Antimicrobial Assay for E. coli BW25113 ∆tolC::kan - Dose Response
There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs. ..more
BioActive Compounds: 1230
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Kim Lewis, Northeastern University
There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs.
E. coli is a gram negative bacterium with an outer membrane barrier and multi-drug resistant efflux pumps that can reduce the intracellular concentrations of potential antibiotics. TolC is an outer membrane porin that contributes to antimicrobial resistance; consequently TolC deletion mutants are expected to be more sensitive to antimicrobials. As a strategy to increase the hit rate for compounds with antimicrobial activity, we screened the NIH Small Molecule Repository using an E. coli strain with mutations in TolC, referred to as E. coli ΔTolC. In the primary screen (AID 573) compounds were tested at a final concentration of 10 uM in a standard bacterial growth assay using optical density as a measure of growth. Actives were defined as compounds that showed >=60.37% growth inhibition in the assay.
Hits from that screen were reordered from the NIH small molecule repository (MLSMR) for confirmation screening in dose response. One thousand two hundred and sixty five compounds that displayed the highest % inhibition and were available for re-order and re-tested in a dose-response screen at concentrations ranging from 0.2 - 100uM (1% final DMSO).
Antimicrobial Assay for E. coli BW25113 ∆TolC:kan : Protocol for 384-well HTS
Preparation of glycerol stock
-Pick a single colony from a plate of E. coli BW25113 ∆TolC:kan provided by Dr. Kim Lewis (Northeastern University, Boston, MA)
-Streak on Luria Broth (LB) agar plate containing 50 ug/ml Kanamycin; incubate overnight (o/n, 16-18 h) at 37C.
-Pour 15 ml LB broth medium + 50 ug/ml Kanamycin into sterile flask
-Transfer a single colony from the overnight streaked agar plate into the flask and incubate o/n in a shaking incubator at 37C, 250 rpm.
-Store aliquots of the culture in 30% glycerol at -80C.
Preparation of assay
-Inoculate LB broth medium containing 50 ug/ml Kanamycin with a small sample from the frozen stock and shake overnight (16-18 h ) in water bath or floor shaker at 250 rpm, 37C.
-Place culture at room temperature until 1.5 h before plating time
-Dilute the culture 1:10 in fresh LB broth medium containing 50 ug/ml Kanamycin and shake for 1.5hrs at 250rpm, 37C.
-After 1.5 h, the culture is then diluted 1:1000 in fresh LB broth medium containing 50 ug/ml Kanamycin and 25 ul is dispensed into 384 well plates (drugs have been added to plates at this point in a 5 ul volume *** ) using a Multidrop micro under a laminar flow hood.
-Place plates in PE bag stacked 2 high.
-Incubate plates at 37C for 20 h.
*** In the case of Z' plates: 5 ul medium/DMSO or Chloramphenicol/DMSO (at 30 uM final) is added with Biomek FX.
-Read optical density at 615 nm on Envision plate reader (Abs folder A615- 30 s shake, 384 general)
LB agar plate prep (100 ml)
-100 ml MilliQ water
-2 g LB broth (Fisher, BP1427-500)
-1.5 g LB agar (Fisher, BP1425-500)
-Autoclave at 121C for 15 min.
-Cool, add Kanamycin to a final concentration of 50 ug/ml, pour plates.
LB broth media (1L)
-1L MilliQ water
-20 g LB broth
-5 g NaCl (Sigma, S3014-500G)
-Autoclave at 121C for 15 min
-Add 50 mg Kanamycin
Kanamycin (Sigma, K1637-5G)
Agar plate stock: Stock was prepared at 5 mg/ml in MilliQ water and filter sterilized. Vials of 1 ml aliquots were frozen. Use 1 vial for each 100 ml of LB agar plates.
Type of plates used
384-well clear flat bottom w/lid, TCT plates (Fisher Corning#3701).
Data were analyzed using IDBS ActivityBase software. Dose response curves were analyzed using XLfit equation 205 for a four parameter logistic fit; the maximum and minimum values were fixed at 100% and 0%.
Possible artifacts in this assay include, but are not limited to, compounds that absorb light at 615 nm or that precipitate.
Outcome: Compounds that showed ≥20% inhibition in the assay at any concentration are Actives. Compounds with less than 20% inhibition at all concentrations are Inactives.
Activity Score ranks compounds based on potency and was calculated as ten times the negative log (base 10) of the molar IC50.
Data Table (Concise)