Bookmark and Share
BioAssay: AID 617

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
_
   
 Tested Compounds
 Tested Compounds
All(1259)
 
 
Active(1230)
 
 
Inactive(29)
 
 
 Tested Substances
 Tested Substances
All(1259)
 
 
Active(1230)
 
 
Inactive(29)
 
 
 Related BioAssays
 Related BioAssays
AID: 617
Data Source: SRMLSC (E. coli tolc dose response)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-03-16
Modify Date: 2007-04-11

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 1230
Related Experiments
AIDNameTypeComment
552Antimicrobial HTS Assay for E. coli BW25113 (wild type)Screeningdepositor-specified cross reference
573Primary Antimicrobial Assay for E. coli BW25113 ∆tolC::kan Protocol for 384-well HTSScreeningdepositor-specified cross reference
635Antimicrobial Assay for E. coli BW25113 (wild type) mutant pool - DRConfirmatorydepositor-specified cross reference
638Antimicrobial Assay for E. coli BW25113 (wild type) - DRConfirmatorydepositor-specified cross reference
710Antimicrobial HTS Assay for E. coli BW25113 (wild type) - Expanded ScreenScreeningdepositor-specified cross reference
Description:
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
Award: X01MH077622


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).
Protocol
Antimicrobial Assay for E. coli BW25113 ∆TolC:kan : Protocol for 384-well HTS
Preparation of glycerol stock
Day 1:
-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.
Day 2:
-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.
Day3:
-Store aliquots of the culture in 30% glycerol at -80C.
Preparation of assay
Day 1:
-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.
Day 2:
-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.
Day 3:
-Read optical density at 615 nm on Envision plate reader (Abs folder A615- 30 s shake, 384 general)
Media Prep
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%.
Comment
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.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 modifierString
2EC50FloatμM
3Hill Slope modifierString
4Hill SlopeFloat
5Normalized Chi-squared modifierString
6Normalized Chi-squaredFloat
7Standard Deviation modifierString
8Standard DeviationFloat
9Inhibition @ 100 uMFloat%
10Inhibition @ 50 uMFloat%
11Inhibition @ 25 uMFloat%
12Inhibition @ 12.5 uMFloat%
13Inhibition @ 6.25 uMFloat%
14Inhibition @ 3.125 uMFloat%
15Inhibition @ 1.563 uMFloat%
16Inhibition @ 0.781 uMFloat%
17Inhibition @ 0.391 uMFloat%
18Inhibition @ 0.195 uMFloat%
19Data VerificationString

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: