Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program

Bringing Back Nature to Drug DiscoveryNatural Molecules in an Antibacterial Program Mark ONeil-Johnson

Parallel Purification TechnologiesState-of-the-art NMR Probe Technology Taxonomically Diverse PlantCollectionHigh-throughput Natural Products ChemistrySequoia Sciences, Inc.

Plants to Compound LibrarySequoia Sciences, Inc.Analysis performed on each sampleNormalized sample weightszzSequoias StrengthsParallel Purification TechnologiesState-of-the-art NMR Probe TechnologyTaxonomically Diverse PlantCollectionFive step purification process to removenon-drug-like compoundsApproximately 90% of plant extracts are composed of non-drug-like compoundsSequoias process removes these non-drug-like compoundsGenerates approximately 110 preparative HPLC fractions (sample) per plant8-channel MUX LC/MS with 8 ELSDsMass SpectrumTIC Chromatogram506.2447.2244.9336.0

Sheet1

Highly Lipophilic Compounds> Log P of 5

Highly Hydrophilic Compounds< Log P of 0

High Molecular Weight Compounds> 1000 Daltons

Miscellaneous Compounds

i.e. Carbohydrates, Tannins

Sheet2

Sheet3

Flow ProbeCoil5 mLFlow cell1.5 mLActive volumeBruker AvanceFlow ProbeCoil5 mLFlow cell1.5 mLActive volume Collaborator rescreens purified compounds Biological Activity to Chemical StructureSequoia Sciences, Inc.Collaborator identifies anActive SampleSequoia acquires NMR spectra on 5 to 100 ug of an Active Compound Structure elucidation of an Active CompoundSequoia delivers compounds to CollaboratorSequoia isolates compounds from an Active SampleSequoia elucidates the chemical structure of an Active Compound600 MHz Magnet34

What can I do with only micrograms of a sample?

High-throughput Natural Products Chemistry Move to Miniaturize100 mL1 L10 mL1 mL1 mL100 mL10 mL

CapNMR Probe

The Ultimate NMR Probe for Samples of Limited Mass

CapNMRTM ProbeHigh-throughput Natural Products Chemistry2 mg Quinidine in MeOH,d42 mg in 550 mL MeOH,d42 mg in 8 mL MeOH,d4; Infuse 5 mL5mm TXI probe @ 900 MHzCapNMR probe @ 600 MHz

CapNMRTM ProbeHigh-throughput Natural Products Chemistry1.5 mlit.(~1 mm)5 mlit. 5 mm std. NMR tube270 mlit.(~17 mm)5 mm Rf Coil

Sequoia Sciences, Inc.High-Throughput Natural Products ChemistrySample Submission Hardware, Solvent and Waste Recoverymini-tube10 ml gas tight syringe96-well plateWaste recoveryvial

CapNMRTM Probe4 mg. Amount isolated: 4 mg. Dilute with 7mL MeOD Infuse 5 mL Center in flow cell with 7 mL

High-throughput Natural Products ChemistrySequoia Sciences, Inc.HPLC / ELSD of Hit 620 of Fraction 8, tube 34

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Active Fractions From Hit 620 Active Fractions for Rescreen and NMR

SF_4329SF_4331SF_4335SF_4351SF_4356

Plant Family: OrchidaceaeSpecies: Phragmipedium calurum

Combine tubes F10, G10 and H10SepFraction 155096 mg total (2 HPLC runs)To NMRPlant Family: AsteraceaeSpecies: Layia platyglossa

512 Scans64 ScansData base peak search

Bacterial BiofilmsNasty Stuff!

Test Summary: Pseudomonas Disinfection and Regrowth in Pipes R. Anderson (Center for Disease Control) filled plastic pipes with water contaminated with two strains of Pseudomonas.

Incubate for 8 weeks

Empty and douse pipes with germ-killing biocides, include chlorine, for7 days

Refill with sterile water and periodically sample clean water

Both strains of Pseudomonas survived and reestablished colonies!

Bacteria attach on surface, begin to secrete polysaccharide matrix

Cells grow into a community, complete with nutrient channels

Community thrives, protected by polysaccharide matrix

Industrial Biofilms

Ship bottoms and pipes Cooling towers Air ventilation systems Drinking water filtration and distribution systems Air filtration units Dental water lines Hemodialysis units Food processing systems Medical Biofilms

Urinary tract infections Lung infections Sinus infections Ear infections Chronic wounds Dental caries Medical device implants Acne

Resistant to antibiotics at concentrations from 1000 to 1500 times higher than conventional use

Involved in two-thirds of human bacterial infections

No specific biofilm inhibitor is commercially available

96 Well plate format for initial screen (initial screen at 10 mg/ml)

Asiatic Acid Corosolic Acid Ursolic Acid

Minimum Inhibitory Concentrations (MIC) against planktonic bacteria not relevant to biofilms

New biofilm susceptibility methods need to be developed: biofilm inhibition conc. (BIC) or minimum biofilm eradication conc. (MBEC)

Variability in BIC and MBEC values suggest characteristics of biofilms is related to model used to grow them in

96 Well plate format Rotating Disk Reactor (RDR)

Bacterial strain: Pseudomonas aeruginosa (PA01)

Ciprofloxacin active against planktonic bacteria at 1 mg/ml

Ciprofloxacin active against a bacterial biofilm at 50 mg/ml

Develop a valid physiological screening assay

Pseudomonas aeruginosa airway infections: leading cause of death in Cystic Fibrosis patients

Chronic infection that is resistant to antibiotic therapies

Standard treatment: tobramycin and azithromycin

RDR, a physiological valid screening method?

Test Sequoia compounds in RDR with tobramycin and azithromycin or ciprofloxacin combination

255+toby 255+water toby control

Chart1

257000

243000

86300

14800000

23000000

113000000

797000

852000

144000

21800000

24700000

38300000

50ug/ml 255

10 C255 4-26-05

Sequoia coupons set up 4-22-05 take down 4-25-05

255 concentration at 10ug/ml for 24 hours

Tobramycin and 255

coupon #

1avg2avg

3141612191314.8111097129.8

log

1.17E+067.73E+059.71E+055.99cfu/cm^2

255 and water

1avg2avg

5141612191515.2232124202121.8

1.20E+081.72E+081.46E+088.16cfu/cm^2

control and Tobramycin

coupon #

1avg2avg

41112971310.435118148.2

8.21E+066.47E+067.34E+066.87cfu/cm^2

control

coupon #

1avg2avg

510141791412.8121612141513.8

1.01E+081.09E+081.05E+088.02cfu/cm^2

10 C255 4-19-05



Tobramycin and 255

coupon #

1avg2avg

37115386.8272524252825.8

log

5.37E+052.04E+061.29E+066.11cfu/cm^2

255 and water

1avg2avg

5131214111613.2141215111814

1.04E+081.10E+081.07E+088.03cfu/cm^2


coupon #

1avg2avg

4363941443739.4573645

3.11E+073.95E+061.75E+077.24cfu/cm^2

control

coupon #

1avg2avg

5353538394037.4373837324137

2.95E+082.92E+082.94E+088.47cfu/cm^2

10 C255 4-9-05



Tobramycin and 255

coupon #

1avg2avg2avg

31091669105812577.41396758

7.89E+055.84E+056.31E+056.68E+055.82cfu/cm^2

255 and water

1avg2avg2avg

58128788.610612978.813109121812.4

6.79E+076.95E+079.79E+077.84E+077.89cfu/cm^2


coupon #

1avg2avg2avg

312710121611.4271516281520.2151720221217.2

9.00E+051.59E+061.36E+061.28E+066.11cfu/cm^2

control

coupon #

1avg2avg2avg

51091171410.281417111813.6221813101215

8.05E+071.07E+081.18E+081.02E+088.01cfu/cm^2

50 C255 2-15-05


255concentration at 50ug/ml for 24 hours

Tobramycin and 255

coupon #

12avg

2383642363737.8362425282427.4

2.98E+052.16E+052.57E+055.41cfu/cm^2

255 and water

12avg

4253127352829.29681278.4

2.30E+076.63E+061.48E+077.17cfu/cm^2


coupon #

12avg

3761191299612131611.2

7.10E+058.84E+057.97E+055.90cfu/cm^2

control

coupon #

12avg

4303941433838.2211513171917

3.01E+071.34E+072.18E+077.34cfu/cm^2

50 C255 2-22-05



Tobramycin and 255

coupon #

12avg

2343632423135272329302426.6

2.76E+052.10E+052.43E+05

255 and water

12avg

4353129373232.8222129302525.4

2.59E+072.00E+072.30E+07


coupon #

12avg

31011981410.49816111211.2

8.21E+058.84E+058.52E+05

control

coupon #

12avg

4353736414438.6282226192524

3.05E+071.89E+072.47E+07cfu per cm2

50 C255 2-26-05



Tobramycin and 255

coupon #

12avg3avg

2131714131213.89910121110.281091078.8

1.09E+058.05E+046.95E+048.63E+044.94cfu/cm^2

255 and water

12avg3avg

5496535.428313020362918107538.6

4.26E+072.29E+086.79E+071.13E+088.05cfu/cm^2


coupon #

12avg3avg

2241722162120262216212722.471516101312.2

1.58E+051.77E+059.63E+041.44E+055.16cfu/cm^2

control

coupon #

12avg2avg

4585359493550.8584449394146.2222624182723.4

4.01E+073.65E+071.85E+073.17E+077.50cfu/cm^2

100 C255 12-6-04



Tobramycin and 255

columncoupon #

12avgLog

15.84E+055.77

31037897.416171112141411.10E+066.04

21.74E+088.24

5.84E+051.10E+068.45E+055.93cfu/cm^228.68E+077.94

31.09E+066.04

31.83E+066.26

255 and PBS48.05E+077.91

12avg47.26E+077.86

519242225202212151210611

1.74E+088.68E+071.30E+088.11cfu/cm^2


coupon #

12avg

313915171513.8232025212723.2

1.09E+061.83E+061.46E+066.16cfu/cm^2

control

coupon #

12avg

51081171510.288119109.2

8.05E+077.26E+077.66E+077.88cfu/cm^2

100 C255 12-6-04

1

Toby + 255 255 + PBS Toby Control

100 C255 12-16-04



Tobramycin and 255

columncoupon #

12avgLog

10

21054625.4252730263528.610

log20

4.26E+042.26E+051.34E+055.13cfu/cm^220

30

30

255 and PBS40

12avg40

4272630262827.4303834322732.2

2.16E+072.54E+072.35E+077.37cfu/cm^2


coupon #

12avg

34107586.8435343.8

5.37E+053.00E+054.18E+055.62cfu/cm^2

control

coupon #

12avg

4393532384437.6374332444740.6

2.97E+073.20E+073.09E+077.49cfu/cm^2

100 C255 12-16-04

0

0

0

0

0

0

0

0

Toby + 255 255 + PBS Toby Control

100 C255 1-8-05



Tobramycin and 255

coupon #

12avg

1171421171416.6101110121411.4

log

1.31E+049.00E+031.10E+044.04cfu/cm^2

255 and PBS

12avg

4161823191818.8221719211418.6

log

1.48E+071.47E+071.48E+077.17cfu/cm^2


coupon #

12avg

4151117151314.2685786.8

log

1.12E+075.37E+068.29E+066.92cfu/cm^2

control

coupon #

12avg

575851071214871110.4

log

5.52E+078.21E+076.87E+077.84cfu/cm^2

200 C255 3-4-05



Tobramycin and 255

coupon #

12avg3avg

2523133374138.88710181010.6515348475049.8

3.06E+058.37E+043.93E+052.61E+055.42cfu/cm^2

255 and water

12avg3avg

59148869964566423423

7.10E+074.74E+072.37E+074.74E+077.68cfu/cm^2


coupon #

12avg3avg

38648107.2151518242619.61211991310.8

5.68E+051.55E+068.52E+059.89E+056.00cfu/cm^2

control

coupon #

12avg2avg

5369475.861159108.24910877.6

4.58E+076.47E+076.00E+075.52E+077.74cfu/cm^2

200 C255 3-8-05



Tobramycin and 255

coupon #

12avg3avg

2282330262526.4182220192220.287116139

2.08E+051.59E+057.10E+041.46E+055.17cfu/cm^2

255 and water

12avg3avg

4484754454247.2283425273229.2321520182221.4

3.73E+072.30E+071.69E+072.57E+077.41cfu/cm^2


coupon #

12avg3avg

3132112151615.4232518222222342526242627

1.22E+061.74E+062.13E+061.69E+066.23cfu/cm^2

control

coupon #

12avg2avg

4423746484142.8293634373233.6394147433841.6

3.38E+072.65E+073.28E+073.01E+077.48cfu/cm^2

200 C255 3-11-05



Tobramycin and 255

coupon #

12avg

212716101211.4141213111613.2

9.00E+041.04E+059.71E+044.99cfu/cm^2

255 and water

12avg

4372827293330.811779139.4

2.43E+077.42E+061.59E+077.20cfu/cm^2


coupon #

12avg

3161613121514.4211719231719.4

1.14E+061.53E+061.33E+066.13cfu/cm^2

control

coupon #

12avg

4443229283032.6463932242833.8

2.57E+072.67E+072.62E+077.42cfu/cm^2

summary plots

16.68E+05

27.84E+07

31.28E+06

41.02E+08

12.57E+05

12.43E+05

18.63E+04

21.48E+07

22.30E+07

21.13E+08

37.97E+05

38.52E+05

31.44E+05

42.18E+07

42.47E+07

43.83E+07

18.45E+05

11.34E+05

11.10E+04

21.30E+08

22.35E+07

21.48E+07

31.46E+06

34.18E+05

38.29E+06

47.66E+07

43.09E+07

46.87E+07

12.61E+05

11.46E+05

19.71E+04

24.74E+07

22.57E+07

21.59E+07

39.89E+05

31.69E+06

31.33E+06

45.52E+07

43.01E+07

42.62E+07

16.68E+05

22.57E+05

22.43E+05

28.63E+04

38.45E+05

31.34E+05

31.10E+04

42.61E+05

41.46E+05

49.71E+04

67.84E+07

71.48E+07

72.30E+07

71.13E+08

81.30E+08

82.35E+07

81.48E+07

94.74E+07

92.57E+07

91.59E+07

111.28E+06

127.97E+05

128.52E+05

121.44E+05

131.46E+06

134.18E+05

138.29E+06

149.89E+05

141.69E+06

141.33E+06

161.02E+08

172.18E+07

172.47E+07

173.83E+07

187.66E+07

183.09E+07

186.87E+07

195.52E+07

193.01E+07

192.62E+07

C255 dose response

C255

10ug/ml50 ug/ml100 ug/ml200 ug/ml

C255 + toby5.825.295.525.23

C255 + water7.897.707.757.47

toby6.115.786.536.13

control8.017.457.777.57

summary plots

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0

50ug/ml 255

0

0

0

0

0

0

0

0

0

0

0

0

100ug/ml results

0

0

0

0

0

0

0

0

0

0

0

0

200ug/ml results



0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

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0

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0

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0

0

0

0

0

0

0

0

0

0

10 50 100 200 10 50 100 200 10 50 100 200 10 50 100 200 Toby+255 255+water Toby Control

10ug/ml 255

0

0

0

0

0

0

0

0

0

0

0

0



Experiment Summary

This experiment uses the standard RDR protocol with PAO1, and doses of 10, 50, 100 and 200 ug/ml C255 with the controls as just water, just tobramycin or just C255. The goal here was to establish that there IS a dose response when using C255. Each treatment was done in duplicate.

Results at 10ug/ml there is a slight reduction from toby alone. at 50ug/ml there is nearly a log reducton from toby.at 100ug/ml there is a log reduction, (some variability though)at 200ug/ml there is a clear log readuction

At all doses water only and C255 only had 1e8 cells/ml and Toby alone had 1e6 cells/ml

Important to note that the controls were run separately for each treatment (obviously) so their were 8 repetitions of toby only, and water only, and there was very little variablity between the two.

Chart1

7.4027.2898.345.822

7.2666.9468.03136.167

7.4566.8957.7896.062

Control Control + Toby Cipro+ Water Cipro+Toby

Log cfu/coupon

Ciprofloxacin 10ug/ml

10-31-05

Log Counts

ControlContro+TobyCipro+ WaterCipro+ Toby

7.407.298.345.82

7.276.958.036.17

7.4566.8957.7896.062

1234

1234

1234

10-31-05

Control Control + Toby Cipro+ Water Cipro+Toby

Log cfu/coupon

Ciprofloxacin 10ug/ml

C = ciprofloxacinT = tobramycin

Chart3

8.05563312436.72970462136.1238516415.0606978404

8.11171047097.33845649365.97312785365.2496059543

Control Cont + T C255 + C C255 + C + T

Log cfu/coupon

C255 50 ug/mL with Cipro 10ug/mL

Summary

Sequoia RDR summary for 10/14/05 to 3/18/06

* Tobramycin post-treatments are all 2 hours of 100 ug/mL

**Average counts are in cells/cm2 of coupon

End of run dateCompound TreatmentControlControl + TobyCompound ControlCompound + Toby

10/14/05Ciprofloxacin 50ug/mL1.15E+071.42E+062.34E+041.10E+03

10/21/05C108 10 ug/mL8.14E+071.78E+069.35E+072.15E+07

10/31/05C108 50 ug/mL2.49E+084.04E+072.70E+082.24E+07

11/17/05C255 10ug + Cipro 10ug4.24E+072.85E+061.87E+067.98E+05

12/9/05C255 50ug + Cipro 10ug9.59E+071.07E+078.96E+051.15E+05

12/16/05C255 100ug + Cipro 10ug1.91E+075.31E+055.25E+061.07E+06

1/14/06C457 10ug/mL1.45E+071.78E+067.85E+068.88E+04

1/21/06C457 50ug/mL2.95E+078.68E+052.60E+072.17E+06

1/27/06C457 10ug + Cipro 10ug1.25E+072.45E+061.63E+051.21E+04

2/5/06C457 50ug + Cipro 10ug7.18E+064.85E+051.42E+061.60E+03

2/18/06C536 10ug/mL9.97E+068.42E+051.18E+072.00E+06

2/28/06C536 50ug/mL8.55E+051.64E+042.89E+050.00E+00

3/4/06C536 50ug/mL3.10E+074.73E+06no countsno counts

3/18/06C536 10ug + Cipro 10ug2.30E+075.12E+062.45E+051.03E+04

3/25/06C536 50ug + Cipro 10ug1.37E+072.13E+054.35E+041.42E+02

Tobramycin log reductions vs. Control

End of run datelog reduction

10/14/050.91

10/21/051.66

10/31/050.79

11/17/051.17

12/9/050.95

12/16/051.56

1/14/060.91

1/21/061.53

1/27/060.71

2/5/061.17

2/18/061.07

2/28/061.72

3/4/060.82

3/18/060.65

3/25/061.81

Average1.16

Std Dev0.380

Summary

0.9098853453

1.6613568805

0.7898707975

1.1727993523

0.9515699196

1.5555244632

0.9097740208

1.5307040828

0.7063703024

1.1707551505

1.0734892316

1.7160033436

0.8173339302

0.6530884474

1.8075183248

log reduction

Tobramycin log reductions

10-14-05

Sequoia plate counts

10/14/05

50 ug/mL Ciprofloxacin and 100 ug/mL Tobramycin

Log counts

Coupon surface area =1.267cm^2ContCont + TobCiproCipro + Tob

Control + water7.166.254.473.15

10^510^68.095.863.343.73

Coupon 1171Coupon 2124

121110

nc136

1.46E+071.23E+08

Average1.46E+07cells/coupon

1.15E+07cells/cm^2

Control + Tobramycin

10^410^4


17956

19265

1.80E+067.30E+05


1.42E+06cells/cm^2

Ciprofloxacin + water1234

10^310^21234


428

4711

2.97E+042.20E+03


2.34E+04cells/cm^2

Ciprofloxacin + Tobramycin

10^210^2

Coupon 120Coupon 2nc

1780

527

1.40E+035.35E+03


1.10E+03cells/cm^2

10-14-05

0000

0000

Control

Cont + tob

Cipro

Cipro + Tob

Control Cont + Toby Cipro Cipro + Toby

Log CFU/coupon

Ciprofloxacin 50ug/mL Treatment

10-21-05


10/21/05

Treatment: 10 ug/mL C108 and 100 ug/mL Tobramycin

Log Counts

Coupon surface area =1.267cm^2ControlCont + TobC108108 + Tob

Control + water7.685.878.157.33

10^610^68.206.587.987.52


36155

51152

4.73E+071.59E+08


8.14E+07cells/cm^2

1234

Control + Tobramycin1234

10^510^5

Coupon 15Coupon 250

1129

634

7.33E+053.77E+06


1.78E+06cells/cm^2

C108 + water

10^610^6


14493

17894

1.41E+089.57E+07


9.35E+07cells/cm^2

C108 + Tobramycin

10^510^6


18826

19835

2.14E+073.30E+07


2.15E+07cells/cm^2

10-21-05

0000

0000

Control

Cont + Tob

Control Cont + Toby C108 C108 + Toby

Log cfu/coupon

Compound 108 10ug/mL Treatment

10-31-05


10/31/05

Treatment: 50 ug/mL C108 and 100 ug/mL Tobramycin

Log Counts


Control + water8.427.718.557.12

10^610^68.567.718.527.64


241347

282373

2.64E+083.67E+081234

Average3.16E+08cells/coupon1234

2.49E+08cells/cm^2


10^510^5


486521

487474

5.13E+075.11E+07


4.04E+07cells/cm^2

C108 + water

10^610^6


309325

372329

3.52E+083.32E+08


2.70E+08cells/cm^2

C108 + Tobramycin

10^510^6


12850

20348

1.31E+074.37E+07


2.24E+07cells/cm^2

10-31-05

0000

0000


Log cfu/coupon

C108 50 ug/mL Treatment

11-17-05


11/17/05

Treatment: 10 ug/mL C255 with 10 ug/mL Cipro for 24 hours

post treatment of 100 ug/mL Tobramycin for two hours

Log Counts


Control + water7.806.576.336.20

10^610^67.656.546.415.65


5947

5650

6.27E+074.47E+071234


4.24E+07cells/cm^2


10^410^4


357374

tntctntc

3.72E+063.50E+06


2.85E+06cells/cm^2

C255 + water

10^410^5


22236

19620

2.15E+062.60E+06


1.87E+06cells/cm^2

C255 + Tobramycin

10^410^4


15946

16646

1.58E+064.47E+05


7.98E+05cells/cm^2

Control reactor10^7

24

35

35


2.47E+08cells/cm^2

11-17-05

0000

0000


Log cfu/coupon


12-9-05


12/9/05



Log Counts


Control + water8.066.736.125.06

10^610^68.117.345.975.25


111146

127128

1.14E+081.29E+08


9.59E+07cells/cm^21234


10^510^5


48218

57204

5.37E+062.18E+07


1.07E+07cells/cm^2

C255 + water

10^410^4


13795

14192

1.33E+069.40E+05


8.96E+05cells/cm^2

C255 + Tobramycin

10^310^3


96167

114154

1.15E+051.78E+05


1.15E+05cells/cm^2

12-9-05

0000

0000


Log cfu/coupon

C255 50 ug/mL with Cipro 10ug/mL

12-16-05


12/16/05



Log Counts


Control + water7.315.796.856.36

10^610^67.455.866.795.63


2228

2034

2.03E+072.80E+071234


1.91E+07cells/cm^2


10^410^4


7161

55

6.20E+057.25E+05


5.31E+05cells/cm^2

C255 + water

10^510^5


6343

8673

7.13E+066.17E+06


5.25E+06cells/cm^2

C255 + Tobramycin

10^410^4


25339

19947

2.29E+064.23E+05


1.07E+06cells/cm^2


12-16-05

0000

0000


Log cuf/coupon

C255 100ug/mL with 10ug/mL Cipro

1-14-06


1/14/06

Treatment: 10 ug/mL C457 for 24 hours

post treatment of 100 ug/mL Tobramycin for two hoursLog Counts

ControlCont + TobC457457 + Tob

Coupon surface area =1.267cm^27.216.136.874.18

Control + water7.316.507.105.32

10^610^6


1731

18171234

1.63E+072.03E+071234


1.45E+07cells/cm^2


10^410^5


15522

15930

1.35E+063.17E+06


1.78E+06cells/cm^2

C457 + water

10^510^5


76120

NCNC

7.45E+061.25E+07


7.85E+06cells/cm^2

C457 + Tobramycin

10^410^4

Coupon 11Coupon 217

25

2NC

1.50E+042.10E+05


8.88E+04cells/cm^2


1-14-06

0000

0000


Log cfu/coupon

C457 10ug/mL Treatment

1-21-06


1/21/06



Log Counts


Control + water7.456.047.526.46

10^610^67.670.006.42


2440

2552

2.80E+074.67E+071234


2.95E+07cells/cm^2


10^410^4


1060

1110

1.10E+060.00E+00


8.68E+05cells/cm^2

C457 + water

10^510^5

Coupon 1320Coupon 2TNTC

328TNTC

342TNTC

3.30E+07NC


2.60E+07cells/cm^2

C457 + Tobramycin

10^410^4


TNTC263

326TNTC

2.86E+062.64E+06


2.17E+06cells/cm^2

Control RDR bulkTreated RDR bulk

10^610^5

112176

113171

121151

Average1.15E+08cells/couponAverage1.66E+07cells/coupon

9.10E+07cells/cm^21.31E+07cells/cm^2

1-21-06

0000

0000


Log cfu/coupon


1-27-06


1/27/06



Log Counts


Control + water7.296.715.364.07

10^610^57.096.025.274.28


22117

23132

1.93E+071.22E+071234


1.25E+07cells/cm^2


10^510^4


30165

5282

5.17E+061.04E+06


2.45E+06cells/cm^2

C457 + Cipro Control

10^410^4


2119

2417

2.27E+051.87E+05


1.63E+05cells/cm^2

C457 + Tobramycin

10^310^3


1318

1218

1.17E+041.90E+04


1.21E+04cells/cm^2

Control RDR bulkTreated RDR bulk

10^610^5

142132

163123

223121

Average1.76E+08cells/couponAverage1.25E+07cells/coupon

1-27-06

0000

0000


Log cfu/coupon


2-5-06


2/5/06



Log Counts


Control + water6.735.576.183.07

10^510^57.115.936.323.46


61158

NCNC

5.40E+061.28E+071234


7.18E+06cells/cm^2


10^410^4


3189

25NC

3.73E+058.55E+05


4.85E+05cells/cm^2

C457 + Cipro Control

10^510^5


921

2228

1.50E+062.10E+06


1.42E+06cells/cm^2

C457 + Tobramycin

10^210^2


1634

823

1.17E+032.90E+03


1.60E+03cells/cm^2


2-5-06

0000

0000


Log cfu/coupon


2-18-06


2/18/06



Log Counts

Coupon surface area =1.267cm^2ControlCont + TobC536536 +Tob

Control + water7.196.227.016.44

10^510^56.995.687.296.37


11789

16289

1.55E+079.77E+06


9.97E+06cells/cm^21234


10^410^4


18642

16359

1.66E+064.77E+05


8.42E+05cells/cm^2

C536 Control

10^510^5


111204

102188

1.03E+071.97E+07


1.18E+07cells/cm^2

C536 + Tobramycin

10^510^5


3622

2327

2.73E+062.33E+06


2.00E+06cells/cm^2


2-18-06

0000

0000


Log cfu/coupon


2-28-06


2/28/06



Log Counts


Control + water6.11394335234.32221929475.5314789170

10^510^55.93785209334.31527043485.59476075260

Coupon 113Coupon 25

nc10

1311

1.30E+068.67E+05


8.55E+05cells/cm^21234

** These control counts are around a log lower than normal.


10^310^3


3021

nc26

2.10E+042.07E+04


1.64E+04cells/cm^2

C536 Control

10^410^4


4425

nc47

3.40E+053.93E+05


2.89E+05cells/cm^2

C536 + Tobramycin

10^210^2** I did not get any colonies at the 10^2 dilution for these coupons.

Coupon 10Coupon 20

00

00

0.00E+000.00E+00


0.00E+00cells/cm^2

2-28-06

0000

0000


Log cfu/coupon


3-4-06


3/4/06**This run will be repeated again



Coupon surface area =1.267cm^2

Control + water

10^610^6


5624

4630

5.53E+072.33E+07


3.10E+07cells/cm^2


10^410^5


80149

NCNC

8.30E+051.12E+07


4.73E+06cells/cm^2

C536 & Cipro Control

10^510^5

Coupon 1TNTCCoupon 2TNTC

TNTCTNTC

TNTCTNTC

0.00E+000.00E+00


0.00E+00cells/cm^2

C536 & Cipro + Tobramycin

10^410^4

Coupon 1TNTCCoupon 2TNTC

TNTCTNTC

TNTCTNTC

0.00E+000.00E+00


0.00E+00cells/cm^2

3-18-06


3/18/06

Treatment: 10 ug/mL C536 + 10 ug/mL Ciprofloxacin for 24 hours


Log Counts


Control + water7.357.015.654.30

10^610^67.566.455.233.78


2535

2428

2.23E+073.60E+071234


2.30E+07cells/cm^2


10^510^5


12020

8528

1.01E+072.83E+06


5.12E+06cells/cm^2


10^410^4


5512

4124

4.50E+051.70E+05


2.45E+05cells/cm^2


10^310^3

Coupon 119Coupon 28**These are low for plate counts

203

217

2.00E+046.00E+03


1.03E+04cells/cm^2

Control RDR Bulk FluidTreated RDR Bulk Fluid

10^710^6

215**These are low for plate counts

273

2.40E+08cells/mL4.00E+06cells/mL

3-18-06

0000

0000


Log cfu/coupon

C536 10 ug/mL with 10ug/mL Cipro

3-25-06


3/25/06

Treatment: 50 ug/mL C536 + 10 ug/mL Ciprofloxacin for 24 hours


Log Counts


Control + water7.365.714.632.56

10^610^67.074.434.830.00


228

2117

2.30E+071.17E+071234


1.37E+07cells/cm^2


10^410^4

Coupon 157Coupon 23

522

453

5.13E+052.67E+04


2.13E+05cells/cm^2


10^310^3


3466

4874

4.27E+046.77E+04


4.35E+04cells/cm^2


10^110^1

Coupon 126Coupon 20

430

390

3.60E+020.00E+00


1.42E+02cells/cm^2

Control RDR Bulk Fluid

10^7

8

11

9.50E+07cells/mL


3-25-06

0000

0000

Control Cont + T C536 +C C536 + C + T

Log cfu/coupon

C536 50 ug/mL with 10ug/mL Cipro


Summary

Asiatic Acid and Corosolic Acid in a physiological relevant biofilm screen are not true biofilm inhibitors but biofilm modulators

Synergistic effect on P. aeruginosa biofilm by Sequoia compounds indicate that they do affect the tolerance of antibiotic to PA

Sequoia compounds not true biofilm inhibitors, suggesting a different MOA by modification of biofilm

P. aeruginosa grown in presence of 10 mg/ml Asiatic Acid makes biofilm sensitive to 10 mg/ml ciprofloxacin

Sequoia Compounds are excellent biofilm potentiator candidates

High-throughput Natural Products ChemistrySequoia Sciences, Inc.Special Thanks to:Bill CostertonGarth James

Documents

Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program