Detection and Inhibition of Bacterial Cell–Cell Communication - Bacterial Pathogenesis: Methods and Protocols - page 60

0.25

0.20

Control
QSI 10 µg/mL
QSI 20 µg/mL

0.15

0.10

0.05

0.00

7 8 22

Time after inoculation (h)

Fig. 2. Elastase activity of Pseudomonas aeruginosa in the presence and absence
of a quorum-sensing inhibitor (QSI). Samples were collected at 7, 8, and 22 h post
inoculation in the presence of a quorum-sensing antagonist at 10 and 20 μg/mL. Elastase
activity was determined on cell-free supernatants from the different time points and
was normalized based on total protein concentration of the supernatants.

Growth is followed spectrophotometrically and aliquots removed at various time
points for the preparation of cell-free supernatants.
2. Cell-free supernatants are prepared by centrifugation of culture aliquots for 5 min
at 6000 × g followed by filtration of the supernatant through a 0.22-μm filter (Pall
acrodisc supor syringe filter) and collection of the supernatant in a sterile 1.5-mL
Eppendorf tube.
3. Cell-free supernatants can be frozen and stored at -20°C until used.

3.3.2. Detection of AI-2 Activity/Inhibition
To test for the presence of AI-2 activity, grow the V. harveyi reporter strain
BB170 for 16 h at 30°C with shaking in AB medium.
1. Dilute cells 1:5000 into 30°C pre-warmed AB medium.
2. Prepare microtiter plates by adding 90 μL of the diluted suspension to wells of
96-well microtiter plates and then add 10 μL of either medium (for negative
controls) or supernatants to be tested.
3. Incubate the microtiter plates at 30°C with shaking at 175 rpm. Hourly determi-
nations of the total luminescence as well as cell density are quantified.
4. Report activity as the percentage of activity obtained from V. harveyi BB152
cell-free supernatant.
5. To test for inhibition of AI-2-mediated cell-cell signaling, add the diluted monitor
strain to the plates as indicated in steps 1-3 , where the supernatant added is
from the positive control strain, V. harveyi BB152. Add the antagonist from
the stock solution, as extracted in Subheading 3.1.1 . Again, it is advisable
to first perform MIC determinations of the inhibitor extract as described in