Staphylococcus aureus biofilm

inhibitors from an elmleaf

blackberry extract

Cassandra L. Quave1, Cesar

Compadre2, Howard Hendrickson2,

Karen Beenken1, and Mark Smeltzer1

University of Arkansas for Medical Sciences, 1College of Medicine,

Department of Microbiology and Immunology; 2College of Pharmacy,

Department of Pharmaceutical Sciences

– Field to pharmacy approach to drug discovery

• Studying biological sources of traditional medical remedies

Ethnopharmacology

Elmleaf Blackberry

• Rubus ulmifolius Schott. (Rosaceae)

†Quave et al. Journal of Ethnobiology & Ethnomedicine. 2008. 4(5)

*Quave et al. Journal of Ethnopharmacology. 2008. 118:418-428

• Traditional uses in S. Italy†: – Leaves: furuncles,

abscesses, and other skin inflammations

– Roots: hair loss

– Fruits: eaten fresh and in marmalades

• Anti-biofilm activity first identified & published in 2008*

Questions

• Can the activity be increased with

fractionation/refinement of the extract?

• Is this effect due to interference with cell growth?

• What is its phytochemical makeup?

• Does it prevent biofilm formation regardless of

genotypic background?

• Does synergy occur when used as an adjuvant to

antibiotics?

Extraction of Rubus ulmifolius roots in EtOH (x 72 hours, twice):

Extract #220

220B (hexane partition)

Liquid-liquid partitioning

Water partition

220C (ethyl acetate partition)

Liquid-liquid partitioning

Water partition

220D (butanol partition)

Liquid-liquid partitioning

220E (water partition)

Column chromatography

220D-F1 (30:70 MeOH: CH2Cl2)

220D-F2 (40:60 MeOH: CH2Cl2)

220D-F3 (50:50 MeOH: CH2Cl2)

220D-F4 (60:40 MeOH: CH2Cl2)

220D-F5 (70:30 MeOH: CH2Cl2)

220D-F6 (80:20 MeOH: CH2Cl2)

220D-F7 (90:10 MeOH: CH2Cl2)

220D-F8 (100% MeOH)

220D-F9 (100% water)

Extr

ac

tio

n P

roto

co

l

300

mg/L

200

mg/L

100

mg/L

50

mg/L

300

mg/L

200

mg/L

100

mg/L

50

mg/L

Untr

ea

ted W

ild T

yp

e (

DM

SO

excip

ien

t)

UA

MS

-1 (

US

A2

00

)

22

0B

(H

exa

ne

pa

rtitio

n o

f 2

20

)

22

0 (

Cru

de

EtO

H e

xtr

act

of

R.

ulm

ifo

lius r

oo

ts)

22

0C

(E

thyl a

ce

tate

pa

rtitio

n o

f 2

20

)

22

0D

(B

uta

no

l p

art

itio

n o

f 2

20

)

22

0E

(W

ate

r p

art

itio

n o

f 2

20

)

22

0D

-F1

(30

:70

Me

OH

:CH

Cl2

fraction

)

22

0D

-F2

(40

:60

Me

OH

:CH

Cl2

fraction

)

Untr

ea

ted s

arA

- m

uta

nt (D

MS

O

excip

ien

t)

UA

MS

-17

82

(U

SA

30

0)

22

0D

-F4

(60

:40

Me

OH

:CH

Cl2

fraction

)

22

0D

-F3

(50

:50

Me

OH

:CH

Cl2

fraction

)

22

0D

-F5

(7

0:3

0 M

eO

H:C

HC

l2

fraction

)

22

0D

-F6

(80

:20

Me

OH

:CH

Cl2

fraction

)

22

0D

-F7

(90

:10

Me

OH

:CH

Cl2

fraction

)

22

0D

-F8

(10

0%

M

eO

H f

raction

)

22

0D

-F9

(1

00

% H

2O

fra

ctio

n)

Questions

• Can the activity be increased with

fractionation/refinement of the extract?

– Yes

• Is the anti-biofilm effect due to interference

with cell growth?

• What is its phytochemical makeup?

• Does it prevent biofilm formation regardless of

genotypic background?

• Does synergy occur when used as an adjuvant to

antibiotics?

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

0 4 8 12 16 20 24

Time (hours)

CF

U/m

l

Growth Curve

200 μg/ml 220D-F2 + UAMS-1 (wt)

UAMS-1 (wt) control

UAMS-929 (ΔsarA)

Questions

• Can the activity be increased with

fractionation/refinement of the extract?

– Yes

• Is the anti-biofilm effect due to interference with cell

growth?

– No

• What is its phytochemical makeup?

• Does it prevent biofilm formation regardless of

genotypic background?

• Does synergy occur when used as an adjuvant to

antibiotics?

25000

30000

35000

40000

45000

50000

0 2 4 6 8 10 12 14 16 18 20

Time (minutes)

To

tal

Co

un

ts (

m/z

290-5

00)

Chromatographic Fingerprint

C20H16O12

C20H18O14

LC-Q/ToFMS

Questions

• Can the activity be increased with fractionation/refinement of the extract? – Yes

• Is the anti-biofilm effect due to interference with cell growth? – No

• What is its phytochemical makeup? – Rich in phenolic glycosides

• Does it prevent biofilm formation regardless of genotypic background?

• Does synergy occur when used as an adjuvant to antibiotics?

Untreated

Wild type

300 μg/ml

200 μg/ml

100 μg/ml

50 μg/ml

Untreated

sarA- mutant

USA

100 USA 200 USA 300

USA

400

USA

500 USA

600

USA

700

USA

800

USA

1000 USA

1100

1893 1

UAMS I.D.

270 601 1894 1625 1782 1790 1039 1895 1896 1897 1898 1899 1900

220D-F2 is active against all PFT

A B C D E

F G J H I

US

A-2

00

(U

AM

S-1

) U

SA

-30

0 (

UA

MS

-17

82

)

Wild type ΔsarA mutant 100 μg/ml of 220D-F2 50 μg/ml of 220D-F2 12.5 μg/ml of 220D-F2

Confocal Laser Scanning Microscopy

Questions

• Can the activity be increased with fractionation/refinement of the extract? – Yes

• Is the anti-biofilm effect due to interference with cell growth? – No

• What is its phytochemical makeup? – Rich in phenolic glycosides

• Does it prevent biofilm formation regardless of genotypic background? – Yes

• Does synergy occur when used as an adjuvant to antibiotics?

Clindamycin (10X MIC)

Treatment Day Treatment Day

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 5 Day 6 Day 7

CF

U/

cath

ete

r

p <0.001

p <0.05 p <0.001

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

CF

U/c

ath

ete

r

Biofilm Control (UAMS-1)

200 μg/ml 220D-F2

5 μg/ml Clindamycin

200 μg/ml 220D-F2 + 5 μg/ml Clindamycin

Daptomycin (10X MIC)

Treatment Day Treatment Day

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 5 Day 6 Day 7

CF

U/

cath

ete

r

p <0.05

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

CF

U/c

ath

ete

r

Biofilm Control (UAMS-1)

200 μg/ml 220D-F2

10 μg/ml Daptomycin

200 μg/ml 220D-F2 + 10 μg/ml Daptomycin

Vancomycin (10X MIC)

Treatment Day Treatment Day

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

CF

U/c

ath

ete

r

Biofilm Control (UAMS-1)

200 μg/ml 220D-F2

20 μg/ml Vancomycin

200 μg/ml 220D-F2 + 20 μg/ml Vancomycin1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 5 Day 6 Day 7

CF

U/

cath

ete

r

p <0.05

Oxacillin (1X MIC)

Treatment Day Treatment Day

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 5 Day 6 Day 7

CF

U/

cath

ete

r

p <0.001

p <0.05

p <0.05

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

CF

U/c

ath

ete

r

Biofilm Control (UAMS-1)

200 μg/ml 220D-F2

0.5 μg/ml Oxacillin

200 μg/ml 220D-F2 + 0.5 μg/ml Oxacillin

Conclusions

• Extract 220D-F2 is effective at:

– Inhibiting biofilm formation in all PFT of S. aureus

– Reducing biofilm biomass when used in combo with

antibiotics

• Increases therapeutic efficacy of Daptomycin,

Vancomycin, Clindamycin, and Oxacillin in biofilm

removal

Future Work

• Animal studies leading to a BDA (botanical drug

application)

– Efficacy/feasibility in prophylactic and therapeutic

applications

– Safety (acute toxicity, genotoxicity, carcinogenicity,

reproductive toxicology)

– Bioavailability

• Other important issues to address:

– Formulation

– MOA

• Long-term goal: clinical trials for treating biofilm-

associated S. aureus infections

Acknowledgements • Funding

– NIH NCCAM #F32AT005040 to C.L. Quave

– NIH NIAID #R01-AI43356 to M.S. Smeltzer

• People – Smeltzer lab team

• Karen Beenken

• Lara Mrak

• Tenita McCoy

• Michelle Griffin

• Aga Zielinska

• Gerren Hobby

• Justin Vaughn

– Compadre lab team • Paola Ordoñez

• Jadirra Ordoñez

– Participating Communities in Italy

http://www.etnobotanica.us/ cassy.quave@gmail.com