Synergy between Vancomycin and Nafcillin against Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model
Citation: Leonard SN (2012) Synergy between Vancomycin and Nafcillin against Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic
Model. PLoS ONE 7(7): e42103. doi:10.1371/journal.pone.0042103
Synergy between Vancomycin and Nafcillin against Staphylococcus aureus in an In Vitro Pharmacokinetic/ Pharmacodynamic Model
Steven N. Leonard 0
Martin Rottman, Harvard Medical School, United States of America
0 1 School of Pharmacy, Bouve College of Health Sciences, Northeastern University , Boston , Massachusetts, United States of America, 2 Department of Pharmacy, Brigham and Women's Hospital , Boston, Massachusetts , United States of America
Introduction: Continued pressure from glycopeptide use has led to non-susceptible strains of Staphylococcus aureus including heterogeneously vancomycin-intermediate S. aureus (hVISA). Infections with hVISA are associated with poor patient outcomes, thus incentivizing novel treatments. Evidence suggests that vancomycin and anti-staphylococcal penicillin susceptibility are inversely related which indicates that the use of this combination may be particularly useful against methicillin-resistant S. aureus with reduced susceptibility to vancomycin, such as hVISA. The aim of this study was to evaluate the potential for synergy between vancomycin and nafcillin against hVISA. Methods: Twenty-five hVISA strains were evaluated for vancomycin and nafcillin minimum inhibitory concentration (MIC) by broth microdilution in duplicate. Potential for synergy was assessed by time-kill at 1/2x MIC in triplicate. Five strains were chosen, representing the range nafcillin MIC's available in the cohort -4, 16, 64, 128, and 256 mg/mL, and were run in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model in duplicate over 72 hours to evaluate the potential of the combination with simulated human pharmacokinetics. In addition, 4 fully glycopeptide susceptible strains of S. aureus including 2 methicillin-susceptible (MSSA) and 2 methicillin-resistant (MRSA) were run in the PK/PD model for comparison. Results: In the time-kill, 92% of strains (23 of 25) displayed synergy with the combination of vancomycin and nafcillin. In the PK/PD model, all five strains of hVISA showed an improvement in overall activity (P#0.004) and organism burden at 72 hours (P#0.001) with the combination compared to either drug alone. The combination was also successful against both MRSA and MSSA in overall activity (P#0.009) and organism burden at 72 hours (P#0.016), though the magnitude of the effect was diminished against MSSA. Conclusions: The combination of vancomycin and nafcillin significantly improved antibacterial activity against hVISA, MRSA, and MSSA compared to either drug alone.
Funding: This work was supported in part by a Northeastern University, Bouve College of Health Sciences Proposal Development Grant. No other specific
funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The author has declared that no competing interests exist.
The worldwide dissemination and poor treatment outcomes of
methicillin-resistant Staphylococcus aureus (MRSA) presents
therapeutic difficulties for clinicians. Historically vancomycin has been
the mainstay of therapy for MRSA infections, however decades of
selective pressure has led to evolutionary changes in S. aureus
diminishing the utility of this agent. [1,2,3,4,5,6,7] Of note is the
emergence of heterogeneously vancomycin intermediate S. aureus
(hVISA); a particularly concerning organism as it is not detected
by traditional susceptibility testing or automated systems
commonly utilized in clinical microbiology laboratories. [4,8,9] Due to
these detection difficulties the true prevalence is difficult to
estimate but generally ranges from 515% (although this varies
widely based on geographic location, testing method used, time
period of isolates tested, etc.). [4,9,10,11] It has also been shown
that the prevalence of hVISA may be rising.  This is concerning
as preliminary studies have found an association between infection
with hVISA and poor treatment outcomes including prolonged
fever and bacteremia, increased length of hospital stay,
vancomycin treatment failure, and longer total duration of antibiotic
The use of combination antimicrobial therapy is a common
occurrence and represents a potential treatment option for
infections caused by hVISA.  Multiple guidelines from the
Infectious Diseases Society of America (IDSA) advocate for the use
of a myriad of combination antimicrobial therapies for different
purposes. [12,13,14,15] The clinical use of combination therapy
for MRSA, outside of the clinical practice guidelines above, has
become ubiquitous and thus there is an ongoing need to
characterize antimicrobial interactions to find the most potentially
useful combinations. Several previous investigations have found
synergy between beta lactams and anti-MRSA agents including
vancomycin, daptomycin, and telavancin against MRSA.
[16,17,18,19,20,21] These combinations have been explored
because, clinically, the use of an antistaphylococcal penicillin is
desirable in the setting where beta lactams have activity. [22,23]
There are also reports showing an inverse relationship between
vancomycin and beta lactam susceptibility, indicating that the use
of beta lactam combinations may be particularly useful against
organisms with reduced susceptibility to vancomycin, such as
hVISA. [24,25,26] The objective of this investigation was to
evaluate the potential for synergy between vancomycin and
nafcillin against hVISA by time kill analysis and further evaluate
the combination with an in vitro
pharmacokinetic/pharmacodynamic (PK/PD) model utilizing realistic drug concentrations and
Materials and Methods
Twenty five clinical isolates of hVISA already proven positive
by population analysis area under the curve ratio using Mu3 as
a positive control (PAP-AUC) (provided by the Anti-Infective
Research Laboratory, Detroit, MI) were utilized for susceptibility
and time kill experiments.  Isolates were collected between 1986
and 2007 from several hospitals throughout metropolitan Detroit
(Detroit Receiving Hospital and Henry Ford Hospital, Detroit,
MI, and William Beaumont Hospital, Royal Oak, MI) and from
the SENTRY antimicrobial surveillance program. Five strains
were selected from the above described cohort of 25 strains and
run in a one compartment in vitro
pharmacokinetic/pharmacodynamic (PK/PD) model. In addition, 4 fully glycopeptide
susceptible clinical isolates of S. aureus including 2 methicillin-susceptible
(MSSA) and 2 methicillin-resistant (MRSA) were run in the PK/
PD model for comparison. These 4 isolates were collected from
Brigham and Womens Hospital, Boston, MA in 2010.
Vancomycin and nafcillin were purchased from a commercial
source (Sigma Chemical Company, St. Louis, MO).
Mueller-Hinton broth (Difco, Detroit, MI) supplemented with
25 mg/L of calcium, 12.5 mg/L magnesium, and 2% sodium
chloride (due to the presence of nafcillin and according to CLSI
recommendations) (SMHB) was used for all susceptibility testing,
time kills, and PK/PD models.  Colony counts were
determined using Tryptic Soy Agar (TSA, Difco, Detroit, MI).
Mueller Hinton Agar (MHA, Difco, Detroit, MI) was used to test
for the emergence of resistance.
Figure 1. Timekill curve analysis of one hVISA (R3003, VAN MIC = 2, NAF MIC = 256 mg/mL) isolate. All antimicrobials are at
a concentration of 0.5x MIC. This isolate shows synergy between vancomycin and nafcillin. Data are presented as a graph of the mean bacteria
remaining vs. time with error bars at the sampling points representing 1 standard deviation from the mean. GC = Growth Control, NAF = Nafcillin,
VAN = Vancomycin.
Figure 2. Activity of vancomycin and nafcillin alone and in combination in the PK/PD model against hVISA strains where the
concentration of nafcillin was below the MIC of the respective organism for 100% of the dosing interval. Graphs shown are of isolate
R5253 (Nafcillin MIC = 16 mg/mL) (A), R1915 (Nafcillin MIC = 64 mg/mL) (B), R2729 (Nafcillin MIC = 128 mg/mL) (C), and R3003 (Nafcillin MIC = 256 mg/
mL) (D). Data are presented as a graph of the mean bacteria remaining vs. time with error bars at the sampling points representing 1 standard
deviation from the mean. GC = Growth Control, NAF = Nafcillin, VAN = Vancomycin.
Minimum inhibitory concentrations (MIC) of study
antimicrobial agents were determined by broth microdilution at ,5.5 log10
CFU/mL according to Clinical and Laboratory Standards
Institute (CLSI) guidelines. .
Potential for synergy with vancomycin plus nafcillin was
determined by time-kill methods in triplicate at a final inoculum
of ,106 CFU/mL. All time-kill experiments were performed at 1/
2x the MIC of the respective antibiotic. Aliquots (0.1 ml) were
removed at 0, 4, 8, and 24 hours, serially diluted in 0.9% sodium
chloride, and plated on TSA plates with a lower limit of detection
of 2 log10 CFU/mL. Time-kill curves were constructed by plotting
mean colony counts (log10 CFU/ml) versus time. Synergy was
defined as $ 2-log10 CFU/mL increase in killing at 24 hours with
the combination, in comparison with the killing by the most active
single drug. Combinations that resulted in $1-log10 bacterial
growth in comparison to the least active single agent were
considered to represent antagonism. All combinations not meeting
the definition of synergy or antagonism were considered
indifferent. All samples were incubated at 37uC for 24 hours.
In Vitro Pharmacokinetic/Pharmacodynamic (PK/PD)
Five strains of hVISA, one from each available nafcillin MIC
in the cohort 4 (isolate R1629), 16 (R5253), 64 (R1915), 128
(R2729), and 256 (R3003) mg/mL, were chosen to be run in an
in vitro PK/PD model consisting of a 125 mL one-compartment
glass apparatus with ports for the addition and removal of media,
antibiotics, and samples. These strains were selected to represent
the full continuum of nafcillin susceptibility available in order to
determine if there was a beta lactam susceptibility ceiling to
any enhanced killing effect observed. In addition, two strains of
methicillin-susceptible S. aureus (MSSA; isolates SNL4 and SNL9)
and two strains of methicillin-resistant S. aureus (MRSA; isolates
SNL96 and SNL98), all fully glycopeptide susceptible, were run
in the PK/PD model as comparators. The model was placed in
a water bath at 37uC throughout the simulation with a magnetic
stir bar for mixing. Fresh media was continuously supplied and
removed via a peristaltic pump (Masterflex, Cole-Parmer
Instrument Company, Chicago, IL) set to simulate the half-lives of
the antibiotics. A starting inoculum of ,107 CFU/mL was used
for all simulations. This higher inoculum was chosen because
hVISA requires a high inoculum to observe the heterogeneous
phenotype and to provide a more rigorous experimental
condition for vancomycin and nafcillin, both of which are
subject to an inoculum effect on their activity. [28,29] Free drug
concentrations were used to simulate regimens of vancomycin
1 g every 12 h (targets: fCmax: 30 mg/mL, fCmin: 7.5 mg/mL,
half-life: 6 h; at 50% protein binding for vancomycin these levels
correspond to a total Cmax of 60 mg/mL and Cmin of 15 mg/mL)
, nafcillin 2 g every 4 h (targets: fCmax: 5.2 mg/mL, fCmin:
0.325 mg/mL, half-life: 1 h; at 87% protein binding these levels
correspond to a total Cmax of 40 mg/mL and Cmin of 2.5 mg/mL)
[31,32], and vancomycin 1 g every 12 h combined with nafcillin
2 g every 4 h. The vancomycin dose was chosen to simulate
a total drug trough of 1520 mg/mL to conform to the recent
vancomycin dosing guidelines stating target trough values should
fall within this range for most infections.  The nafcillin dose
is the standard dose used to treat serious staphylococcal
infections. Model simulations involving two drugs with different
half-lives were performed using a previously validated method.
 All models were done in duplicate to ensure reproducibility.
Samples (approximately 1 mL each) were drawn from each
model at 0, 1, 2, 4, 8, 24, 28, 32, 48, 56, and 72 h, serially diluted
in 0.9% sodium chloride, and plated on TSA plates for
quantification with a lower limit of detection of 2 log10 CFU/
mL. Antibiotic carryover was accounted for using serial dilutions.
The total reduction in log10 CFU/mL was determined by plotting
time-kill curves of the number of remaining organisms over the 72
hour time period. Bactericidal activity was defined as $ 3 log10
CFU/mL (99.9%) reduction in colony count from initial
inoculum. The time to achieve a 99.9% bacterial load reduction
(T99.9) was determined by linear regression (r2$0.95) or by visual
Pharmacokinetic samples were obtained, through the injection
port over 72 h for verification of target antibiotic concentrations.
Concentrations of vancomycin were measured by bioassay
utilizing Bacillus subtilis ATCC 6633.  Nafcillin concentrations
were measured by bioassay utilizing Micrococcus luteus ATCC 9341
as previously described.  The elimination half-lives (t1/2), areas
under the curve (AUC), peaks (fCmax), and troughs (fCmin) were
determined using WinNonlin PK/PD modeling software program
(Pharsight, Cary, NC, USA).
Development of resistance was evaluated at multiple time points
throughout the simulation at 24, 48, and 72 hours. One hundred
mL samples from each time point were plated on Mueller Hinton
agar plates containing 3 fold the MIC of the respective antibiotic
to assess the development of resistance. Plates were then examined
for growth after 48 hours of incubation at 35uC. The MIC for
observed growth was measured by broth microdilution. In
Figure 3. Activity of vancomycin and nafcillin alone and in combination in the PK/PD model against the hVISA strain with a nafcillin
MIC of 4 mg/mL (R1629). Data are presented as a graph of the mean bacteria remaining vs. time with error bars at the sampling points
representing 1 standard deviation from the mean. GC = Growth Control, NAF = Nafcillin, VAN = Vancomycin.
Figure 4. Activity of vancomycin and nafcillin alone and in combination in the PK/PD model against one MSSA isolate (SNL9) (A)
and one MRSA isolate (SNL96) (B). Data are presented as a graph of the mean bacteria remaining vs. time with error bars at the sampling points
representing 1 standard deviation from the mean. GC = Growth Control, NAF = Nafcillin, VAN = Vancomycin.
addition, growth from quantification plates at 24, 48, and 72 h
was subjected to MIC testing by broth microdilution.
Overall activity of regimens over the 72 hour period was
compared by calculating the area under the killing curve (AUC)
for each regimen using SigmaPlot software (version 11.1, Systat
Software Inc., San Jose, CA). The AUCs were then compared
using analysis of variance (ANOVA) with Tukeys post-hoc test.
Additionally, changes in log10 CFU/mL at the 72 hour time point
were compared using ANOVA with Tukeys post-hoc test. All
statistical comparisons were done with IBM SPSS Statistics
(Version 19.0, SPSS Inc., Chicago, IL). A P value of #0.05 was
For the 25 isolates of hVISA the vancomycin MIC50
(MIC50 = median MIC) and MIC90 (MIC90 = MIC at which
90% of strains were inhibited) were both 2 mg/mL (range 12 mg/
mL) and the nafcillin MIC50 and MIC90 were 128 and 256 mg/
mL respectively (range 4256 mg/mL). In time kill analysis the
addition of nafcillin to vancomycin showed synergy in 92% of
strains (23/25) with the remaining strains showing indifference. An
example time kill graph is displayed in figure 1.
All 5 hVISA strains selected for the PK/PD model had
a vancomycin MIC of 2 mg/mL, and, as described above, were
selected to represent the full range of nafcillin MIC available in
the cohort of 25 strains. All 5 strains displayed synergy in time
kill analysis. Both MRSA isolates used in the PK/PD model
had a vancomycin MIC of 1 mg/mL while one (MRSA SNL96)
had a nafcillin MIC of 32 mg/mL and the other (MRSA
SNL98) had a nafcillin MIC of 128 mg/mL. Likewise, both
MSSA isolates used had a vancomycin MIC of 1 mg/mL and
both had a nafcillin MIC of 0.5 mg/mL. Pharmacokinetic
analysis demonstrated the accuracy of the models performed
with PK parameters within 10% of targeted values. The free
peak (fCmax), trough (fCmin), and half-life of vancomycin
obtained in the PK/PD model were (all data presented as
Results are presented as mean 6 standard deviation.
mean 6 standard deviation throughout) 32.863.2 mg/mL,
8.262.3 mg/mL (corresponding with a total drug trough mean
of 16.4 mg/mL), and 6.260.5 h respectively. The free peak
(fCmax) and half-life of nafcillin were 4.860.7 mg/mL and
In the PK/PD model against the 5 hVISA strains (figures 2 and
3); vancomycin alone demonstrated similar activity between
isolates resulting in maximal killing of between 23 log10 CFU/
mL from baseline between 24 and 32 hours and thereafter
showing regrowth. Likewise, nafcillin alone displayed generally
similar activity against 4 of the 5 isolates displaying 12 log10
CFU/mL kill in the first 4 hours, followed by regrowth (figure 2).
Against the isolate with the lowest nafcillin MIC (MIC = 4 mg/mL;
figure 3) nafcillin displayed similar activity to vancomycin alone
(P = 0.54) resulting in maximal kill at 8 hours followed by slow
regrowth over the remaining 64 hours of the experiment. Several
changes in MIC were noted with vancomycin alone and with
nafcillin alone. For 3 of the 5 strains (those with baseline nafcillin
MICs of 16, 64, and 128 mg/mL) the vancomycin MIC changed
from 2 mg/mL to 4 mg/mL by 72 hours. Three of the 5 strains
also showed a one dilution step increase in nafcillin MIC by 72
hours (strains with an original nafcillin MIC of 4, 64, and 128 mg/
mL changed to 8, 128, and 256 mg/mL respectively). Although all
of these MIC changes were confirmed through replicates of testing
as described above, it is generally considered that one dilution step
is within the standard margin of error for an MIC test and
therefore these results should be interpreted with caution. Against
all 5 isolates, the combination of vancomycin and nafcillin was
superior to either drug alone overall (P#0.004 for all comparisons)
and at the 72 hour time point (P#0.001 for all comparisons). The
time to bactericidal activity for the combination was 7.760.4 h
while no individual drug achieved bactericidal activity alone. No
changes in MIC were observed for the combination of vancomycin
and nafcillin for either drug or any of the 5 strains over the 72
hour period. Two of 5 strains (R1915 and R2729) were killed to
detection limits (2 log10 CFU/mL) at 72 hours.
Against the MRSA and MSSA strains (Figure 4), the activity of
vancomycin alone was similar across all 4 strains, and indeed was
similar to the activity of vancomycin alone against the hVISA
strains. The difference, however, was that no changes in MIC were
observed for any of the MRSA and MSSA over the 72 hours for
either vancomycin or for nafcillin with any experimental regimen.
As expected, the activity of nafcillin alone against MRSA was
minimal while the activity of nafcillin against MSSA was
significantly better than that of vancomycin alone overall
(P#0.003) and at the 72 hour time point (P#0.001). The
combination of vancomycin and nafcillin was significantly better
than either vancomycin or nafcillin alone against both MRSA
(P#0.001 for overall activity, P#0.005 at the 72 hour time point)
and both MSSA (P#0.009 for overall activity, P#0.016 at the 72
hour time point). For MRSA the time to bactericidal activity for
the combination was 6.360.5 h while no individual drug achieved
bactericidal activity alone. Against MSSA the time to bactericidal
activity for the combination was 10.262.2 h and was 14.360.8 h
for nafcillin alone. Vancomycin alone was not bactericidal against
MSSA. Kill to detection limits at 72 hours was achieved for one
MSSA strain (SNL9) and neither MRSA strain. All AUC values
from PK/PD model experiments are displayed in table 1.
Staphylococcus aureus remains a common cause of a variety of
infections causing high morbidity and mortality.  Throughout
much of the world, including such places as the United States,
several European countries, much of South America, Australia,
and Japan, the prevalence of methicillin resistance in S. aureus is
quite high.  Due to the high prevalence in these areas, beta
lactams cannot be used empirically and therefore vancomycin is
generally considered the standard of care for suspected
staphylococcal infections. This level of vancomycin selective pressure has
led to an increasing problem with decreased susceptibility to
vancomycin in S. aureus, including hVISA.
Consistent with previous investigations, we found vancomycin
to be mostly ineffective against hVISA strains, failing to produce
bactericidal activity and resulting in MIC elevations in several
strains. [28,37,38] However, when vancomycin was combined
with nafcillin, strong enhancement of bacterial killing was
observed for all 5 strains examined. This is in spite of nafcillin
concentrations being below the MIC of the organism for most
(strain with nafcillin MIC 4 mg/mL) to all (all remaining strains) of
the dosing interval. This is a peculiar because the time drug
concentrations are above the MIC of the organism is what drives
antibacterial activity for beta lactams.  One potential reason
for this observation is that the hindrance of peptidoglycan
synthesis, in this case by vancomycin, can reduce beta lactam
resistance.  Though this is the case, the exact mechanism of
synergy between beta lactams and glycopeptides has not been
precisely elucidated to date.
Likewise, against MRSA and MSSA we observed this same
enhancement in killing with the combination of vancomycin and
nafcillin. Against all of these strains, similar to hVISA, vancomycin
alone failed to produce bactericidal activity while nafcillin was not
effective at all against MRSA and was quite effective alone against
MSSA, as expected. This strong activity of nafcillin against MSSA
accounts for the fact that, while the combination of vancomycin
and nafcillin was better than either drug alone, the magnitude of
the difference was much less for MSSA than it was for both MRSA
and hVISA strains that were not susceptible to beta lactams.
The observation of synergy between beta lactams and
vancomycin is not new, though the combinations have only been
described once previously using simulated human
pharmacokinetics.  In that study vancomycin was combined with cefazolin
against 2 MRSA strains, one hVISA strain, and one vancomycin
intermediate S. aureus strain and the combination of the 2 drugs
was found to improve overall activity, but not bacterial density at
the end of the experiments (48 and 72 hours). One potential
reason for this disparity is that the two studies used different
vancomycin dosing regimens. In their investigation, they used
every 8 hour dosing as opposed to every 12 hours, however, given
that the trough values in both studies were similar and that massive
increases in AUC/MIC ratio have been shown not to result in
improvements in vancomycin activity,  this seems unlikely to
be the reason. Another difference was that they used a different
beta lactam agent (cefazolin vs. nafcillin) which may have been less
active than nafcillin against S. aureus. A final possibility is that the
starting inoculums differed between the two studies, with a higher
starting inoculum in the present investigation. Given that both
glycopeptides and beta lactams are subject to an inoculum effect,
[28,29] where the killing effect of an antibiotic is lessened as the
inoculum of organism increases, this could have led to the
diminished activity of vancomycin in this study compared to their
investigation where vancomycin displayed much more kill alone.
In conclusion, the combination of vancomycin and nafcillin
significantly improved overall antibacterial activity, rate of
bacterial killing, and remaining organism burden at 72 hours
against MSSA, MRSA, and hVISA isolates over either drug alone.
This improvement was seen even when the isolate was very
resistant to nafcillin (susceptible breakpoint for S. aureus is MIC
#2 mg/mL) and the nafcillin time above MIC was zero. These
data support the continued evaluation of this combination, and its
potential role in the treatment of S. aureus infections.
A portion of this work was presented at the 49th Interscience Conference
on Antimicrobial Agents and Chemotherapy (ICAAC), San Francisco, CA,
USA 2009 (Abstract E-1451) and the 50th ICAAC, Boston, Massachusetts,
USA 2010 (Abstract A1-1350). The author would like to thank Dr. Michael
J. Rybak, Anti-Infective Research Laboratory, Wayne State University,
Detroit, MI, USA for the kind contribution of the isolates used in this work.
Conceived and designed the experiments: SNL. Performed the
experiments: SNL. Analyzed the data: SNL. Contributed reagents/materials/
analysis tools: SNL. Wrote the paper: SNL.
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