monotherapy
(5, 14). This poor clinical response has been supported by
previous in vitro data and is confirmed in the rabbit model of
experimental endcarditis as described in this report. We examined
several properties of staphylococci with reduced susceptibility to
vancomycin in an attempt to determine their contribution to the lack of
effectiveness of vancomycin in the treatment of clinical GISA infections.
First, vancomycin resistance among staphylococci is expressed in a
heterogeneous or heterotypic manner. This heterotypic expression may
lead to difficulty in the detection of the phenotype by usual antimicrobial susceptibility testing and may be a partial explanation for the failure of antimicrobial therapy. Among methicillin-resistant staphylococci, the heterogeneous expression of methicillin resistance has been shown to be associated with the failure of -lactams in
experimental endocarditis models due to the selection of highly resistant subpopulations among vegetation material following exposure to -lactams (6). One of the simplest means of
demonstrating heterotypic expression is through the use of population
analysis profiles. The population analysis profiles of clinical GISA
isolates completed for this report demonstrate the heterotypic
expression of vancomycin resistance, with a small proportion of cells
expressing resistance to vancomycin at levels above the MIC. However,
our data generated with rabbits with experimental endocarditis suggest that the failure of vancomycin therapy is not associated with the
selection of highly resistant subpopulations among the entire heteroresistant population following treatment. In our endocarditis model, after treatment of infections caused by three clinical GISA
isolates (isolates HIP5827, HIP5836, and MU50) with vancomycin, we
found no significant change in the level of vancomycin resistance among
cultured bacteria, as measured by population analysis profiles.
The second observed property of staphylococci associated with decreased
susceptibility to vancomycin is the tendency to develop stepwise
increases in resistance following short-term exposure to vancomycin or
other glycopeptides. For laboratory isolates of both coagulase-negative
and coagulase-positive staphylococci, increases in vancomycin MICs can
be seen following a single overnight passage in the presence of
subinhibitory concentrations of vancomycin (step passage). The
development of vancomycin resistance following passage is particularly
prominent among coagulase-negative staphylococci such as S. haemolyticus (26). In fact, the first
vancomycin-resistant clinical staphylococcal isolate was an MRSH strain
reported by Schwalbe et al. (25) in 1987. Additionally,
vancomycin susceptibility as measured by MICs is lower among both MRSE
and MRSH isolates in comparison to that among S. aureus
isolates (1). Among clinical isolates, exposure to
glycopeptides appears to be a prerequisite to the development of
resistance because all current clinical GISA isolates have been
isolated following vancomycin or teicoplanin treatment of patients.
Although the in vitro development of vancomycin resistance occurs in a
relatively short time frame, it is unclear over what time period in
vivo selection of higher levels of vancomycin resistance occurs among
S. aureus strains. In our endocarditis model, we did not
observe in vivo selection of higher levels of vancomycin resistance
following a 3-day treatment regimen. However, the isolation of clinical
GISA isolates has followed relatively long periods of glycopeptide
exposure ranging from 12 days to several months (5, 14, 22).
The third property of staphylococci relevant to decreased
susceptibility to vancomycin is the poor bactericidal activity of this
antibiotic. It has long been known that vancomycin is less rapidly
bactericidal than -lactams in the treatment of serious staphylococcal infections such as endocarditis (15).
Vancomycin tolerance as an explanation for therapeutic failures has
also been proposed by some investigators (13). Among GISA
isolates, there was also a poor bactericidal response to vancomycin.
Time-kill experiments demonstrated that vancomycin at concentrations up to 4× the MIC for GISA isolates had no bactericidal activity. This may
be related to the fact that vancomycin does not display concentration-dependent killing (16). The current practice
of intermittent dosing of vancomycin may hinder its action, since more
effective killing may be achieved with continuous infusion, by which
levels in serum are maintained above the vancomycin MIC. The lack of
bactericidal activity of vancomycin was confirmed with the endocarditis model.
Although our report demonstrates that vancomycin montherapy is
ineffective in the treatment of GISA infections, vancomycin still may
have a role in the therapy of this emerging resistance phenotype. Our
data indicate that vancomycin given in combination with
antistaphylococcal -lactams demonstrates synergistic activity against a variety of staphylococcal isolates with reduced
susceptibility to glycopeptides. Synergy was demonstrated among both
coagulase-negative and coagulase-positive staphylococci by the broth
microdilution checkerboard, time-kill, disk diffusion, and agar
dilution techniques. The synergy between vancomycin and a number of
-lactams with antistaphylococcal activity (resistance to
staphylococcal -lactamase) correlated with the glycopeptide
resistance levels of the isolates. In fact, the development of
vancomycin resistance in previously vancomycin-sensitive isolates was
associated with the emergence of synergy. This was clearly demonstrated
among step-passage isolates 27619VR, 27280-2, and 27280-3. Although the
FIC indices for the vancomycin-susceptible parents 27619 and 27280 indicated only additive effects with the combination of vancomycin and
oxacillin, for the step passage isolates the FIC indices were lower,
indicating the presence of synergy between the same combinations of
antibiotics (Fig. 1). Synergy between vancomycin and -lactams was
also associated with selective killing of the most vancomycin-resistant
subpopulations (Fig. 3).
The observation that vancomycin and -lactams may have synergistic
activities against methicillin-resistant staphylococci has been made
previously, as determined from the results of antimicrobial susceptibility testing (2, 12, 23, 31). Vancomycin has been
demonstrated to have synergistic activity when it is combined with
cephalothin (23), imipenem (2), cefazolin
(31), and oxacillin (12) in in vitro tests. In
addition, Shlaes et al. (26) noted that the combination of
cefotaxime and teicoplanin showed synergism against the
teicoplanin-resistant derivative S. aureus 12873. The data
from our endocarditis model represent the first validation of these in
vitro observations in an in vivo model of infection. For the treatment
of experimental aortic valve endocarditis due to two separate strains
of GISA (HIP5827 and MU50), the combination of vancomycin and nafcillin
was significantly better than either drug given singly. Therapy with
the combination of vancomycin and nafcillin also showed a higher rate
of sterilization of kidney abscesses following treatment.
A detailed explanation for the observed synergism between vancomycin
and -lactams against staphylococcal isolates is confounded by an
incomplete knowledge of the mechanisms responsible for vancomycin resistance, the expression of methicillin resistance, and the complex
regulation of peptidoglycan synthesis in staphylococci. Glycopeptide-resistant isolates of staphylococci have been described to
have the following characteristics: thicker cell walls (14, 27-29), slower growth (14, 28), decreased autolysis
(27, 28), decreased peptidoglycan cross-linking following
exposure to vancomycin, as evidenced by the lack of higher oligomer
muropetide components of intact peptidoglycan cell wall by
high-pressure liquid chromatographic analyses (28, 29), and
the ability to trap vancomycin at the periphery of the cell wall
(28, 29). These observations have supported the theory that
resistance to the action of vancomycin involves the production of a
thicker cell wall with an increased proportion of free pentapeptide
D-Ala-D-Ala termini capable of binding to
vancomycin. However, the ability to bind to vancomycin is not the sole
explanation for resistance because the vancomycin binding capacity of
glycopeptide-resistant step-passage isolates correlates poorly with the
vancomycin MIC (29). Additional alterations of tertiary
peptidoglycan structure, cell wall synthesis regulation, and cell wall
porosity (29) are likely to contribute to vancomycin resistance.
The observed synergism between vancomycin and -lactams against
methicillin-resistant staphylococci may be related to the substrate
specificity of PBP 2a. In the presence of -lactams, PBP 2a is
assumed to perform all cross-linking of the pentaglycine cross-bridges
by transpeptidation of the terminal D-Ala. High-pressure liquid chromatographic analysis of methicillin-resistant
staphylococci has shown that PBP 2a has a specific substrate
specificity for monomeric disaccharide pentapeptides and is unable to
cross-link to higher oligomeric muropeptides (3, 10, 11).
Following exposure of GISA to both vancomycin and -lactams,
competition for monomeric muropeptide components of the developing
peptidoglycan may be occurring. This mechanism is supported by the
observation that a greater degree of synergy was seen against
staphylococci with reduced vancomycin susceptibility and that synergy
occurred only with higher concentrations of vancomycin, indicating
concentration-dependent kinetics and a requirement for saturation of
the available target.
The data presented in this report would indicate that future clinical
isolates of GISA should undergo additional susceptibility testing for
the presence of synergy between vancomycin and -lactams. We conclude
that combination therapy may be a reasonable alternative in the
treatment of infections caused by staphylococcal isolates that
demonstrate reduced susceptibility to glycopeptides.
| |
ACKNOWLEDGMENTS |
We thank Geri Hale-Cooper and Elizabeth Hanners for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: McGuire Veterans
Affairs Medical Center, 1201 Broad Rock Blvd., Section 111-C, Richmond, VA. Phone: (804) 675-5018. Fax: (804) 675-5437. E-mail:
Climo.Michael{at}Richmond.va.gov or
MWCLIMO{at}aol.com.
| |
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Abstract
Introduction
