as an activator of the reporter assay. DNA sequencing of the
agrDgene can be utilized to identify the specificagrgroup a
strain belongs to; however, this will not confirm that the strain
is an AIP producer.- A sufficient number of AIP concentrations should be used to
generate a smooth EC 50 curve. A minimum of eight different
concentrations (0.1, 0.5, 1, 10, 50, 250, 500, and 1000 nM) is
recommended. N.B. the AIP added to the plate must be 20
times more concentrated. For example, to generate a final
concentration of 50 nM in the plate, 10μlofa1μM AIP
stock solution must be used. - High concentrations of solvents such as DMSO can affect the
luxreaction. For that reason, the DMSO concentration is
maintained constant in all samples (0.5%) in the microtiter
plate assay. - The 60 internal wells of a 96-well plate are used for samples.
This limits the loss of sample volume by dehydration that
occurs in the outside wells of the 96-well plate. Outside wells
should be filled with sterile media. If you are attempting to
quantify AIP, a standard curve must be generated using syn-
thetic AIP for each individual plate/experiment. - Chloramphenicol is not required to maintain the pAgrC(X)A
plasmid from this point onwards in the assay. - Plate readers from different manufacturers have different sen-
sitivities; therefore detection parameters should be optimized
to suit the specific equipment available. - We routinely use the software package Graphpad Prism for data
analysis.
References
- Chambers HF, Deleo FR (2009) Waves of
resistance:Staphylococcusaureusin the antibi-
otic era. Nat Rev Microbiol 7:629–641 - Clatworthy AE, Pierson E, Hung DT (2007)
Targeting virulence: a new paradigm for
antimicrobial therapy. Nat Chem Biol
3:541–548 - Williams P (2002) Quorum sensing: an
emerging target for antibacterial chemother-
apy? Expert Opin Ther Targets 6:257–274 - Chan WC, Coyle BJ, Williams P (2004) Viru-
lence regulation and quorum sensing in staph-
ylococcal infections: competitive AgrC
antagonists as quorum sensing inhibitors. J
Med Chem 47:4633–4641 - Novick RP, Geisinger E (2008) Quorum sens-
ing in staphylococci. Annu Rev Genet
42:541–564
6. Ji G, Beavis RC, Novick RP (1995) Cell density
control of staphylococcal virulence mediated
by an octapeptide pheromone. Proc Natl Acad
Sci U S A 92:12055–12059
7. Geisinger E, George EA, Chen J, Muir TW,
Novick RP (2008) Identification of ligand
specificity determinants in AgrC, theStaphylo-
coccusaureusquorum-sensing receptor. J Biol
Chem 283:8930–8938
8. Koenig RL, Ray JL, Maleki SJ, Smeltzer MS,
Hurlburt BK (2004) Staphylococcusaureus
AgrA binding to the RNAIII-agrregulatory
region. J Bacteriol 186:7549–7555
9. Ji G, Beavis R, Novick RP (1997) Bacterial
interference caused by autoinducing peptide
variants. Science 276:2027–2030 - Wright JS 3rd, Jin R, Novick RP (2005) Tran-
sient interference with staphylococcal quorum
Detection of AIPs Produced byS. aureus 95