2
150 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Hennard et al.
final concentration used (generally 64 µg/mL). A range of
concentrations was obtained by successively diluting by a
factor of 2 this first solution. To 1 mL of each diluted solution
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9 mL of agar medium (to which 25 mg/L Mg2+, 50 mg/L Ca2+
and the nutrient medium, M u¨ ller-Hinton or succinate, were
added) maintained at 50 °C was added, and the mixture was
poured in a Petri dish. The dishes were inoculated with 1 µL
7
of bacterial suspension (10 cells/mL) using a Denley multi-
points instrument, then incubated 18 h at 37 °C.
(
b) An tibiotic Assa ys in Liqu id Med ia . Each compound
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40-850.
to be tested was solubilized in water at a concentration twice
as high as the highest final concentration used (generally 64
µg/mL). 200 µL of this solution was placed in the first row of
an ELISA plate. The successive dilutions by a factor 20 were
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solution + 100 µL of the culture medium). The same instru-
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°
C and monitored measuring the OD550nm using a microplate
head. After comparison of the measurements before and after
incubation, the concentration for which the increase in the OD
was less than 10% with respect to the blank without antibiotic
was determined.
For the MIC assays, the concentration of adducts NPS (4),
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quinolone equivalents.
Gr ow th Kin etics. Ten milliliters of bacterial suspension
at the end of the exponential phase of growth was centrifuged
for 4 min at 12000 rpm, then resuspended in succinate culture
medium to an OD600nm between 0.02 and 0.04. To 2 mL of this
suspension placed in disposable spectrophotometric cuvettes
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(the medium devoid of bacteria being taken as the reference
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3
to the ligand.
All these experiments were performed in sterile conditions.
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acetate membranes of 0.2 µm porosity. The cuvettes and the
stirring magnets were soaked in ethanol overnight then dried
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Ack n ow led gm en t. We thank Prof. Maurice Goeld-
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Pharmacy of Strasbourg) for his kind help in providing
us the use of his laboratory for performing some of the
radiolabeling experiments and for his very stimulating
discussions. We also thank Mr. Roland Graff for deter-
mination of the NMR spectra. We gratefully thank Dr.
Fran c¸ ois-Xavier Bernard for stimulating discussions
and Ms. Nadine Bertheau and her assistants (Rh oˆ ne
Poulenc Rorer) for their expert technical assistance.
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