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Antimicrobial Agents and Chemotherapy, July 1998, p. 1574-1577, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vitro Activities of Co-Amoxiclav at Concentrations Achieved
in Human Serum against the Resistant Subpopulation of
Heteroresistant Staphylococcus aureus: a Controlled
Study with Vancomycin
J.
Prieto,1
L.
Aguilar,2,*
M. J.
Giménez,2
D.
Toro,1
M. L.
Gómez-Lus,1
R.
Dal-Ré,2 and
I. P.
Balcabao1
Microbiology Department, School of Medicine,
Universidad Complutense,1 and
Medical Department, SmithKline Beecham
Pharmaceuticals,2 Madrid, Spain
Received 3 November 1997/Returned for modification 22 March
1998/Accepted 27 April 1998
 |
ABSTRACT |
The effects of concentrations that simulated those in human serum
after a single intravenous dose of amoxicillin (2 g),
amoxicillin-clavulanic acid (2,000 and 200 mg, respectively), or
vancomycin (500 mg), on the viability and 
-lactamase
activity of two isogenic (-lactamase and non--lactamase producer)
heteroresistant Staphylococcus aureus strains were studied
in an in vitro pharmacodynamic model. A reduction of 
97% of the
initial inoculum was obtained with vancomycin and amoxicillin-clavulanic acid against both strains, with respect to
the total bacterial population and the oxacillin-resistant subpopulation. The same pattern was observed with amoxicillin and
the -lactamase-negative strain. -Lactamase activity in the -lactamase-positive strain changed over time parallel to viability, decreasing with amoxicillin-clavulanic acid or vancomycin and increasing in the amoxicillin and control groups. Clavulanic acid concentrations achievable in serum that changed over time allowed amoxicillin to act against the -lactamase-producing
methicillin-resistant S. aureus to a similar extent as
vancomycin.
| |
INTRODUCTION |
Infections due to
methicillin-resistant Staphylococcus aureus (MRSA) became
epidemiological and clinical problems in the 1980s (15) and
remain so in the current decade (23). MRSA is considered resistant to all penicillins, cephalosporins, carbapenems, and monobactams (24), vancomycin being the standard treatment
for infections due to MRSA. Despite the cross-resistance that MRSA exhibits to all -lactams, the majority of strains exhibit
heteroresistance, with only a small subpopulation expressing obvious
resistance while the majority remain susceptible (12).
Induction by -lactams of the gene mecA is seen in those
strains with inducer-repressors, and the resistance phenotype is
induced by environmental conditions (such as temperature and
osmolarity) (5); thus, the promotion of growth of the
resistant subpopulation is favored by incubation at cooler temperatures
(30 to 35°C) and the presence of NaCl (2 to 4%) (15).
The production of PBP 2A (encoded by mecA) with low
-lactam affinity provides, in the absence of -lactamase
production, resistance to most types of -lactams (3). But
interestingly, traditional -lactams such as penicillin G,
amoxicillin, and ampicillin, have relatively good affinity for PBP 2A,
amoxicillin showing 10 times greater affinity than methicillin,
oxacillin, and clavulanate (which do not overcome the subpopulation
resistance) (10). In addition, most MRSA strains also
contain the -lactamase resistance mechanism, contributing to
elevated MICs (10).
Due to the higher affinity of amoxicillin for PBP 2A and the
-lactamase-blocking effect exerted by clavulanate, the clinical efficacy of the combination in the treatment of MRSA infections has
been hypothesized, this being supported by the reported therapeutic efficacy in rat models of endocarditis (10). The presence of 2 to 4 µg of clavulanic acid per ml allows concentrations of 8 to 25 µg of amoxicillin per ml to inhibit 50 to 100% of MRSA strains, these concentrations being unachievable in humans by the oral route but
achievable with the intravenous formulation (25).
In this study, we assessed the in vitro effects of co-amoxiclav,
amoxicillin, and vancomycin versus control, using concentrations simulating those in human serum over time, on the viability of the
total population and the resistant subpopulation of two isogenic strains (one producing penicillinase and the other not) of
MRSA. In addition, the effect on the -lactamase activity of the
corresponding strain was measured.
| |
MATERIALS AND METHODS |
The two isogenic strains used in this study were kindly supplied
by P. Moreillon (CHUV, Lausanne, Switzerland). The parent strain (1. Paris, 1984) and its penicillinase-negative derivate, together with the
method of obtaining it, were fully described by Franciolli et al.
(10).
The MICs of amoxicillin, co-amoxiclav (2:1), and vancomycin for these
two strains were determined in Mueller-Hinton (MH) broth by a standard
method (19) and also in MH broth with 2% NaCl incubated at
35°C.
The pharmacodynamic simulation method used has been previously
described for S. aureus (1, 17). The
concentrations used were similar to those obtained in serum after an
intravenous dose of 2 g of amoxicillin combined with 200 mg of
clavulanic acid (6, 26) or after 500 mg of vancomycin
(8, 16). The initial inoculum was obtained by diluting an
overnight culture of MH broth (Difco Laboratories, Detroit, Mich.) with
2% NaCl (to allow the growth of the resistant subpopulation) in new
fresh broth, which was incubated in a 35°C shaking bath until an
A580 of 0.3 (Hitachi U 1,100 spectrophotometer)
was reached. The culture was further diluted 1:10, resulting in a final
inoculum of approximately 107 CFU/ml in a 4-ml volume of MH
broth with 2% NaCl containing the initial co-amoxiclav, amoxicillin,
or vancomycin concentrations (Table 1).
Incubation was performed with Centriprep-10 concentrator tubes
(membrane pore size, 10,000 Da; Amicon, Beverly, Mass.) at 35°C in a
shaking bath for the first incubation period listed in Table 1. After
incubation, aliquots of 200 µl were diluted in 1,800 µl of sterile
saline solution, and appropriate decimal solutions were prepared in MH
agar plates with 4% NaCl for colony counting of the total population
and in MH agar plates with 4% NaCl and 25 µg of oxacillin per ml for
colony counting of the resistant subpopulation, with incubation at
35°C for 48 h. The limit of detection was 50 CFU/ml.
Centrifugation-filtration for 10 min at 2,000 × g was
then performed, and the supernatant above the filter was carefully
collected for further determination of -lactamase activity. A
bacterial suspension volume of approximately 500 µl remained below
the filter. New MH broth (4 ml) with the subsequent corresponding drug
concentration was added to the bacterial suspension. The tubes were
incubated under the conditions described above for the ensuing
incubation period. This method was used throughout five incubation
periods. Experiments were performed three times for each experimental
group (co-amoxiclav, amoxicillin, and vancomycin). As a control, the
initial inoculum in MH broth without antibiotics was processed
according to the same centrifugation-filtration, colony counting, and
supernatant collection procedures for the five incubation periods.
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TABLE 1.
Concentrations of vancomycin and co-amoxiclav simulating
those in human serum for various incubation periods
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|
The antibiotic carryover effect, which occurred in the 500-µl
bacterial suspensions remaining after the centrifugation-filtration steps, was determined. The amounts of antibiotics in the 500-µl bacterial suspensions after each incubation period were calculated by
the equation y = 0.5x, where x is
the initial amount of antibiotic per milliliter in the 4-ml broth
(total antibiotic amount in 4 ml = 4x) and y
is the resulting amount in the 500-µl suspension. Concentrations for
each incubation period were calculated by adding the carried-over
antibiotic amount in the 500-µl suspension to the antibiotic
concentration in the new broth that was added. The final concentrations
calculated are listed in Table 1. -Lactamase activity was measured
by adding 25 µl of a 500-µl/ml solution of nitrocefin (Glaxo Ltd.,
Greenford, Middlesex, England) to 225 µl of each broth supernatant
previously collected, which was incubated for 30 min at 37°C
(21). Afterwards, 1.5 ml of phosphate buffer (0.05 M) was
added, and A482 was read with the
spectrophotometer (17).
Viability rate (percent decrease) with respect to the initial inoculum
(I) in terms of CFU per milliliter was calculated by the
equation 100 
(It = x h × 100/It = 0 h), with the viability of the
initial inoculum (t = 0 h) set at 100%.
To detect the different trends over time, the repeated-measures
multivariate analysis of variance was used. The time lags used in the
total population comparisons lie between 0 and 4. In the subpopulation
comparisons, the time lags are between 0 and 2. To check the
assumptions of the analysis, the sphericity test of Anderson was used.
When the Wilks P value was significant (P < 0.05), contrast between groups was made with the Tukey-Kramer test to
adjust the type I error.
| |
RESULTS |
MICs of amoxicillin, co-amoxiclav (2:1), and vancomycin for the
-lactamase-positive strain were 32, 1, and 1 µg/ml, respectively, in the absence of NaCl and with 37°C incubation and 128, 16, and 1 µg/ml, respectively, in the presence of 2% NaCl and with 35°C
incubation. For the -lactamase-negative strain, values were 1, 1, and 1 µg/ml, respectively, in the absence of NaCl and with 37°C
incubation and 16, 16, and 1 µg/ml, respectively, in the presence
of 2% NaCl and with 35°C incubation.
After an overnight incubation in MH broth with 2% NaCl at 35°C to
allow expression of the PBP 2A and at the appropriate dilutions, initial inoculum at time zero of the pharmacodynamic simulation was
7.2 × 106 and 9.8 × 106 CFU/ml for
the -lactamase-positive and -negative strains, respectively. Initial
counts for the resistant subpopulation were 6.6 × 103 and 2.8 × 104 CFU/ml for the
-lactamase-positive and -negative strains, respectively. At time
zero, the rates of resistant CFU per milliliter with respect to the
total population were 1/1,000 for the -lactamase-positive strain and
3/1,000 for the -lactamase-negative strain.
The viability rates at the different incubation times for the
-lactamase-positive strain are shown in Table
2 for the total population and in Table
3 for the resistant subpopulation.
Significant differences were found among amoxicillin (P = 0.03), vancomycin (P = 0.01), and co-amoxiclav
(P = 0.01) versus control as well as between
vancomycin and amoxicillin (P = 0.03) and co-amoxiclav versus amoxicillin (P = 0.01), due to a regrowth in the
latter group from 2 h on. The same growth patterns were observed
in the subpopulation, with regrowth in the amoxicillin group from
2 h on. Significant differences were found in the initial inoculum reduction rate over time with respect to the resistant
subpopulation between amoxicillin and vancomycin (P = 0.03). As in the total population, significant differences were
also found between antibiotic groups and control
(P 
0.003), where bacterial counts rose from 6.6 × 103 CFU/ml at time zero to 4 × 105
CFU/ml at 8 h. The resistant subpopulation counts were below the
limit of detection in the vancomycin and co-amoxiclav groups from
3 h on.
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TABLE 3.
Viability rates (percent decreases) of
-lactamase-positive MRSA subpopulation at the different
incubation times
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|
Tables 4 and
5 show the viability rates at the
different incubation times for the total population and the resistant
subpopulation of the -lactamase-negative strain, respectively. Both
populations showed similar patterns of reduction of the initial
inoculum with significant differences with respect to control
(P = 0.01 for the total population and any of the three
antibiotics and P = 0.0001 for the resistant
subpopulation and any of the three antibiotic groups). In the control
group, the counts of the resistant subpopulation increased from
2.8 × 104 to 2 × 106 CFU/ml. No
differences were found between the different antibiotic groups with
respect to the total population reduction rates over time, but
differences became statistically significant in comparing the change
over time of the resistant subpopulation inocula between vancomycin and
amoxicillin (P = 0.004) or co-amoxiclav
(P = 0.007) and between co-amoxiclav and amoxicillin
(P = 0.0003). In the amoxicillin and vancomycin groups,
the colony counts of this subpopulation were under the limit of
detection from 3 h on, whereas in the co-amoxiclav group this
occurred from 2 h on.
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TABLE 5.
Viability rates (percent decreases) of
-lactamase-negative MRSA subpopulation at the different
incubation times
|
|
Comparative -lactamase activities (means ± standard
deviations) for the four groups at the different incubation times are shown in Table 6. From 0.5 h on, all
antibiotic groups presented statistically significant differences
versus control (P < 0.05). From 2 h on, a
significant difference was found between co-amoxiclav or vancomycin and
amoxicillin; there were no statistically significant differences
between co-amoxiclav and vancomycin.
| |
DISCUSSION |
Vancomycin is the treatment of choice for infections due to MRSA,
but resistance could emerge (13), and so alternatives to
vancomycin should be explored (22).
The aim of the experiment was to study the effect of concentrations
simulating those after an intravenous dose of 500 mg of vancomycin or
2,000/200 mg (amoxicillin and clavulanic acid, respectively) of
co-amoxiclav on the viability of the methicillin-resistant subpopulation. Dosing intervals for these doses (8 h for amoxicillin or
co-amoxiclav and 6 h for vancomycin) fit the experimental time (8 h to include both dosing intervals). These doses fulfil the following
two conditions: (i) peak concentration/MIC ratio of 10 and (ii) time
over MIC of 75% (6 h) of dosing interval (when the MIC is determined
with standard conditions: MH broth and 37°C incubation). These are
important pharmacodynamic facts because the two antibiotics tested
exhibit a time-dependent action. In the case of amoxicillin, levels are
over the MIC during 6 h (8 h for vancomycin); theoretically, the
rest of the experiment time (dosing interval) would be covered due to
the postantibiotic effect on susceptible S. aureus
(7). To detect the effect of the antibiotics, with these
achieved pharmacokinetic-in vitro susceptibility values, on the
resistant subpopulation viability, the medium osmolarity and the
temperature of incubation were changed in order to allow the expression
of PBP 2A. A high inoculum (approximately 107 CFU/ml) was
used in order to obtain measurable numbers of CFU of the resistant
subpopulation, despite the inoculum effect having been described for
aminopenicillins against S. aureus but not for vancomycin
(2); the effect might influence the results with the total
population due to the favorable conditions for the glycopeptide. On the
other hand, allowing PBP 2A expression through osmolarity and
temperature of incubation increases the MIC of co-amoxiclav for both
strains from 1 to 16 µg/ml but not the MIC of vancomycin, which
remains 1 µg/ml.
With respect to the total population, with the -lactamase-negative
strain, no differences were found between antibiotic groups due to the
pharmacodynamic conditions being fulfilled and the standard MICs being
the same. Differences in MICs became relevant when, with the expression
of gene mecA, only vancomycin maintained its value. Due to
this, as expected, significant differences in the reduction of the
resistant subpopulation initial inoculum over time were found between
vancomycin and amoxicillin, because of the higher and earlier killing
with vancomycin (59 versus 95%) in the first incubation period.
Interestingly, when clavulanic acid is added, amoxicillin
acquires an initial killing capability similar to that of
vancomycin (97 versus 95%). This increase in earlier killing by
clavulanic acid is also extended to the time needed to reduce the
initial inoculum 100-fold (9) in the resistant subpopulation: 2 h for co-amoxiclav and 3 h for vancomycin.
In evaluating the effect on the total population, with respect to the
-lactamase-positive strain, significant differences were found
between amoxicillin and vancomycin or co-amoxiclav, because of the
regrowth obtained in the amoxicillin group due to the regrowth of the
resistant subpopulation. No differences were found between vancomycin
and co-amoxiclav in considering either the total population or the
resistant subpopulation, with 100-fold reduction in the initial
inoculum being obtained after 3 h in all cases. -Lactamase
activity over time changed in parallel with viability, increasing with
the increase of viability (amoxicillin or control) and decreasing with
the decrease of viability in the co-amoxiclav and vancomycin
group, without statistically significant differences between
them.
With both strains, amoxicillin or co-amoxiclav concentrations from
6 h on are subinhibitory despite a maintenance of the more than
99% reduction of the initial inoculum in both groups except in the
amoxicillin group and with the -lactamase-producing strain. This
postantibiotic effect, detected and maintained due to a
post--lactamase inhibitory effect in the co-amoxiclav group with the
-lactamase-producing strain, has been previously described for
methicillin-susceptible S. aureus (1, 17).
These findings on the effects of concentrations achievable in serum may
contribute to explaining the efficacy in vivo of the combination
aminopenicillin--lactamase inhibitor in infections caused by MRSA,
both in animal models (4, 10, 18, 27) and in the clinical
setting (11, 20). Despite vancomycin remaining the drug of
choice for MRSA infections, the increase of vancomycin resistance and
the Centers for Disease Control and Prevention recommendation
(14) for prudent use of vancomycin have encouraged the
search for alternatives. Aminopenicillin--lactamase inhibitor may be one of them, adding to its wide spectrum this activity, which
may be considered for empirical treatment in the clinical setting, if
further in vitro studies and animal models confirm these results with
other MRSA strains.
| |
ACKNOWLEDGMENTS |
We thank P. Moreillon (CHUV, Lausanne, Switzerland) for his
critical review of the manuscript and J. J. García
(Cibest, Madrid, Spain) for performing the statistical analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Medical
Department, SmithKline Beecham Pharmaceuticals, Valle de la
Fuenfría, 3, 28034 Madrid, Spain. Phone: 34-91-3345275. Fax: 34-91-3345141. E-mail: lorenzo.aguilar{at}sb.com.
| |
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Antimicrobial Agents and Chemotherapy, July 1998, p. 1574-1577, Vol. 42, No. 7
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
