ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2010, p. 804–810 Vol. 54, No.

2
0066-4804/10/$12.00 doi:10.1128/AAC.01190-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Comparison of the Activity of a Human Simulated, High-Dose, Prolonged

Infusion of Meropenem against Klebsiella pneumoniae Producing the
KPC Carbapenemase versus That against Pseudomonas aeruginosa
in an In Vitro Pharmacodynamic Model䌤
Catharine C. Bulik,1 Henry Christensen,1 Peng Li,1 Christina A. Sutherland,1
David P. Nicolau,1,2 and Joseph L. Kuti1*

Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI

Center for Anti-Infective Research and Development, Hartford Hospital, Hartford, Connecticut,1 and Department of Medicine,
Division of Infectious Diseases, Hartford Hospital, Hartford, Connecticut2
Received 21 August 2009/Returned for modification 10 October 2009/Accepted 26 November 2009

We have previously demonstrated that a high-dose, prolonged-infusion meropenem regimen (2 g every 8 h

[q8h]; 3-hour infusion) can achieve 40% free drug concentration above the MIC against Pseudomonas aerugi-
nosa with MICs of <16 ␮g/ml. The objective of this experiment was to compare the efficacy of this high-dose,
prolonged-infusion regimen against carbapenemase-producing Klebsiella pneumoniae isolates with the efficacy
against P. aeruginosa isolates having similar meropenem MICs. An in vitro pharmacodynamic model was used
to simulate human serum concentrations. Eleven genotypically confirmed K. pneumoniae carbapenemase
(KPC)-producing isolates and six clinical P. aeruginosa isolates were tested for 24 h, and time-kill curves were
constructed. High-performance liquid chromatography (HPLC) was used to verify meropenem concentrations
in each experiment. Meropenem achieved a rapid >3 log CFU reduction against all KPC isolates within 6 h,
followed by regrowth in all but two isolates. The targeted %fT>MIC (percent time that free drug concentra-
tions remain above the MIC) exposure was achieved against both of these KPC isolates (100% fT>MIC versus
MIC ⴝ 2 ␮g/ml, 75% fT>MIC versus MIC ⴝ 8 ␮g/ml). Against KPC isolates with MICs of 8 and 16 ␮g/ml that
did regrow, actual meropenem exposures were significantly lower than targeted due to rapid in vitro hydrolysis,
whereby targeted %fT>MIC was reduced with each subsequent dosing. In contrast, a >3 log CFU reduction
was maintained over 24 h for all Pseudomonas isolates with meropenem MICs of 8 and 16 ␮g/ml. Although KPC
and P. aeruginosa isolates may share similar meropenem MICs, the differing resistance mechanisms produce
discordant responses to a high-dose, prolonged infusion of meropenem. Thus, predicting the efficacy of an
antimicrobial regimen based on MIC may not be a valid assumption for KPC-producing organisms.

Enteric Gram-negative bacteria are frequently responsible
for serious nosocomial infections, such as bacteremia and
pneumonia, in critically ill hospitalized patients. The increase
in resistance among these bacteria, most notably Klebsiella spp.
and Escherichia coli, due to the production of extended-spec-
trum ␤-lactamases (ESBLs) has led to the frequent use of
carbapenem antibiotics. Carbapenems are considered by many
to be the agents of choice for serious infections caused by
ESBL-producing bacteria that may also display resistance to
fluoroquinolones and aminoglycosides (23). Resistance to car-
bapenems among these bacteria remains remarkably rare in
most countries. However, Klebsiella spp. that produce serine-
based carbapenemase enzymes, referred to as Klebsiella pneu-
moniae carbapenemases (KPCs), have been identified in re-
cent years (5).
The first KPC (KPC-1) was detected in a K. pneumoniae
strain isolated in North Carolina in 2001 (28). Subsequently,
KPC-2 and KPC-3 were identified from isolates from the mid-
Atlantic and New England regions of the United States (1, 26,
27). Since the discovery of these initial KPC enzymes, numer-
* Corresponding author. Mailing address: Center for Anti-Infective
Research and Development, Hartford Hospital, 80 Seymour Street, Hart-
ford, CT 06102. Phone: (860) 545-3612. Fax: (860) 545-3992. E-mail:
jkuti@harthosp.org.
䌤
Published ahead of print on 7 December 2009.
ous other variants (KPC-4 to KPC-7) have been reported in-
ternationally, as well as in other bacterial species, such as E.
coli, Enterobacter spp., and Pseudomonas aeruginosa (9, 11, 13,
21, 25). Surveillance studies of the New York area have un-
covered trends in the emergence of this resistance genotype
(13). When comparing isolates gathered from the years 1999,
2001, and 2006, K. pneumoniae isolates have shown an alarm-
ing decline in antimicrobial susceptibility. The percentage of
isolates that are resistant to carbapenems rose to 25%, and
KPCs were detected in 38% of isolates. The number of mul-
tidrug-resistant K. pneumoniae isolates also increased to 59%
in 2006, with 20% being resistant to all ␤-lactams, fluoroquino-
lones, and amikacin. These decreases in antimicrobial sus-
ceptibility have limited the number of therapeutic choices.
In some studies, only polymyxins and tigecycline retain con-
sistent susceptibility against KPC-producing Klebsiella spp.
(2, 11).
While most KPC-producing K. pneumoniae isolates have
carbapenem MICs above the breakpoint for susceptibility,
some isolates can have imipenem and meropenem MICs as low
as 1 to 2 ␮g/ml, and a number of them have meropenem MICs
of 8 ␮g/ml and 16 ␮g/ml (5, 9, 21, 25). Standard dosages of
meropenem (1 g every 8 h [q8h]) and imipenem (500 mg q6h)
would not be expected to have clinical utility against organisms
with MICs of 8 to 16 ␮g/ml.

804
VOL. 54, 2010 HUMAN SIMULATED MEROPENEM VERSUS KPC ISOLATES
Due to the lack of new antimicrobials with activity against
multidrug-resistant, Gram-negative bacteria, thought leaders
have suggested the application of pharmacodynamic principles
to currently available antibiotics as our best opportunity to
address these difficult-to-treat organisms (6). For ␤-lactams, a
combination of administering higher doses and extending the
infusion leads to increases in the time that free drug concen-
trations remain above the MIC (fT⬎MIC). In animal infection
models, carbapenems require at least 40% fT⬎MIC to achieve
maximal bactericidal activity against P. aeruginosa (6). A clin-
ical pharmacodynamic study of patients with respiratory tract
infections also identified a similar target (54% fT⬎MIC) as
predictive of microbiological success (16). Higher dosages (2 g
q8h) and prolonged infusions (3 h) have been applied to mero-
penem in numerous reports to improve the probability
of achieving optimal bactericidal exposure against resistant
Pseudomonas aeruginosa, Acinetobacter spp., and Burkholderia
cepacia (7, 19) isolates. In pharmacokinetic simulation models,
a 2-g q8h dose regimen of meropenem, with each dose infused
over 3 h, has demonstrated a high probability of attaining 40%
fT⬎MIC against organisms with meropenem MICs of up to 16
␮g/ml (12). In theory, this dosage should maintain bactericidal
activity against all organisms with MICs of up to 16 ␮g/ml,
including KPC-producing isolates. Given the scarcity of new
antibiotic options against Klebsiella spp. that harbor the KPC
carbapenemase and its inevitable spread across hospitals as a
cause of serious infections, the objective of this study was to
assess the performance of a pharmacodynamically optimized
meropenem regimen against KPC-producing Klebsiella isolates
compared with P. aeruginosa isolates having the same mero-
penem MIC.
(This study was presented at the 49th Interscience Confer-
ence on Antimicrobial Agents and Chemotherapy, San Fran-
cisco, CA, 2009.)
MATERIALS AND METHODS
Bacterial strains and susceptibility testing. Clinical K. pneumoniae isolates
with KPC genotypes, all Hodge test positive and blaKPC positive (courtesy of
Steve Jenkins, Mount Sinai Medical Center), and clinical P. aeruginosa isolates
from our institution (Hartford Hospital) were screened for inclusion. Mero-
penem MICs for all isolates were determined by broth microdilution in triplicate
according to Clinical Laboratory Standards Institute (CLSI) methodology, and
the modal MIC was used for calculations in all experiments. Based on mero-
penem MICs, 11 K. pneumoniae isolates with the following MICs were selected:
2 ␮g/ml (n ⫽ 1); 8 ␮g/ml (n ⫽ 3); 16 ␮g/ml (n ⫽ 3); 32 ␮g/ml (n ⫽ 3); and 64
␮g/ml (n ⫽ 1). Six P. aeruginosa isolates with MICs of 8 ␮g/ml (n ⫽ 3) and 16
␮g/ml (n ⫽ 3) were included for comparison.
Modified Hodge test. To confirm that the six P. aeruginosa isolates did not
produce a carbapenemase, the modified Hodge test was applied as previously
described (22). Briefly, the indicator organism, Escherichia coli ATCC 25922, at
a turbidity of a 0.5 McFarland standard, was used to inoculate the surface of a
Mueller-Hinton agar plate. After the plate was allowed to dry at room temper-
ature for 3 to 10 min, a 10-␮g meropenem susceptibility disk was placed in the
center of the plate. Each of the six P. aeruginosa test isolates was then heavily
streaked from the edge of the disk to the plate periphery on individual plates.
Klebsiella pneumoniae ATCC BAA-1706 was used as the negative control, and
Klebsiella pneumoniae ATCC BAA-1705 was used as the positive control. The
plates were incubated overnight at 35°C in ambient air for 16 to 24 h. After
incubation, the plates were examined for a cloverleaf type of indentation at the
intersection of the test organism and the E. coli ATCC 25922 within the zone of
inhibition of the carbapenem susceptibility disk.
Antibiotics. Meropenem for intravenous injection (50 mg/ml; Merrem; Astra-
Zeneca Pharmaceuticals) was obtained from the pharmacy at Hartford Hospital
(lot PH0082; expiration date, February 2011; Novaplus).
805
Simulated meropenem exposure. The free drug concentrations for a mero-
penem dose of 2 g q8h administered as a 3-hour infusion over a 24-hour period
(i.e., three doses) were simulated. Concentration-time profiles were based on
median parameter estimates from a population pharmacokinetic analysis of
meropenem in infected patients (15). Median estimates were entered into Win-
NonLin Professional version 5.0.1 (Pharsight Corporation, Mountain View, CA)
to simulate steady-state exposure. Protein binding was applied by multiplying
each concentration by 0.97 over the 24-hour period. The simulated peak con-
centration for each dose at 3, 11, and 19 h was 38.4 ␮g/ml, and the trough at 0,
8, 16, and 24 h was 3.04 ␮g/ml.
In vitro pharmacodynamic model. The in vitro pharmacodynamic model used
in the study has been described previously (10). Each experiment consisted of
three independent models (two experimental treatment models and one growth
control model), which ran simultaneously for each isolate. The models were
Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI
placed in a 37°C circulating water bath for optimal temperature control, and
magnetic stir bars were used in each model to ensure adequate mixing of con-
tents. Cation-adjusted Mueller-Hinton broth (CAMHB) (Becton, Dickinson and
Company, Sparks, MD) was used as the bacterial growth medium in all in vitro
model experiments. The volume of growth medium used for meropenem in each
of the models was approximately 1,000 ml, based on the half-life and flow rate
calculations. Three independent starting inoculums of 106 CFU/ml were set up
from an overnight culture of the test isolate for all model experiments. To ensure
that the bacteria were in logarithmic growth phase prior to antimicrobial expo-
sure, antibiotic experiments were started 0.5 h after inoculation of bacteria into
models.
In order to simulate the rising concentrations of a 3-hour infusion, meropenem
was injected into the model in 30-min intervals from time zero to 3 h while the
infusion pump was off. After 3 h, fresh CAMHB was continuously pumped into
each of the models by a peristaltic pump (Masterflex L/S, model no. 7524-40;
Cole-Parmer Instrument Company) at a rate that simulated the distribution
half-life of meropenem obtained from the above-mentioned study. At 4.5 h, the
rate of the peristaltic pump was slowed to simulate the terminal half-life of
meropenem. At 8 and 16 h, the peristaltic pump was stopped, and the dosing
process was repeated for the 24-h experiment.
To assess bacterial density over time, samples were obtained from each model
and serially diluted in normal saline. Aliquots of each diluted sample were plated
for quantitative culture. The volume of the aliquots used for bacterial counts was
either 10 or 100 ␮l, depending on the dilution used. Trypticase soy agar plates
(100-mm diameter) with 5% sheep blood were used for quantitative determina-
tions. After 18 to 24 h of incubation at 37°C, the change in log10 CFU/ml over the
24-h interval was calculated, and time-kill curves were constructed by plotting
log10 CFU/ml against time. The lower limit of detection for bacterial density was
101 CFU/ml. MIC testing in triplicate was repeated for select KPC isolates to
determine whether an MIC change had occurred over the 24-h experiment.
Antibiotic concentration determinations. Samples of CAMHB taken from
each of the treatment models were assayed for meropenem at 0, 0.5, 1, 1.5, 2, 2.5,
3, 4.5, 6, 7, 8, 9.5, 11, 14, 16, 17.5, 19, 22, and 24 h. All samples were immediately
stored at ⫺80°C until analyses. Samples were analyzed by a high-performance
liquid chromatography (HPLC) method as described previously (8), validated for
broth. The meropenem HPLC assay was linear (r ⬎ 0.997) over a concentration
range from 0.25 to 40 ␮g/ml. The intraday quality control samples (n ⫽ 10 each)
of 0.5 and 30 ␮g/ml had a percentage coefficient of variation (%CV) of 3.46 and
4.85, respectively. The interday quality control samples (n ⫽ 26) of 0.5 and 30
␮g/ml had a %CV of 3.09 and 3.16, respectively.
RESULTS
Meropenem exposures. Prior to bacterial experiments (i.e.,
in bacterium-free models), we developed a specific dosing ad-
ministration strategy that consistently resulted in the targeted
meropenem concentration-time profile depicted in Fig. 1.
However, in infected models, meropenem concentration-time
profiles differed depending on the baseline MIC and isolate
being tested. This resulted in achieving intended fT⬎MIC ex-
posures for all P. aeruginosa isolates but only 3 of the 11
KPC-producing K. pneumoniae isolates (Table 1). The overall
desired %fT⬎MIC of ⱖ40% was achieved for each dosing
interval only in the K. pneumoniae isolate with an MIC of 2
␮g/ml and two of the three isolates with a meropenem MIC of
8 ␮g/ml. Rapid in vitro hydrolysis of the meropenem ␤-lactam
806 BULIK ET AL. ANTIMICROB. AGENTS CHEMOTHER.

DISCUSSION

Carbapenems have historically been considered the agents

of choice to treat infections caused by multidrug-resistant En-
terobacteriaceae, but the increasing prevalence of isolates pro-
ducing KPC carbapenemase has compromised the effective-
ness of this class (21). Therapeutic options for infections
caused by KPC-producing Klebsiella are scarce, with only the
polymyxin antibiotics and tigecycline demonstrating consistent
susceptibility. However, these antibiotics are not without their
own limitations, thus additional treatment options are still
needed for the successful treatment of these organisms. Since

Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI

a number of KPC-producing K. pneumoniae isolates can have
meropenem MICs in the susceptible (ⱕ4 ␮g/ml), intermediate
(8 ␮g/ml), or low-level resistant (16 ␮g/ml) ranges, this study
sought to determine whether a human simulated, high-dose,
prolonged-infusion meropenem regimen could produce and
FIG. 1. Targeted concentration-time profile for 2-g q8h mero- maintain a bactericidal effect comparable with that of P. aerugi-
penem regimen, as a 3-hour infusion.
nosa having the same meropenem MICs. The free drug expo-
sure from a meropenem dose of 2 g q8h, with each dose
infused over 3 h, was selected for this study because this dosing
regimen has been used clinically (7, 20) and is not greater than
ring was presumed to be the cause for lower-than-intended the maximum approved daily dose recommended by the pack-
meropenem concentrations, because in many of the experi- age insert, which has demonstrated safety and tolerability, and
ments, no meropenem peak was noted on HPLC after dosing importantly, because previous Monte Carlo simulation studies
to simulate the second and third intervals. Importantly, while have demonstrated that this dosing regimen has a high prob-
targeted meropenem fT⬎MIC exposures during the first 8-h ability of achieving at least 40% fT⬎MIC at MICs of up to 16
dose interval were achieved against all KPC isolates with MICs ␮g/ml, which is the exposure that produced maximal bacteri-
of 8 ␮g/ml, drug exposure fell short of the target during the cidal activity against P. aeruginosa in murine infection models
second and third 8-h dose intervals. A similar decline with each (12, 19).
subsequent dosing was also noted for two of the three KPC Despite administering meropenem in dosages consistent
isolates with MICs of 16 ␮g/ml. As a result, we were unable to with achieving the targeted exposure for each isolate, this op-
achieve our targeted exposure of 47% fT⬎MIC during any of timized regimen failed to maintain bactericidal activity against
the experiments for these isolates. 9 of the 11 studied KPC-producing K. pneumoniae isolates.
Bactericidal activity. The average bacterial density of the Although rapid bactericidal exposure was observed over the
starting inoculum was (1.96 ⫾ 0.47) ⫻ 106 CFU/ml and (7.54 ⫾ first 6 h against all KPC isolates, all but two (MICs of 2 ␮g/ml
0.52) ⫻ 105 CFU/ml for K. pneumoniae and P. aeruginosa
isolates, respectively. KPC control isolates grew to (3.06 ⫾
1.18) ⫻ 108 at 24 h, while P. aeruginosa control isolates grew to
TABLE 1. Meropenem MIC, targeted fT⬎MIC exposure, and
(3.29 ⫾ 2.35) ⫻ 107. Figure 2 summarizes the time-kill curves achieved fT⬎MIC exposure for KPC-producing K. pneumoniae
for K. pneumoniae isolates, defined by baseline meropenem and P. aeruginosa isolates tested
MIC. Meropenem achieved a rapid ⬎3 log CFU reduction
Achieved fT⬎MIC
within 6 h against all K. pneumoniae isolates, followed by re- for each dosing
MIC Targeted
growth in all but two isolates. Targeted fT⬎MIC exposure was Isolate Organism
(␮g/ml) fT⬎MIC (%) interval (%)
achieved during each of the 8-h dosing intervals for these 0-8 h 8-16 h 16-24 h
isolates (KPC isolate 377, MIC ⫽ 2 ␮g/ml; KPC isolate 328A,
377 K. pneumoniae 2 100 100 100 100
MIC ⫽ 8 ␮g/ml). Although a fT⬎MIC exposure greater than
328A K. pneumoniae 8 69 69 72 78
40% was achieved for each of the dosing intervals against KPC 351 K. pneumoniae 8 69 69 62 50
isolate 351 (MIC, 8 ␮g/ml), this isolate regrew back to control 354 K. pneumoniae 8 69 68 56 3
levels. In contrast, meropenem was observed to have rapid and 353 K. pneumoniae 16 47 38 59 0
357 K. pneumoniae 16 47 12 0 0
sustained bactericidal activity (ⱖ3 log reduction) against all P.
360 K. pneumoniae 16 47 0 0 0
aeruginosa isolates tested (MIC, 8 ␮g/ml and 16 ␮g/ml) over 334 K. pneumoniae 32 16 0 0 0
the 24-h experiment. Figures 3a and b summarize the resultant 368 K. pneumoniae 32 16 0 0 0
time-kill curves against P. aeruginosa. 375 K. pneumoniae 32 16 0 0 0
Changes in meropenem MIC. Five of the 11 KPC isolates 361 K. pneumoniae 64 0 0 0 0
1030 P. aeruginosa 8 69 75 78 78
were selected to have the MIC for the organisms that regrew 1038 P. aeruginosa 8 69 94 100 82
after exposure to meropenem retested. These isolates had 1177 P. aeruginosa 8 69 75 78 78
baseline MICs of 8 ␮g/ml, 16 ␮g/ml (n ⫽ 2), and 32 ␮g/ml (n ⫽ 762 P. aeruginosa 16 47 50 63 56
2). All isolates demonstrated increased meropenem MICs to 896 P. aeruginosa 16 47 50 63 56
988 P. aeruginosa 16 47 47 50 38
⬎32 ␮g/ml after 24 hours of exposure to meropenem.
 Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI

FIG. 2. Mean bacterial density over 24 hours for all KPC-producing Klebsiella pneumoniae isolates grouped by meropenem MIC. The thick solid black
lines represent the control groups, the broken lines represent different KPC isolates at each MIC, and the lower limit of detection is set at 101 CFU/ml.

807
808 BULIK ET AL.
FIG. 3. Mean bacterial density over 24 hours for all Pseudomonas aeruginosa isolates grouped by meropenem MIC. The solid black lines
represent the control groups, the broken lines represent different P. aeruginosa isolates at each MIC, and the lower limit of detection is set at 101
CFU/ml.
and 8 ␮g/ml) regrew back to control isolate levels by 12 to 16 h.
The simulated meropenem dosing regimen aimed at achieving
100% fT⬎MIC against the organism with an MIC of 2 ␮g/ml.
This exposure was accomplished, resulting in bactericidal ac-
tivity within 1 h of dosing that was retained throughout the
24-h period (Table 1). The second isolate that responded to
this meropenem regimen had an MIC of 8 ␮g/ml; 69% fT⬎MIC
was targeted and attained against this isolate, resulting in a
bactericidal response that, albeit slower, was maintained over
the 24-h experiment (Table 1).
Unexpectedly, the remaining two KPC isolates having a
meropenem MIC of 8 ␮g/ml regrew to control levels (Fig. 2).
Moreover, a substantial loss of meropenem was observed in
the models as analyzed by HPLC, likely due to hydrolysis of
the carbapenem structure. While the targeted exposure (69%
fT⬎MIC) was achieved against all isolates with an MIC of 8
␮g/ml during the first 8-h dosing interval, meropenem expo-
sure began to decline with subsequent doses for KPC isolates
351 and 354. Although one of these isolates (KPC isolate 351)
was still exposed to meropenem concentrations that remained
above the MIC for 40% fT⬎MIC, this organism regrew. KPC
isolate 354 (MIC of 8 ␮g/ml) was exposed to meropenem
concentrations for 56% and 3% of the dosing interval after the
second and third dosages, respectively. Not surprisingly, this
isolate also regrew to control levels and had a 24-h repeat MIC
that was now ⬎32 ␮g/ml. A similar observation was noted for
the three isolates with meropenem MICs of 16 ␮g/ml. Targeted
exposure was 47% fT⬎MIC, which was not achieved during
any of the dosing intervals, with the majority of the exposures
being 0% fT⬎MIC. All three of these isolates regrew to con-
trol levels at 24 h, and repeat MICs increased to ⬎32 ␮g/ml in
the two isolates that were retested. As expected, all KPC iso-
lates with baseline meropenem MICs of 32 ␮g/ml and 64 ␮g/ml
regrew to control levels, as targeted exposure was 16%
fT⬎MIC and 0% fT⬎MIC, respectively. However, despite ad-
ministering the same amount of meropenem to the models as
in all other experiments, detectable meropenem concentra-
ANTIMICROB. AGENTS CHEMOTHER.
Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI
tions above the MIC for these organisms were not observed.
Because of these observations, these experiments were re-
peated independently several times on different days, with con-
sistent findings in each.
Antimicrobial regimens are usually driven by the phenotypic
profile rather than the genotypic profile of the organisms.
Previous work by our group with a murine thigh infection
model has demonstrated that regardless of the presence of an
ESBL, which demonstrated a significant in vitro inoculum ef-
fect on cefepime MIC, the desired bactericidal effect was still
achieved as long as the cefepime exposure was ⱖ40% fT⬎MIC
(18). This would suggest that in organisms with increased
meropenem MICs, resistance mechanisms, such as the produc-
tion of ␤-lactamases, can be overcome with the same fT⬎MIC
exposure as that required for wild-type isolates. Clearly, this
was not the case in the current study against the KPC-produc-
ing K. pneumoniae isolates, where two of the three isolates at 8
␮g/ml regrew to control levels despite achieving ⬎40%
fT⬎MIC for the first and second dosing intervals. Our obser-
vations may reflect an in vitro/in vivo paradox, since previous
work conducted by Craig and colleagues (3) suggested that the
presence of KPCs in Enterobacteriaceae had no impact on the
T⬎MIC required to produce bacteriostasis with doripenem,
meropenem, or imipenem in the murine infection model; how-
ever, many of those isolates had much lower meropenem MICs
than those of the clinical isolates from our study. Similarly, we
observed bacteriostatic effects when examining a human sim-
ulated doripenem dose of 1 g q8h administered as a 4-h infu-
sion against these same clinical isolates (doripenem MICs, 4 to
32 ␮g/ml) in the murine thigh infection model. In contrast, this
doripenem regimen has previously demonstrated consistent 2
to 3 log reductions in P. aeruginosa CFU with doripenem MICs
of up to 8 ␮g/ml, and some variable killing against isolates with
MICs as high as 16 ␮g/ml (4).
Against the six clinical P. aeruginosa isolates with MICs of 8
and 16 ␮g/ml, the simulated meropenem regimen achieved a
⬎3 log reduction in CFU over the first 8 to 12 h, with minimal
VOL. 54, 2010 HUMAN SIMULATED MEROPENEM VERSUS KPC ISOLATES
regrowth of the isolates by 24 h (Fig. 3). Furthermore, free
meropenem concentrations remained above the MIC for at
least the targeted 69% and 47% of the dosing interval, respec-
tively. Importantly, isolates from these experiments did begin
to regrow by 24 h, but colony counts were still well below that
of the control, and were significantly different from the KPC
isolates at MICs of 8 and 16 ␮g/ml.
Nevertheless, it is notable that the killing profile of this
simulated meropenem regimen differed between the KPC and
P. aeruginosa isolates even though the baseline MICs were
similar. This divergence may be in part due to the differing
resistance mechanisms of these species. The predominant
mechanisms of carbapenem resistance within P. aeruginosa iso-
lates arise from the interaction between efflux systems, ampC
expression, and loss of outer membrane porins (deletions in
oprD expression) (17, 24). Furthermore, we demonstrated that
the six P. aeruginosa isolates tested did not produce a carba-
penemase enzyme according to the modified Hodge test. In
contrast, meropenem resistance among these K. pneumoniae
isolates is due predominantly to the production of the serine
carbapenemase. Importantly, other resistance mechanisms, such
as the production of additional ␤-lactamases (i.e., ESBLs),
ampC expression, and loss of OmpK36, can contribute to car-
bapenem resistance in Klebsiella spp. (14). A limitation to our
experiment is the absence of data about detailed resistance
mechanisms of these Klebsiella isolates other than their known
expression of blaKPC. It has been demonstrated that many of
the clinical KPC isolates from New York City produce several
␤-lactamases, in addition to the serine carbapenemase (9).
These additional resistance mechanisms may have been re-
sponsible for the discordance in meropenem effect among the
three KPC isolates with an MIC of 8 ␮g/ml. Further work is
required to determine which resistance mechanisms may have
contributed to increasing meropenem MICs among the tested
KPC isolates during the experiment.
In conclusion, carbapenemase-producing K. pneumoniae can
have a meropenem MIC similar to that of P. aeruginosa but
exhibits markedly different bactericidal responses to a pharma-
codynamically optimized regimen, due to the different geno-
typic resistance mechanisms. Despite the proposed effective-
ness of the optimized meropenem regimen against organisms
with MICs of up to 16 ␮g/ml, a reliable reduction in the
bacterial density is not seen in the presence of serine-based
carbapenemases. While the phenotypic profile of an organism
has historically driven the efficacy of the antimicrobial regimen,
these assumptions may no longer be valid in the case of car-
bapenemase-producing K. pneumoniae isolates.
ACKNOWLEDGMENTS
This study was funded by a research grant from AstraZeneca Phar-
maceuticals, Inc., Wilmington, DE. D.P.N. and J.L.K. have received
research support and consultancy support and are members of the
speakers’ bureau for AstraZeneca.
We acknowledge Stephen Jenkins for kindly providing KPC-positive
K. pneumoniae isolates.
REFERENCES
1. Bradford, P. A., S. Bratu, C. Urban, M. Visalli, N. Mariano, D. Landman,
J. J. Rahal, S. Brooks, S. Cebular, and J. Quale. 2004. Emergence of
carbapenem-resistant Klebsiella species possessing the class A carbapenem-
hydrolyzing KPC-2 and inhibitor-resistant TEM-30 beta-lactamases in New
York City. Clin. Infect. Dis. 39:55–60.
809
2. Bratu, S., P. Tolaney, U. Karumudi, J. Quale, M. Motty, S. Nichani, and D.
Landman. 2005. Carbapenamase-producing Klebsiella pneumoniae in Brook-
lyn, NY: molecular epidemiology and in vitro activity of polymyxin B and
other agents. J. Antimicrob. Agents 56:128–132.
3. Craig, W. A., S. Kethireddy, D. R. Andes, T. Stamstad, K. Marchilo, J.
Asbeck, and R. N. Jones. 2008. Impact of KPCs on the in vivo activity of
three carbapenems in the neutropenic mouse-thigh infection model. Ab-
str. 48th Intersci. Conf. Antimicrob. Agents Chemother., abstr. A-029.
4. Crandon, J. C., C. C. Bulik, and D. P. Nicolau. 2009. In vivo efficacy of 1- and
2-gram human simulated prolonged infusions of doripenem against Pseudo-
monas aeruginosa. Antimicrob. Agents Chemother. 53:4352–4356.
5. Deshpande, L. M., P. R. Rhomberg, H. S. Sader, and R. N. Jones. 2006.
Emergence of serine carbapenemases (KPC and SME) among clinical
strains of Enterobacteriaceae isolated in the United States Medical Cen-
ters: report from the MYSTIC program (1999–2005). Diagn. Microbiol.
Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI
Infect. Dis. 56:367–372.
6. Drusano, G. L. 2003. Prevention of resistance: a goal for dose selection for
antimicrobial agents. Clin. Infect. Dis. 36(Suppl. 1):S42–S50.
7. Drusano, G. L., F. Sorgel, J. Quinn, B. Mason, and D. Melnick. 2005. Impact
of pharmacodynamic dosing of meropenem on emergence of resistance
during treatment of ventilator-associated pneumonia (VAP): a prospective
clinical trial. Abstr. 45th Intersci. Conf. Antimicrob. Agents Chemother.,
abstr. K-127.
8. Elkhaili, H., S. Niedergang, D. Pompei, L. Linger, K. Leveque, and F. Jehl.
1996. High-performance liquid chromatographic assay for meropenem in
serum. J. Chromatogr. B. 686:19–26.
9. Endimiani, A., A. M. Hujer, F. Perez, C. R. Bethel, K. M. Hujer, J. Kroeger,
M. Oethinger, D. L. Paterson, M. D. Adams, M. R. Jacobs, D. J. Diekema,
G. S. Hall, S. G. Jenkins, L. B. Rice, F. C. Tenover, and R. A. Bonomo. 2009.
Characterization of blaKPC-containing Klebsiella pneumoniae isolates de-
tected in different institutions in the eastern USA. J. Antimicrob. Che-
mother. 63:427–437.
10. Garrison, M. W., K. Vance-Bryan, T. A. Larson, J. P. Toscano, and J. C.
Rotschafer. 1990. Assessment of effects of protein binding on daptomycin
and vancomycin killing of Staphylococcus aureus by using an in vitro phar-
macodynamic model. Antimicrob. Agents Chemother. 34:1925–1931.
11. Hussein, K., H. Sprecher, T. Mashiach, I. Oren, I. Kassis, and R. Finkel-
stein. 2009. Carbapenem resistance among Klebsiella pneumoniae isolates:
risk factors, molecular characteristics, and susceptibility patterns. Infect.
Control Hosp. Epidemiol. 30:666–671.
12. Kuti, J. L., P. K. Dandekar, C. H. Nightingale, and D. P. Nicolau. 2003. Use
of a Monte Carlo simulation to design an optimized pharamcodynamic
dosing strategy for meropenem. J. Clin. Pharmacol. 43:1116–1123.
13. Landman, D., S. Bratu, S. Kochar, M. Panwar, M. Trehan, M. Doymaz, and
J. Quale. 2007. Evolution of antimicrobial resistance among Pseudomonas
aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in Brooklyn,
NY. J. Antimicrob. Chemother. 60:78–82.
14. Landman, D., S. Bratu, and J. Quale. 2009. Contribution of OmpK36 to
carbapenem susceptibility in KPC-producing Klebsiella pneumoniae. J. Med.
Microbiol. 58(Pt 10):1303–1308.
15. Li, C., J. L. Kuti, C. H. Nightingale, and D. P. Nicolau. 2006. Population
pharmacokinetic analysis and dosing regimen optimization of meropenem in
adult patients. J. Clin. Pharmacol. 46:1171–1178.
16. Li, C., X. Du, J. L. Kuti, and D. P. Nicolau. 2007. Clinical pharmacodynamics
of meropenem in patients with lower respiratory tract infections. Antimi-
crob. Agents Chemother. 51:1725–1730.
17. Livermore, D. M. 2001. Of Pseudomonas, porins, pumps and carbapenems. J.
Antimicrob. Chemother. 47:247–250.
18. Maglio, D., C. Ong, M. A. Banevicius, Q. Geng, C. H. Nightingale, and D. P.
Nicolau. 2004. Determination of the in vivo pharmacodynamic profile of
cefepime against extended-spectrum-beta-lactamase-producing Escherichia
coli at various inocula. Antimicrob. Agents Chemother. 48:1941–1947.
19. Mattoes, H. M., J. L. Kuti, G. L. Drusano, and D. P. Nicolau. 2004. Opti-
mizing antimicrobial pharmacodynamics: dosage strategies for meropenem.
Clin. Ther. 26:1187–1198.
20. Nicasio, A. M., K. J. Eagye, D. P. Nicolau, E. Shore, M. Palter, J. Pepe, and
J. L. Kuti. 2009. Pharmacodynamic-based clinical pathway for empiric anti-
biotic choice in patients with ventilator-associated pneumonia. J. Crit. Care
[Epub ahead of print.] doi:10.1016/j.jcrc.2009.02.014.
21. Nordmann, P., G. Cuzin, and T. Naas. 2009. The real threat of Klebsiella
pneumoniae carbapenemase-producing bacteria. Lancet Infect. Dis. 9:228–
236.
22. Noyal, M. J. C., G. A. Menezes, B. N. Harish, S. Sujatha, and S. C. Parija.
2009. Simple screening tests for detection of carbapenemases in clinical
isolates of nonfermentative Gram-negative bacteria. Indian J. Med. Res.
129:707–712.
23. Paterson, D. L., and R. A. Bonomo. 2005. Extended-spectrum beta-lacta-
mases: a clinical update. Clin. Microbiol. Rev. 18:657–686.
24. Quale, J., S. Bratu, J. Gupta, and D. Landman. 2006. Interplay of efflux
system, ampC, and oprD expression in carbapenem resistance of Pseudomo-
nas aeruginosa clinical isolates. Antimicrob. Agents Chemother. 50:1633–
1641.
810 BULIK ET AL.
25. Rhomberg, P. R., L. M. Deshpande, J. T. Kirby, and R. N. Jones. 2007.
Activity of meropenem as serine carbapenemases evolve in US medical
centers: monitoring report from the MYSTIC program (2006). Diagn. Mi-
crobiol. Infect. Dis. 59:425–432.
26. Smith Moland, E., N. D. Hanson, V. L. Herrera, J. A. Black, T. J. Lockhart,
A. Hossain, J. A. Johnson, R. V. Goering, and K. S. Thomson. 2003. Plasmid-
mediated, carbapenem-hydrolysing ␤-lactamase, KPC-2, in Klebsiella pneu-
moniae isolates. J. Antimicrob. Chemother. 51:711–714.
27. Woodford, N., P. M. Tierno, K. Young, L. Tysall, M. I. Palepou, E. Ward,
ANTIMICROB. AGENTS CHEMOTHER.
R. E. Painter, D. F. Suber, D. Shungu, L. L. Silver, K. Inglima, J. Kornblum,
and D. M. Livermore. 2004. Outbreak of Klebsiella pneumoniae producing a
new carbapenem-hydrolyzing class A ␤-lacatamase, KPC-3, in a New York
medical center. Antimicrob. Agents Chemother. 48:4793–4799.
28. Yigit, H., A. M. Queenan, G. J. Anderson, A. Domenich-Sanchez, J. W.
Biddle, C. D. Steward, S. Alberti, K. Bush, and F. C. Tenover. 2001. Novel
carbapenemase-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-re-
sistant strain of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 45:
1151–1161.
Downloaded from http://aac.asm.org/ on February 2, 2015 by GAZI UNIVERSITESI