Primary Antimicrobial Assay for E. coli BW25113 ∆tolC::kan Protocol for 384-well HTS
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: 1420
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 that has an outer membrane barrier and multidrug efflux pumps (MDRs) that can reduce the intracellular concentrations of potential antibiotics. TolC is an outer membrane porin that contributes to antimicrobial resistance because the major MDRs use it as an outward channel; 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. Compounds were screened 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. A mathematical algorithm was used to determine nominally inhibitory compounds in the primary screen. Two values were calculated: (1) the average percent growth inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % growth inhibition than the cutoff parameter was declared active. Compounds were screened in single dose and there is an inherent uncertainty associated with a single determination.
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 LB agar plate containing 50 ug/ml Kanamycin; incubate overnight (o/n, 16-18hrs) 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 media containing 50 ug/ml Kanamycin with a small sample from the frozen stock and shake overnight (16-18hrs) in water bath or floor shaker at 250 rpm, 37C.
-Place culture in fridge at 4C until 1.5 hrs before plating time
-Dilute the culture 1:10 in fresh LB (define) broth media containing 50 ug/ml Kanamycin and shake for 1.5hrs at 250rpm, 37C.
-After 1.5 hrs, the culture is then diluted 1:1000 in fresh LB broth media 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 Multidrop micro in hood.
-Place plates in PE bag stacked 2 high.
-Incubate plates at 37C for 20 hours.
*** In the case of Z' plates: 5 ul media/DMSO or Chloramphenicol/DMSO (at 30 uM final) is added with Biomek FX.
-Read A615 on Envision (Abs folder A615- 30s shake, 384 general)
LB agar plate prep (100 ml)
100 ml MilliQ water
2g LB broth (Fisher, BP1427-500)
1.5g 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 MQ water
20g LB broth
5g 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 5mg/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.
LB broth stock: Stock was prepared at 50 mg/ml in MilliQ water and filter sterilized. Vials of 1 ml aliquots were frozen. Use 1 vial for 1 liter of LB broth.
Type of plates used
384 well clear flat bottom w/lid, TCT plates (Fisher Corning 3701).
Possible artifacts in this assay include, but are not limited to, compounds that absorb at OD615 or precipitate.
Outcome: Actives were defined as compounds that showed ≥60.37% inhibition in the assay. Compounds that were less than 60.37% inhibitory were Inactives.
Because of the inherent error in all high throughput screens, compounds that were active in this single dose screen were assigned a score of 100. All other compounds were assigned a score of 0.
Data Table (Concise)