bringing back nature to drug discovery natural molecules in an antibacterial program
DESCRIPTION
Bringing Back Nature to Drug Discovery Natural Molecules in an Antibacterial Program Mark O’Neil-Johnson. High-throughput Natural Products Chemistry. Sequoia Sciences, Inc. State-of-the-art NMR Probe Technology. Taxonomically Diverse Plant Collection. Parallel Purification Technologies. - PowerPoint PPT PresentationTRANSCRIPT
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Bringing Back Nature to Drug DiscoveryNatural Molecules in an Antibacterial Program Mark ONeil-Johnson
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Parallel Purification TechnologiesState-of-the-art NMR Probe Technology Taxonomically Diverse PlantCollectionHigh-throughput Natural Products ChemistrySequoia Sciences, Inc.
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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
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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
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What can I do with only micrograms of a sample?
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High-throughput Natural Products Chemistry Move to Miniaturize100 mL1 L10 mL1 mL1 mL100 mL10 mL
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CapNMR Probe
The Ultimate NMR Probe for Samples of Limited Mass
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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
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CapNMRTM ProbeHigh-throughput Natural Products Chemistry1.5 mlit.(~1 mm)5 mlit. 5 mm std. NMR tube270 mlit.(~17 mm)5 mm Rf Coil
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Sequoia Sciences, Inc.High-Throughput Natural Products ChemistrySample Submission Hardware, Solvent and Waste Recoverymini-tube10 ml gas tight syringe96-well plateWaste recoveryvial
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CapNMRTM Probe4 mg. Amount isolated: 4 mg. Dilute with 7mL MeOD Infuse 5 mL Center in flow cell with 7 mL
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High-throughput Natural Products ChemistrySequoia Sciences, Inc.HPLC / ELSD of Hit 620 of Fraction 8, tube 34
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High-throughput Natural Products ChemistrySequoia Sciences, Inc.HPLC / ELSD of Hit 620 of Fraction 8, tube 34
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High-throughput Natural Products ChemistrySequoia Sciences, Inc.Active Fractions From Hit 620 Active Fractions for Rescreen and NMR
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SF_4329SF_4331SF_4335SF_4351SF_4356
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Plant Family: OrchidaceaeSpecies: Phragmipedium calurum
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Combine tubes F10, G10 and H10SepFraction 155096 mg total (2 HPLC runs)To NMRPlant Family: AsteraceaeSpecies: Layia platyglossa
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512 Scans64 ScansData base peak search
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Bacterial BiofilmsNasty Stuff!
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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!
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Bacteria attach on surface, begin to secrete polysaccharide matrix
Cells grow into a community, complete with nutrient channels
Community thrives, protected by polysaccharide matrix
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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
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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
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96 Well plate format for initial screen (initial screen at 10 mg/ml)
Asiatic Acid Corosolic Acid Ursolic Acid
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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)
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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
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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
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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
Sequoia coupons set up 4-15-05 take down 4-18-05
255 concentration at 10ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 4-05-05 take down 4-08-05
255 concentration at 10ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 2-11-05 take down 2-14-05
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
control and Tobramycin
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
Sequoia coupons set up 2-18-05 take down 2-21-05
255concentration at 50ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 2-22-05 take down 2-25-05
255concentration at 50ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 12-03-04 take down 12-06-04
255 concentration at 100ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 12-13-04 take down 12-16-04
255 concentration at 100ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 1-4-05 take down 1-7-05
255 concentration at 100ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 2-28-05 take down 3-03-05
255concentration at 200ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 3-04-05 take down 3-07-05
255concentration at 200ug/ml for 24 hours
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
control and Tobramycin
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
Sequoia coupons set up 3-07-05 take down 3-10-05
255concentration at 200ug/ml for 24 hours
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
control and Tobramycin
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
0
0
0
0
0
0
0
0
0
0
0
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
255+toby 255+water toby control
255+toby 255+water toby control
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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0
0
0
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0
0
0
0
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
255+toby 255+water toby control
255+toby 255+water toby control
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.
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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
Coupon 1168Coupon 298
17956
19265
1.80E+067.30E+05
Average1.80E+06cells/coupon
1.42E+06cells/cm^2
Ciprofloxacin + water1234
10^310^21234
Coupon 138Coupon 227
428
4711
2.97E+042.20E+03
Average2.97E+04cells/coupon
2.34E+04cells/cm^2
Ciprofloxacin + Tobramycin
10^210^2
Coupon 120Coupon 2nc
1780
527
1.40E+035.35E+03
Average1.40E+03cells/coupon
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
Sequoia plate counts
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
Coupon 155Coupon 2170
36155
51152
4.73E+071.59E+08
Average1.03E+08cells/coupon
8.14E+07cells/cm^2
1234
Control + Tobramycin1234
10^510^5
Coupon 15Coupon 250
1129
634
7.33E+053.77E+06
Average2.25E+06cells/coupon
1.78E+06cells/cm^2
C108 + water
10^610^6
Coupon 1102Coupon 2100
14493
17894
1.41E+089.57E+07
Average1.19E+08cells/coupon
9.35E+07cells/cm^2
C108 + Tobramycin
10^510^6
Coupon 1255Coupon 238
18826
19835
2.14E+073.30E+07
Average2.72E+07cells/coupon
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
Sequoia plate counts
10/31/05
Treatment: 50 ug/mL C108 and 100 ug/mL Tobramycin
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC108108 + Tob
Control + water8.427.718.557.12
10^610^68.567.718.527.64
Coupon 1269Coupon 2381
241347
282373
2.64E+083.67E+081234
Average3.16E+08cells/coupon1234
2.49E+08cells/cm^2
Control + Tobramycin
10^510^5
Coupon 1565Coupon 2538
486521
487474
5.13E+075.11E+07
Average5.12E+07cells/coupon
4.04E+07cells/cm^2
C108 + water
10^610^6
Coupon 1374Coupon 2341
309325
372329
3.52E+083.32E+08
Average3.42E+08cells/coupon
2.70E+08cells/cm^2
C108 + Tobramycin
10^510^6
Coupon 163Coupon 233
12850
20348
1.31E+074.37E+07
Average2.84E+07cells/coupon
2.24E+07cells/cm^2
10-31-05
0000
0000
Control Cont + Toby C108 C108 + Toby
Log cfu/coupon
C108 50 ug/mL Treatment
11-17-05
Sequoia plate counts
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
Coupon surface area =1.267cm^2ControlCont + TobC255255 + Tob
Control + water7.806.576.336.20
10^610^67.656.546.415.65
Coupon 173Coupon 237
5947
5650
6.27E+074.47E+071234
Average5.37E+07cells/coupon1234
4.24E+07cells/cm^2
Control + Tobramycin
10^410^4
Coupon 1386Coupon 2325
357374
tntctntc
3.72E+063.50E+06
Average3.61E+06cells/coupon
2.85E+06cells/cm^2
C255 + water
10^410^5
Coupon 1227Coupon 222
22236
19620
2.15E+062.60E+06
Average2.38E+06cells/coupon
1.87E+06cells/cm^2
C255 + Tobramycin
10^410^4
Coupon 1148Coupon 242
15946
16646
1.58E+064.47E+05
Average1.01E+06cells/coupon
7.98E+05cells/cm^2
Control reactor10^7
24
35
35
Average3.13E+08cells/coupon
2.47E+08cells/cm^2
11-17-05
0000
0000
Control Cont + Toby C255 C255 + Toby
Log cfu/coupon
C255 10 ug/mL Treatment
12-9-05
Sequoia plate counts
12/9/05
Treatment: 50 ug/mL C255 with 10 ug/mL Cipro for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC255255 + Tob
Control + water8.066.736.125.06
10^610^68.117.345.975.25
Coupon 1103Coupon 2114
111146
127128
1.14E+081.29E+08
Average1.22E+08cells/coupon1234
9.59E+07cells/cm^21234
Control + Tobramycin
10^510^5
Coupon 156Coupon 2232
48218
57204
5.37E+062.18E+07
Average1.36E+07cells/coupon
1.07E+07cells/cm^2
C255 + water
10^410^4
Coupon 1121Coupon 295
13795
14192
1.33E+069.40E+05
Average1.14E+06cells/coupon
8.96E+05cells/cm^2
C255 + Tobramycin
10^310^3
Coupon 1135Coupon 2212
96167
114154
1.15E+051.78E+05
Average1.46E+05cells/coupon
1.15E+05cells/cm^2
12-9-05
0000
0000
Control Cont + T C255 + C C255 + C + T
Log cfu/coupon
C255 50 ug/mL with Cipro 10ug/mL
12-16-05
Sequoia plate counts
12/16/05
Treatment: 100 ug/mL C255 with 10 ug/mL Cipro for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC255255 + Tob
Control + water7.315.796.856.36
10^610^67.455.866.795.63
Coupon 119Coupon 222
2228
2034
2.03E+072.80E+071234
Average2.42E+07cells/coupon1234
1.91E+07cells/cm^2
Control + Tobramycin
10^410^4
Coupon 160Coupon 284
7161
55
6.20E+057.25E+05
Average6.73E+05cells/coupon
5.31E+05cells/cm^2
C255 + water
10^510^5
Coupon 165Coupon 269
6343
8673
7.13E+066.17E+06
Average6.65E+06cells/coupon
5.25E+06cells/cm^2
C255 + Tobramycin
10^410^4
Coupon 1234Coupon 241
25339
19947
2.29E+064.23E+05
Average1.36E+06cells/coupon
1.07E+06cells/cm^2
C = ciprofloxacinT = tobramycin
12-16-05
0000
0000
Control Cont + T C255 + C C255 + C + T
Log cuf/coupon
C255 100ug/mL with 10ug/mL Cipro
1-14-06
Sequoia plate counts
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
Coupon 114Coupon 213
1731
18171234
1.63E+072.03E+071234
Average1.83E+07cells/coupon
1.45E+07cells/cm^2
Control + Tobramycin
10^410^5
Coupon 190Coupon 243
15522
15930
1.35E+063.17E+06
Average2.26E+06cells/coupon
1.78E+06cells/cm^2
C457 + water
10^510^5
Coupon 173Coupon 2129
76120
NCNC
7.45E+061.25E+07
Average9.95E+06cells/coupon
7.85E+06cells/cm^2
C457 + Tobramycin
10^410^4
Coupon 11Coupon 217
25
2NC
1.50E+042.10E+05
Average1.13E+05cells/coupon
8.88E+04cells/cm^2
C = ciprofloxacinT = tobramycin
1-14-06
0000
0000
Control Cont + Toby C457 C457 + Toby
Log cfu/coupon
C457 10ug/mL Treatment
1-21-06
Sequoia plate counts
1/21/06
Treatment: 50 ug/mL C457 for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC457457 + Tob
Control + water7.456.047.526.46
10^610^67.670.006.42
Coupon 135Coupon 248
2440
2552
2.80E+074.67E+071234
Average3.73E+07cells/coupon1234
2.95E+07cells/cm^2
Control + Tobramycin
10^410^4
Coupon 1113Coupon 20
1060
1110
1.10E+060.00E+00
Average1.10E+06cells/coupon
8.68E+05cells/cm^2
C457 + water
10^510^5
Coupon 1320Coupon 2TNTC
328TNTC
342TNTC
3.30E+07NC
Average3.30E+07cells/coupon
2.60E+07cells/cm^2
C457 + Tobramycin
10^410^4
Coupon 1245Coupon 2264
TNTC263
326TNTC
2.86E+062.64E+06
Average2.75E+06cells/coupon
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
Control Cont + Toby C457 C457 + Toby
Log cfu/coupon
C457 50 ug/mL Treatment
1-27-06
Sequoia plate counts
1/27/06
Treatment: 10 ug/mL C457 with 10 ug/mL Cipro for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC457457 + Tob
Control + water7.296.715.364.07
10^610^57.096.025.274.28
Coupon 113Coupon 2118
22117
23132
1.93E+071.22E+071234
Average1.58E+07cells/coupon1234
1.25E+07cells/cm^2
Control + Tobramycin
10^510^4
Coupon 173Coupon 265
30165
5282
5.17E+061.04E+06
Average3.10E+06cells/coupon
2.45E+06cells/cm^2
C457 + Cipro Control
10^410^4
Coupon 123Coupon 220
2119
2417
2.27E+051.87E+05
Average2.07E+05cells/coupon
1.63E+05cells/cm^2
C457 + Tobramycin
10^310^3
Coupon 110Coupon 221
1318
1218
1.17E+041.90E+04
Average1.53E+04cells/coupon
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
Control Cont + T C457 + C C457 + C + T
Log cfu/coupon
C457 10ug/mL with 10ug/mL Cipro
2-5-06
Sequoia plate counts
2/5/06
Treatment: 50 ug/mL C457 with 10 ug/mL Cipro for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC457457 + Tob
Control + water6.735.576.183.07
10^510^57.115.936.323.46
Coupon 147Coupon 298
61158
NCNC
5.40E+061.28E+071234
Average9.10E+06cells/coupon1234
7.18E+06cells/cm^2
Control + Tobramycin
10^410^4
Coupon 156Coupon 282
3189
25NC
3.73E+058.55E+05
Average6.14E+05cells/coupon
4.85E+05cells/cm^2
C457 + Cipro Control
10^510^5
Coupon 114Coupon 214
921
2228
1.50E+062.10E+06
Average1.80E+06cells/coupon
1.42E+06cells/cm^2
C457 + Tobramycin
10^210^2
Coupon 111Coupon 230
1634
823
1.17E+032.90E+03
Average2.03E+03cells/coupon
1.60E+03cells/cm^2
C = ciprofloxacinT = tobramycin
2-5-06
0000
0000
Control Cont + T C457 + C C457 + C + T
Log cfu/coupon
C457 50ug/mL with 10ug/mL Cipro
2-18-06
Sequoia plate counts
2/18/06
Treatment: 10 ug/mL C536 for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC536536 +Tob
Control + water7.196.227.016.44
10^510^56.995.687.296.37
Coupon 1186Coupon 2115
11789
16289
1.55E+079.77E+06
Average1.26E+07cells/coupon1234
9.97E+06cells/cm^21234
Control + Tobramycin
10^410^4
Coupon 1148Coupon 242
18642
16359
1.66E+064.77E+05
Average1.07E+06cells/coupon
8.42E+05cells/cm^2
C536 Control
10^510^5
Coupon 197Coupon 2198
111204
102188
1.03E+071.97E+07
Average1.50E+07cells/coupon
1.18E+07cells/cm^2
C536 + Tobramycin
10^510^5
Coupon 123Coupon 221
3622
2327
2.73E+062.33E+06
Average2.53E+06cells/coupon
2.00E+06cells/cm^2
C = ciprofloxacinT = tobramycin
2-18-06
0000
0000
Control Cont + Toby C536 C536 + Toby
Log cfu/coupon
C536 10 ug/mL Treatment
2-28-06
Sequoia plate counts
2/28/06
Treatment: 50 ug/mL C536 for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC536536 + Tob
Control + water6.11394335234.32221929475.5314789170
10^510^55.93785209334.31527043485.59476075260
Coupon 113Coupon 25
nc10
1311
1.30E+068.67E+05
Average1.08E+06cells/coupon1234
8.55E+05cells/cm^21234
** These control counts are around a log lower than normal.
Control + Tobramycin
10^310^3
Coupon 112Coupon 215
3021
nc26
2.10E+042.07E+04
Average2.08E+04cells/coupon
1.64E+04cells/cm^2
C536 Control
10^410^4
Coupon 124Coupon 246
4425
nc47
3.40E+053.93E+05
Average3.67E+05cells/coupon
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
Average0.00E+00cells/coupon
0.00E+00cells/cm^2
2-28-06
0000
0000
Control Cont + Toby C536 C536 + Toby
Log cfu/coupon
C536 50 ug/mL Treatment
3-4-06
Sequoia plate counts
3/4/06**This run will be repeated again
Treatment: 50 ug/mL C536 for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Coupon surface area =1.267cm^2
Control + water
10^610^6
Coupon 164Coupon 216
5624
4630
5.53E+072.33E+07
Average3.93E+07cells/coupon
3.10E+07cells/cm^2
Control + Tobramycin
10^410^5
Coupon 186Coupon 274
80149
NCNC
8.30E+051.12E+07
Average5.99E+06cells/coupon
4.73E+06cells/cm^2
C536 & Cipro Control
10^510^5
Coupon 1TNTCCoupon 2TNTC
TNTCTNTC
TNTCTNTC
0.00E+000.00E+00
Average0.00E+00cells/coupon
0.00E+00cells/cm^2
C536 & Cipro + Tobramycin
10^410^4
Coupon 1TNTCCoupon 2TNTC
TNTCTNTC
TNTCTNTC
0.00E+000.00E+00
Average0.00E+00cells/coupon
0.00E+00cells/cm^2
3-18-06
Sequoia plate counts
3/18/06
Treatment: 10 ug/mL C536 + 10 ug/mL Ciprofloxacin for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC536536 + Tob
Control + water7.357.015.654.30
10^610^67.566.455.233.78
Coupon 118Coupon 245
2535
2428
2.23E+073.60E+071234
Average2.92E+07cells/coupon1234
2.30E+07cells/cm^2
Control + Tobramycin
10^510^5
Coupon 199Coupon 237
12020
8528
1.01E+072.83E+06
Average6.48E+06cells/coupon
5.12E+06cells/cm^2
C536 & Cipro Control
10^410^4
Coupon 139Coupon 215
5512
4124
4.50E+051.70E+05
Average3.10E+05cells/coupon
2.45E+05cells/cm^2
C536 & Cipro + Tobramycin
10^310^3
Coupon 119Coupon 28**These are low for plate counts
203
217
2.00E+046.00E+03
Average1.30E+04cells/coupon
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
Control Cont + T C536 + C C536 + C + T
Log cfu/coupon
C536 10 ug/mL with 10ug/mL Cipro
3-25-06
Sequoia plate counts
3/25/06
Treatment: 50 ug/mL C536 + 10 ug/mL Ciprofloxacin for 24 hours
post treatment of 100 ug/mL Tobramycin for two hours
Log Counts
Coupon surface area =1.267cm^2ControlCont + TobC536536 + Tob
Control + water7.365.714.632.56
10^610^67.074.434.830.00
Coupon 126Coupon 210
228
2117
2.30E+071.17E+071234
Average1.73E+07cells/coupon1234
1.37E+07cells/cm^2
Control + Tobramycin
10^410^4
Coupon 157Coupon 23
522
453
5.13E+052.67E+04
Average2.70E+05cells/coupon
2.13E+05cells/cm^2
C536 & Cipro Control
10^310^3
Coupon 146Coupon 263
3466
4874
4.27E+046.77E+04
Average5.52E+04cells/coupon
4.35E+04cells/cm^2
C536 & Cipro + Tobramycin
10^110^1
Coupon 126Coupon 20
430
390
3.60E+020.00E+00
Average1.80E+02cells/coupon
1.42E+02cells/cm^2
Control RDR Bulk Fluid
10^7
8
11
9.50E+07cells/mL
C = ciprofloxacinT = tobramycin
3-25-06
0000
0000
Control Cont + T C536 +C C536 + C + T
Log cfu/coupon
C536 50 ug/mL with 10ug/mL Cipro
C = ciprofloxacinT = tobramycin
-
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