Transcript in an In Vitro Pharmacokinetic Bacteremia Model

Carbapenem Activity Against Acinetobacter calcoaceticus-baumanii complex
(ACBC) in an In Vitro Pharmacokinetic Bacteremia Model (PKM)
Eric G Sahloff, Pharm.D., Diane M. Cappelletty, Pharm.D., & Steven J. Martin, Pharm.D., BCPS, FCCM
College of Pharmacy, University of Toledo, 2801 W. Bancroft St.,
Toledo, Ohio 43606
ABSTRACT
Our ICU has experienced an outbreak of multi-drug resistant AC, with
isolates susceptible only to carbapenems and aminoglycosides. Imipenem
(IC) has become an empiric choice for therapy in nosocomial pneumonia and
bacteremia in our ICU. Meropenem (M) has become a financially attractive
option to IC. The purpose of this study was to compare the bactericidal
effects of IC to M against AC clinical isolates using an in vitro PKM.
Four AC clinical isolates were tested in a PKM simulating M 1 g q8 hrs and IC
500 mg q6 hrs over 48 hrs. All isolates were susceptible to M and IC with
MICs of 1 g/ml and 0.25-0.5 g/ml, respectively. Samples were taken from
the model at several time points over the 48 hrs to evaluate killing and PK.
MIC and MBCs were determined at time 0, 24 hrs, and 48 hrs. The rates of
bacterial killing and area under the bactericidal curves (AUBC) were
compared. Isolates were screened for resistant subpopulations (RS) using
antibiotic-containing medium at baseline, 24 and 48 hr.
For M and IC, greater than 99.9% kill was seen by 3-4 hrs in 3/4 isolates,.
However, regrowth occurred by 24 hours for all isolates against M. For IC,
regrowth occurred in 2/4 isolates by 24 hours and between 30-48 hrs for the
remaining 2 isolates. AUBC was lower (greater extent of kill) for IC in 3/4
isolates at 12, 24, and 48 hours. Low-level RS were identified at baseline,
while high-level resistance was noted for regrowth at 48 hr.
M and IC had similar rates of killing with both agents being bactericidal in 3/4
isolates. IC had superior duration of bacterial killing compared to M against
AC. Despite initial bactericidal activity, significant regrowth occurred for all
isolates against both drugs by 48 hours. This suggests carbapenem
monotherapy may select for resistant populations or induce resistance and
coul lead to potential drug failure. The presence of resistant subpopulations
may support the use of combination therapy in AC infections.
Each drug was tested alone against each isolate. Drug was administered into
the model at desired peak concentrations through an access port. Fresh sterile
medium pumped into and drug-containing medium flows out of the model at a
rate simulating the half-life achieved by human pharmacokinetics. All were run
in duplicate at 37o C.
Pharmacokinetic/Pharmacodynamic Resistance Determinations
Serial samples (1.0 ml) were drawn to measure colony counts and drug
concentrations To prevent antibiotic carryover, samples were centrifuged, the
supernatant removed, and the bacteria resuspended in 1 ml normal saline.
Samples were plated by spiral plating techniques. Antibiotic-containing agar
plates at 2x and 4x MIC were evaluated at 24 and 48 hrs to detect and quantify
the emergence of resistance. Endopoints were rate and extent of killing, and
the development and frequency of resistance (regrowth). MIC/MBCs were
performed on any regrowth for both agents to determine if resistance was drugor class-specific. Drug concentrations will be determined by bioassay.
Isolate 9
Isolate 9
8
Imipenem Run 1A
Imipenem Run 2A
Imipenem Run 1B
Imipenem Run 2B
7
6
6
5
5
4
4
Isolate
4
7
9
10
Drug
Meropenem
Im ipenem
Meropenem
Im ipenem
Meropenem
Im ipenem
Meropenem
Im ipenem
Meropenem Run 1B
Meropenem Run 2B
3
3
2
2
1
1
0
0
2
4
6
8 10 1224
30
36
42
48
2
4
6
8 10 1224
30
36
42
48
Time (hrs)
Time (hrs)
RESULTS
Resistant Subpopulations (% of Total P opulation
24 hr
48 hr
2x M IC
4x M IC
2x M IC
4x M IC
< 0.01
NG
NR
<0.01
NG
NG
<0.01
NG
<0.01
<0.01
NR
<0.01
<0.01
NG
<0.01
NG
0.14
<0.01
NR
<0.01
NG
NG
<0.01
NG
<0.01
NG
NR
<0.01
<0.01
NG
NR
<0.01
Meropenem Run 2A
8
7
Isolate 10
Table 1. Frequency of Resistant Subpopulations
Meropenem Run 1A
Meropenem
8
Imipenem
7
6
MIC/MBC were unchanged from
baseline when evaluated from
samples directly from the model or
from growth on drug-free agar at 24
and 48 hr wfor Isolates 9 and 10 for
both M and IC
No growth was noted on antibioticcontaining medium at 2x or 4x MICs
for Isolates 9 or 10 at 24 or 48 hrs.
5
4
3
2
1
0
2
4
6
8 10 1224
Time (hrs)
30
36
42
48
Isolate 4
CONCLUSIONS
BACKGROUND
Acinetobacter spp. Has been documented as the cause of numerous
nosocomial outbreaks and has been associated with increased lengths of
stay in the ICU as well as increased mortality in infected ICU patients. In our
institution the number of isolates cultured nearly doubled from 1998-2000
compared to 2001 with over one-third being multi-drug resistant.
Susceptibility to commonly used antibiotics including cefepime
piperacillin/tazobactam, and ampicillin/ sulbactam decreased while
imipenem/cilistatin and tobramycin susceptibility remained stable.
Antimicrobial resistance makes drug selection difficult. Inappropriate
antimicrobial selection can lead to increased mortality, while exposure to
broad spectrum antimicrobials is associated with development of resistance.
Initial selection of antimicrobials at optimal dosing may have a significant
effects on patient outcome antimicrobial resistance. Carbapenems alone or
in combination with other antimicrobials are considered to be a therapy-ofchoice for Acinetobacter infections. The purpose of this study was to
compare the bactericidal effects of imipenem/cilistatin (IC) and meropenem
(M) against AC clinical isolates using an in vitro PK bacteremia model.
Meropenem 3/4 runs
Meropenem 1/4 runs
Imipenem
8
7
6
5
4
Table 2. MIC/MBC for Meropenem and Imipenem
Against Isolate 4 After Exposure to Meropenem
3
2
1
0
2
4
6
8 10 1224
30
36
42
48
MIC/MBC (mcg/ml)
48 hr.
24 hr.
48 hr. antibiotic
48 hr.
Isolate Model
Drug
Baseline drug-free model
plate
drug-free
plate
sample
(4x
plate
MIC)
Meropenem
1/1
1/>16
>16/ND 128/ND
128/ND
A
Im ipenem
0.5/0.5
1/1
>16/ND >128/ND >128/ND
4
Meropenem
1/1
NG
NG
NG
NG
B
Im ipenem
0.5/0.5
NG
NG
NG
NG
Time (hrs)
Isolate 7
METHODS
Organisms/Susceptibilities.
Four AC clinical isolates were tested. Baseline data collected for M an IC
against isolate included:
MIC/MBC by microtiter methodologies following NCCLS protocol.
Ppresence of resistant subpopulations on antibiotic containing plates
representing 2x and 4x the MIC. Frequency of resistance = the number of
organisms growing on the antibiotic-containing medium divided by number of
antibiotic-free agar plates.
Meropenem 5/6 runs
Meropenem 1/6 runs
Imipenem
8
7
6
5
4
3
2
1
0
In vitro pharmacokinetic/pharmacodynamic model.
Isolates were tested over 48 hours against M and IC in a glass one
compartment in vitro PK model emulating a bloodstream infection. Drug
regimens and human PK parameters to be simulated include:
1) Imipenem/cilistatin 500 mg every six hours simulating a peak
concentration of 40 mcg/ml and a half-life of 1 hour.
2) Meropenem 1 gram every 8 hours simulating a peak concentration of
60 mcg/ml and a half-life of 1 hour.
Resistant subpopulations were seen in all isolates for IC and M (Table 1).
Both agents provided bactericidal killing against 3/4 isolates. Regrowth
occurred earlier for M in 3/4 isolates with IC maintaining killing at or below
2 log for ≥ 30 hours in 2/4 isolates. Despite regrowth occurring, no
changes in MIC/MBC or growth on antibiotic-containing media were noted
for isolates 9 and 10 for either M or IC. In contrast, MIC/MBC changes did
occur with isolates 4 and 7. Tables 2 and 3 describe examples of these
changes. For isolate 4, selection of a resistant clone likely occurred
leading to regrowth in 3/4 model runs. High level resistance was seen
when sampling directly from the model as well as off the antibioticcontaining medium. One of 4 runs did not regrow and may be attributed to
lack of selection of a resistant clone. MIC/MBC changes occurring in
isolate 7 are more difficult to explain. Selection of a resistant clone
explains the regrowth occuring in 5/6 runs. No change in MIC/MBC
occurred when evauating growth from the 24 hr drug-free plates. Induction
of resistance with the addition of the antimicrobial likely occurred followed
by reversion back to the susceptible form with the removal of drug
pressure. Highly resistant MICs at 48 hours from samples from the model
are expected. Tolerance, low MICs and elevated MBCs, is seen when
evaluating growth from the 48 hr antibiotic-containing medium as well as
with one run of the 48 drug-free plates. A second example (model C) at 48
hrs. maintained high level resistance despite removal of drug pressure.
2
4
6
8 10 1224
30
36
42
48
Table 3. MIC/MBC for Meropenem and Imipenem
Against Isolate 7 After Exposure to Meropenem
MIC/MBC (mcg/ml)
48 hr.
24 hr.
48 hr. antibiotic
48 hr.
Isolate Model
Drug
Baseline drug-free model
plate
drug-free
plate
sample
(4x
plate
MIC)
Meropenem
1/1
2/2
>16/ND
4/64
128/ND
C
Im ipenem
0.5/0.5
1/1
>16/ND
1/32
>128/ND
7
Meropenem
1/1
2/2
>16/ND
4/64
4/32
D
Im ipenem
0.5/0.5
1/1
>16/ND
0.5/32
1/16
Time (hrs)
M and IC had similar rates of killing with both agents being bactericidal in
3/4 isolates. IC had superior duration of bacterial killing compared to M
against AC. Despite initial bactericidal activity, significant regrowth
occurred for all isolates against both drugs by 48 hours. This suggests
carbapenem monotherapy may select for resistant populations or induce
resistance and coul lead to potential drug failure. The presence of
resistant subpopulations may support the use of combination therapy in
AC infections.