Transcript Stenotrophomonas (Xanthomonas) maltophilia

In Vitro Activity of Trimethoprim/Sulfamethoxazole Against
Stenotrophomonas maltophilia (Sm) Using a Whole Blood Assay
Sandy J. Close, Pharm.D. and Steven J. Martin, Pharm.D., BCPS, FCCM
College of Pharmacy, University of Toledo, 2801 W. Bancroft St.,
Toledo, Ohio 43606
ABSTRACT
Background: S.maltophila is an opportunistic pathogen in the ICU. TMP/SMX is a
drug-of-choice for this organism, producing good clinical outcomes. Due to the
bacteristatic activity of TMP/SMX against S.maltophilia, it is difficult to use in vitro
testing to predict appropriate doses and dosing regimens for TMP/SMX. We
hypothesize the immune system plays an important role enhancing the activity of
TMP/SMX in vivo. The objective of this study was to determine if the addition of
single donor, whole anticoagulated human blood (the immune component) to time
kill or pharmacokinetic model assays would more accurately predict the in vivo
effect of TMP/SMX against S.maltophilia infections.
Methods: Time kill was performed with TMP/SMX alone (peak concentration 5/95
mg/ml), blood alone (75% v/v), T/S + blood, and control over 24 hours against 3
TMP/SMX-susceptible strains of S.maltophilia. The starting inoculum was 105
cfu/ml. A 2-compartment PK model was used to simulate a 7.5 mg/kg Q12H dose
(peak 5.67mg/ml, t1/2 9.6 hrs). PK models were run using control, blood,
TMP/SMX, and TMP/SMX + blood similar to time kill testing. All experiments were
performed in duplicate. Samples were obtained for all assays at 0, 6, 12 and 24
hours for colony counts and PK. MICs were performed pre/post model runs.
Results: Time kill and pharmacokinetic models produced similar results. TMP/SMX
alone was bacteristatic against all isolates. Blood alone produced a 2.5 log
reduction in colony count at 6 hrs with regrowth by 24 hrs. TMP/SMX + blood was
similar to blood at 6 hrs, however at 24 hrs the combination was synergistic with a
reduction of colony counts to lowest limit of detection (log 2.3) and no regrowth.
Conclusions: Immune system activity plays an important role in the activity of
TMP/SMX in clinical S.maltophilia infections. Traditional time kill or
pharmacokinetic model in vitro testing does not accurately predict TMP/ SMX in
vivo activity. Performing these in vitro tests with the addition of whole
anticoagulated human blood produces a more accurate simulation of in vivo
conditions.
BACKGROUND
METHODS
Stenotrophomonas maltophilia is a common opportunistic pathogen in
immunocompromised and critically ill patients. The majority of infections
caused by S. maltophilia are respiratory. While colonization with this
organism is common, the mortality rate is significant from true infections.
Three clinical S. maltophilia strains were used in testing. TMP-SMX
(TMP Sigma lot #68H1403, SMX Sigma lot #107H1111) were used
for all testing. Mueller-Hinton broth (Difco) and Mueller-Hinton agar
plates (Remel) were used to maintain the organisms, and for colony
counts. Microdilution broth MICs were determined according to the
guidelines of the National Committee for Clinical Laboratory
Standards.7 MICs were performed before all experiments, and
following the model runs for organisms with residual growth at 24
hours.
S. maltophilia is resistant to most beta-lactam, aminoglycoside, and
fluoroquinolone antibiotics. Trimethoprim/sulfamethoxazole (TMP/SMX) is
the drug-of-choice for this organism. There are few data regarding the
pharmacokinetics or optimal dosing strategies of TMP/SMX for these
infections. Dosages from 8 to 15 mg/kg/day (TMP) given in two to four
divided doses have been recommended, to treat serious infections but
without clinical validation.1-4
In vitro susceptibility tests for S. maltophilia have shown varying activity of
TMP/SMX both alone and in combination with other agents.4-6 Lack of
reliability of the results of in vitro testing makes application to the clinical
setting difficult. We hypothesized that adding a component of the human
immune system to traditional in vitro testing methods may help to more
accurately describe the activity of TMP/SMX against S. maltophilia.
Time kill assays were performed with TMP/SMX alone (peak
concentration 5/95 mg/ml), blood alone (75% v/v), TMP/SMX +
blood, and control over 24 hours against each of the three
TMP/SMX-susceptible strains of SM. The starting inoculum was 105
cfu/ml. Whole anticoagulated human blood was obtained from a
single donor on the day of each experiment.
An in vitro two compartment glass infection model was used to
simulate human pharmacokinetics. In this model, an outer “central”
compartment simulates central blood circulation, and an inner
“peripheral” or infection compartment simulates tissue infection. Each
organism was tested in the model. Models were given a 7.5 mg/kg
Q12H dose (peak 5.67mg/ml, t1/2 9.6 hrs). Models were run using
control, blood, TMP/SMX, and TMP/SMX + blood similar to time kill
testing. All experiments were performed in duplicate. Samples were
obtained for all assays at 0, 6, 12 and 24 hours for colony counts and
drug concentrations. MICs were performed pre/post model runs.
RESULTS
DISCUSSION
In both time kill testing and pharmacokinetic models, TMP/SMX alone did not exhibit significant activity
against three strains of S. maltophilia. The addition of whole anticoagulated human blood to TMP/SMX
did show bactericidal activity against these three organisms.
Time kill and pharmacokinetic models produced similar results. Time kill
curves and results from pharmacokinetic model runs for all three isolates
are shown in the figures below. TMP/SMX alone was bacteristatic against
all isolates. Blood alone produced at 2.5 log reduction in colony count at 6
hours with regrowth by 24 hrs. TMP/SMX + blood was similar to blood at 6
hours, however at 24 hours the combination was synergistic with a
reduction of colony counts to lowest limit of detection (log 2.3) and no
regrowth.
Isolate 1
Growth Control
Drug
Blood
Blood+Drug
Log cfu/ml
Log cfu/ml
Time Kill: All isolates
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1
0
lower lim it of detection
0
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One of the main disadvantages of using in vitro laboratory studies to evaluate the potential clinical
efficacy of a drug regimen is the inability to take into consideration the innate activity of the human
immune system against the organism causing infection. The use of TMP/SMX in treating S. maltophilia
infections is an excellent example of this problem, because while TMP/SMX often appears to fail in
traditional in vitro testing, it is known to work in the clinical setting.
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Growth Control
Drug
Blood
Drug+Blood
lower lim it of detection
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While TMP/SMX has been the drug of choice for S. maltophilia infections, little work has been done to
show appropriate dosing regimens. With increasing reports of S. maltophilia isolates that are resistant to
TMP/SMX, more work is important to evaluate the possibilities of more effective dosing regimens as
well as possible combination regimens with other agents that maintain activity against S. maltophilia
such as ticarcillin/clavulanate. The addition of whole anticoagulated human blood to our in vitro model
allows for a more accurate simulation of in vivo conditions under which these experiments can be
conducted.
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