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Antimicrobial Agents and Chemotherapy-2016-Oshima-e02056-16.full
Antimicrobial Agents and Chemotherapy-2016-Oshima-e02056-16.full
Antimicrobial Agents and Chemotherapy-2016-Oshima-e02056-16.full
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January 2017 Volume 61 Issue 1 e02056-16 Antimicrobial Agents and Chemotherapy aac.asm.org 1
Oshima et al. Antimicrobial Agents and Chemotherapy
FIG 1 Survival of mice infected with 3 ⫻ 107 (clinical isolate 1) or 1 ⫻ 108 (clinical isolate 2) CFU of MEPM-resistant
RESULTS
High-dose MEPM treatment protects mice from pneumonia induced by MEPM-
resistant P. aeruginosa. The survival of the mice was observed for 7 days after
infection. As shown in Fig. 1, the survival of mice treated with high-dose MEPM was
significantly restored relative to that of untreated mice in the model of pneumonia and
FIG 3 Numbers of viable bacteria in lungs and of inflammatory cells in BALF. Mice were inoculated with 3 ⫻ 106 CFU of MEPM-resistant
P. aeruginosa. At 38 h after infection (24 h after the first dose of MEPM), mice treated four times a day with MEPM at 500 mg/kg or 150
significantly lower than those in both the untreated group and the group receiving 150
mg/kg MEPM four times a day. In the comparison between the group receiving 500
mg/kg MEPM four times a day and the untreated group, viable bacterial counts in the
lungs were 2.61 ⫾ 0.33 log CFU/ml versus 5.11 ⫾ 0.30 log CFU/ml, respectively (P ⬍
0.001), with clinical isolate 1 and 3.56 ⫾ 0.15 log CFU/ml versus 5.28 ⫾ 0.19 log CFU/ml,
respectively (P ⬍ 0.001), with clinical isolate 2. In the comparison between the two
groups treated with MEPM four times a day (the 500-mg/kg and 150-mg/kg groups),
viable bacterial counts in the lungs were 2.61 ⫾ 0.33 log CFU/ml versus 4.28 ⫾ 0.31 log
CFU/ml, respectively (P ⬍ 0.01), with clinical isolate 1 and 3.56 ⫾ 0.15 log CFU/ml versus
4.41 ⫾ 0.15 log CFU/ml, respectively (P ⬍ 0.05), with clinical isolate 2. However, there
was no significant difference between the untreated group and the group receiving
150 mg/kg MEPM four times a day.
High-dose MEPM treatment inhibits the pulmonary inflammation induced by
MEPM-resistant P. aeruginosa. Quantitative lung cultures and granulocyte counts in
bronchoalveolar lavage fluid (BALF) were evaluated for the control group (Fig. 3A and
B) over time. The number of bacteria in the lungs and the number of granulocytes in
BALF at the evaluation point (38 h after infection) were higher than the corresponding
numbers at the previous two time points (6 and 14 h after infection). The number of
inflammatory cells in BALF (Fig. 3C) was evaluated 38 h after infection for all groups in
the pneumonia model. The group receiving 500 mg/kg MEPM four times a day showed
significantly lower numbers of inflammatory cells in BALF than the untreated group
(5.44 ⫾ 0.11 log cells/ml versus 6.13 ⫾ 0.13 log cells/ml, respectively [P ⬍ 0.01]).
However, there was no difference between the group receiving 150 mg/kg MEPM four
times a day and the untreated group.
Histopathological examination. As shown in Fig. 4, histopathological analysis of
lungs stained with hematoxylin and eosin (HE) at 38 h after infection revealed that
treatment with 500 mg/kg MEPM four times a day was more effective than treatment
with 150 mg/kg MEPM four times a day.
Kinetics of free drug concentrations in the ELF of mice administered high-dose
MEPM. Free MEPM concentrations in epithelial lining fluid (ELF) were evaluated for
both infected and uninfected mice and were found to not exceed 16 g/ml even when
a dose of 500 mg/kg was administered to uninfected mice (Fig. 5A). Conversely, in
infected mice, free drug concentrations exceeded 16 g/ml for 85 min after the
administration of MEPM at 500 mg/kg; however, they never exceeded 16 g/ml after
the administration of MEPM at 150 mg/kg. The percentage of the time that free drug
concentrations remained above the MIC (fTAM) in infected mice receiving 500 or 150
mg/kg MEPM four times a day was 23.6% or 0%, respectively (Fig. 5B). These data
suggest that increased penetration of the airway by high-dose MEPM might underlie
the protection against pulmonary infection with MEPM-resistant P. aeruginosa.
DISCUSSION
The efficacy of -lactam drugs, including MEPM, is generally predicted by compar-
ison between the MIC for the causative bacterium and the concentration of unbound
drug in the extracellular fluid. The efficacy of MEPM can be discussed by considering
the unbound drug concentration in the extracellular fluid to be equivalent to the total
concentration of the drug in plasma, because the plasma protein binding rate of MEPM
in humans is as low as 2%. In contrast, the plasma protein binding rate of MEPM is as
high as 10% in mice; thus, the efficacy of MEPM should be discussed by taking this
difference into consideration. In addition, a number of reports have suggested that the
antibacterial drug concentration at the topical infected site reflects the efficacy of the
FIG 5 Free MEPM concentrations in ELF at 5, 15, 30, and 60 min after the administration of four doses
of MEPM to noninfected mice (A) and to mice infected with 3 ⫻ 106 CFU of MEPM-resistant P. aeruginosa
(B). For noninfected mice, free MEPM concentrations in the ELF did not reach 16 g/ml even when MEPM
was administered at 500 mg/kg. For infected mice, free MEPM concentrations in the ELF were higher than
16 g/ml for 85 min in the group treated with 500 mg/kg of MEPM; however, they never exceeded 16
g/ml in the group treated with 150 mg/kg of MEPM.
drug. Although the topical infected site is indicative of intracellular substances in the
lungs, in pneumonia, because of the presence of extracellular respiratory tract patho-
gens, including P. aeruginosa, bacteria can exist on the alveolar surface as well. Thus,
the drug concentration in ELF is also an important factor that needs to be considered
in discussing therapeutic efficacy (20–22). Hence, we measured the changes in MEPM
concentrations in ELF over time in order to evaluate their association with therapeutic
efficacy.
A noninfection mouse model and a mouse model of P. aeruginosa infection were
used to measure MEPM concentrations in ELF. Because the migration of MEPM into ELF
was found to be poor in the noninfection model, the concentration of unbound MEPM
in ELF did not exceed 16 g/ml in either group receiving MEPM four times a day (at 150
or 500 mg/kg per dose). In the P. aeruginosa infection model, however, for the group
TABLE 1 fTAMs of MEPM dose regimens for humans and mice with pneumonia caused
by MEPM-resistant P. aeruginosa
Host Dose regimen fTAM (%)a
Human 2 g, 3 times/day 25.1
Mouse 500 mg/kg, 4 times/day 24.8
Human 1 g, 3 times/day 15.4
Mouse 150 mg/kg, 4 times/day 17.4
afTAM, the percentage of the time that free drug concentrations remain above the MIC.
CLSI guidelines (42). Isolates stored in Trypticase soy broth with 10% glycerol stocks maintained at ⫺80°C
at Nagasaki University Hospital were spread on LB agar (Sigma, Tokyo, Japan) and were incubated
overnight at 37°C under 5% CO2 prior to use in the experiments. The mechanism of MEPM resistance was
3.5 m; inside diameter, 4.6 mm; length, 20 mm; Nihon Waters K.K., Tokyo, Japan) with methanol–5 mM
sodium dihydrogen phosphate (pH 7.0) (3:17) as the mobile phase delivered at 1.0 ml/min. The HPLC
system (LC-2010C; Shimadzu Co., Kyoto, Japan) was controlled by a Class VP workstation (Shimadzu), and
the wavelength for MEPM detection was 300 nm. Five-point standard curves (0.1 to 10 g/ml) were
linear, with an r2 of ⬎0.98. The lower limit of quantitation was 0.1 g/ml. The concentration of MEPM in
ELF was calculated as follows: (concentration in BALF) ⫻ [(urea in serum)/(urea in BALF)]. Calculating the
fTAM in ELF is the same as calculating the fTAM in plasma [see the “Pharmacokinetic (PK) studies and
determination of dosing regimen” paragraph above]. Serum samples were also collected just before BAL
fluid was obtained from the same mice used for the urea assay.
Urea assay. The rate of decline in NADH levels induced by NH3 in the samples was measured. Urea
was hydrolyzed by urease to produce NH3. The NH3 produced reacted with ␣-ketoisohexanoic acid and
NADH by the action of leucine dehydrogenase to form leucine and NAD. The rate of decline in NADH
levels at this point was measured optically, and the urea content in the sample was calculated by
subtracting the rate of decline resulting from the endogenous ammonia reaction.
Statistical analysis. All data were analyzed by using Prism 5 (GraphPad Software) and were
ACKNOWLEDGMENTS
This work was supported by a grant from the Program for Nurturing Global Leaders
in Tropical and Emerging Communicable Diseases, Graduate School of Biomedical
Sciences, Nagasaki University, Nagasaki, Japan.
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