This is the keynote lecture given at the University of Illinois at Chicago Garden Walk event in the department of Pharmacognosy. The objectives of the talk were: ·Discuss the role of medical ethnobotany in drug discovery efforts ·Explore state-of-the-art research techniques that examine the activity of botanical natural products with next generation antibiotic discovery efforts focused on “alternative targets”, such as bacterial communication systems ·Provide examples of current research underway by her group both in the field (especially through fieldwork in the Mediterranean) and the lab (natural product research on multidrug resistant bacteria).

1. Medical Ethnobotany & the
Discovery of New Drugs for
Antibiotic Resistant Infections
Cassandra L. Quave, Ph.D.
Assistant Professor of Dermatology & Human Health
Curator, Emory University Herbarium
E-mail: cquave@emory.edu
Lab Website: http://etnobotanica.us/
Twitter: @QuaveEthnobot

2. Plants as a source of medicine
Willow
Aspirin
Foxglove
Digoxin/Digitoxin
Mayapple
Podophyllin/ Etoposide
Poppy
Codeine/ Morphine

3. Overview
• Ethnobotanical approach to drug discovery
• Antibiotic resistant bacteria as a target
• New strategies for evaluating old remedies
▫ Quorum quenchers
 Chestnut
▫ Biofilm inhibitors
 Elmleaf Blackberry
• Next generation of anti-infectives

4. The Science of Ethnobotany
• Ethnobotany (from
ethnology, study of culture,
and botany, study of
plants) is the scientific
study of the relationships
that exist between peoples
and plants.
• Ethnobotany is the
science of survival. Ethnobotanical-directed study of plants
used for infectious disease is often more
effective than a random approach.

5. Ethnobotany is Multidisciplinary
Ethnobotany
Botany
Chemistry
Microbiology
Anthropology
Linguistics
Pharmacology
• Medicine
• Conservation
• Food security

6. Deter other
plant
species
from
growing
nearby
Fight off
microbial
invasion &
infection
Attract pollinatorsDefense against
herbivory
Why do plants make medicinal compounds?

7. How to find new medicines?

8. Approach to New Lead Identification
Field-work&Lit.Reviewof
MedicinalPlantsforSSTI
Plant collection &
Taxonomic
identification
Process materials &
prepare extracts
Isolation/elucidation
of active leads or
marker compounds
Animal Studies
DM/PK and
Formulation
Studies
Proof of Concept
Clinical Trials
Bioassay-guided
fractionation
MIC/MBC
Quorum Sensing
Cytotoxicity
Biofilms

9. Study Sites in the Mediterranean

10. Mount Vulture – Basilicata, Italy

13. Plant Collecting

14. Plant Collecting

15. Plant Collecting

16. Origanum heracleoticum L., Lamiaceae

17. Plant Collecting
DNA barcoding
Herbarium Vouchers

18. Plant Extraction
Dried 48-72 hrs
Vacuum-sealed with
silica packets
Pulverized with a
grinder

19. 1:10 extraction in
95% EtOH or MeOH
for 72 hrs. or boiled
in water for 30
minutes
Plant materials
separated from
extract with
vacuum filtration
Solvent removed
under reduced
pressure with a
rotary evaporator
Plant Extraction

20. After freezing at -80°C,
extracts are lyophilized Dried extracts
scraped out and
weighed
DMSO added,
creating a drug
solution for
antibacterial
testing
Plant Extraction

21. Laboratory studies
• Antibacterial testing
• Antibiotic resistance
• Bacterial communication
pathways
• Research targets:
▫ MRSA
▫ Eczema
▫ Acne
▫ Pneumonia
▫ Ear infections
▫ Skin infections
▫ Medical device infections

22. Clinical Relevance
• On the precipice of the post-
antibiotic era?
▫ 2M serious infections, 23k fatalities
linked to MDR infection in US
• Intrinsic vs. acquired resistance
▫ 17M new biofilm infections/year
in US = 550k fatalities
• Antibiotic pipeline nearly empty
• We already face a high economic
burden for infectious disease
(HAI’s in US = $28.4-45 B)

23. Staphylococcus aureus as a Pathogen
• Opportunistic pathogen
• Colonizes nasal passages of 30%
healthy adults in US
• Leading cause of:
▫ Bacteremia
▫ Sepsis
▫ Brain abscesses
▫ Medical device infections
▫ Skin and soft tissue infections (SSTI)
• Commonly implicated in:
▫ Bone and joint infections
▫ Surgical site infections
▫ Pneumonia
▫ Endocarditis
• HA-MRSA vs. CA-MRSA

24. S. aureus exotoxins cause serious disease
Toxic Shock
Syndrome Toxin
(TSST-1)
Pyrogenic Toxin
Superantigens
Scalded Skin
Syndrome
Exfoliative Toxins
Abscesses,
Necrosis, Sepsis
Hemolytic Toxins,
Proteases, Lipases

25. S. aureus immune evasion mechanisms
Rigby and De Leo. 2012 Semin. Immunopathology 34:237-29

26. S. aureus immune evasion mechanisms
Rigby and De Leo. 2012 Semin. Immunopathology 34:237-29

27. Quorum Quenching Approach
• Quorum quenching
▫ “Disarming” bacteria
▫ Protect the host
▫ Adjuvant to existing lines
of antibiotics
• Accessory gene regulator
(agr) system
▫ controls virulence
Be Toxic!
Be Toxic!
Be Toxic!
Be Toxic!

28. Quorum Quenching Approach
• Quorum quenching
▫ “Disarming” bacteria
▫ Protect the host
▫ Adjuvant to existing lines
of antibiotics
• Accessory gene regulator
(agr) system
▫ controls virulence
X
X
X
X X
Not
Bactericidal
Be Toxic!

29. RNAII
AgrB AgrC
agrA agrC agrD agrB P3P2 RNAIII
PSMα1-4, β1-2
agrD
Extracellular environment
Intracellular environment
AIP
AIP
AIP
AIP
AIP
AIP
agrA PagrA
Cell wall associated proteins
fibronectin binding proteins, Protein A
Exoproteins:
TSST-1, haemolysins, exfoliative toxins,
enterotoxins, proteases
Accessory Gene Regulator (agr) system

30. Castanea sativa Mill. (Fagaceae)

31. 224
224C
224C-F2
Fractionation
Scheme
agr P3-GFP reporters used to guide fractionation

32. Quorum quenching by multiple small molecules
0.5
0.7
0.9
1.1
0 12 24 36 48 60 72 84 96
OD600
Retention Time (min)
Growth Inhibition
0
50
100
150
200
250
300
0 12 24 36 48 60 72 84 96
Fluorescence
Retention Time (min)
Quorum Sensing
DMSO control
Test concentration = 25 μg/ml
1 standard deviation
2 standard deviations

33. 224C-F2 inhibits agr in a nonbiocide manner
X X
Not
Bactericidal
Quorum
Quenching
Growth (OD)
agr
(Fluorescence)

34. 224C-F2 blocks d-toxin production

35. 224C-F2-treated S. aureus does not
harm human keratinocytes

36. 224C-F2-treated S. aureus does not
produce alpha-hemolysin or lyse RBCs

37. 224C-F2 is non-toxic to human cells & murine skin

38. 224C-F2 reduces dermatopathology & morbidity

39. Passaging experiments do not yield
resistant mutants

40. RT: 0.00 - 109.62
0 10 20 30 40 50 60 70 80 90 100
Time (min)
0
10
20
30
40
50
60
70
80
90
100 NL:
2.27
Bas
FTM
Full
[150
1500
050
35
36
1
2
3
37, 38
39, 40
41
42
43
44
46
45
47
48
49
50
57
52
54
53
55
58
56
51
59
60
61
62
63
64
65
66
Time (min)
RelativeAbundance
224C-F2 LC-MS Chromatogram
94

41. Core Structures
ursene oleanene
plus

42. Mechanism of action?

43. Conclusions
Ethnobotanical Lead Effective and Safe
Common Core Structure &
New Compounds
Further Isolation and NMR
Quave et al., Submitted

44. Elmleaf Blackberry
• Traditional uses in S. Italy:
▫ Leaves: furuncles, abscesses,
and other skin inflammations
▫ Roots: hair loss
▫ Fruits: eaten fresh and in
marmalades
• One of 116 remedies related
to SSTIs and other topical
dermatological treatments
identified
Quave et al. Journal of Ethnobiology & Ethnomedicine. 2008. 4(5)
Quave et al. Journal of Ethnopharmacology. 2008. 118:418-428
Rubus ulmifolius Schott. (Rosaceae):
The source of the bioactive composition
“220D-F2”.

45. Biofilms and Intrinsic Resistance
• 5-step Process:
1. Initial attachment
2. Irreversible attachment
3. Maturation I
4. Maturation II
5. Dispersion
• Uni- or Poly-microbial
• Heightened gene exchange
• Slow growth/metabolism
• Matrix presents a physical
barrier to host immune
response and antibiotic
therapy
James et al. (2008) Biofilms in chronic wounds. Wound Repair Regen. 16(1):37-44

46. Models for Biofilm
Formation & Dispersal
microtiter plate
catheters in vivo
(Imaging with IVIS)
flow cells
catheters in vitro

47. Bioassay-Guided Fractionation Scheme

48. Following bioassay-guided fractionation steps,
ellagitannins and glycosylated ellagic acid derivatives
were isolated from the extract.
Quave et al., PLoS One. 2012: 7(1)

49. 220D-F2 is effective against all clonal lineages of
S. aureus, regardless of antibiotic resistance
profile and is nontoxic to mammalian cell lines.
Quave et al., PLoS One. 2012: 7(1)

50. 220D-F2 improves response to functionally distinct
classes of antibiotics, including daptomycin,
clindamycin, vancomycin, and oxacillin.
Quave et al., PLoS One. 2012: 7(1)

51. 220D-F2 kills Streptococcus pneumoniae
C
Talekar et al., PLoS One. 2014: 9(5)

52. Talekar et al., PLoS One. 2014: 9(5)
220D-F2 disrupts Streptococcus
pneumoniae biofilms & kills cells

53. Conclusions
Ethnobotanical Lead Effective and Safe
Common Core Structure Further Isolation and NMR
Quave et al., PLoS ONE 2012,; Talekar et al., PLoS ONE 2014

54. Traditional Medicine
for SSTI
Herbarium
voucher
Plant DNA
Botanical
Extracts
δ-hemolysin
RBC lysis
Biofilm formation
Planktonic growth
Quorum sensing
In vivo abscess
ValidationofTM
Cytotoxicity
Antibiotic Combos

55. Acknowledgements
Quave Lab:
James T. Lyles, PhD
Kate Nelson
Eugenia Addie-Noye
Matt Dorian
Avni Hajdari
Parth Jarivala
Rina Lee
Tracy Li
Emily Mapelli
Nami Mottoghi
Amelia Muhs
Alex Pijeaux
Bledar Pulaj
Paula Tyler
Horswill Lab:
Alexander Horswill
Heidi Crosby
Kristopher Heilmann
Jeffery Kavanaugh
Corey Parlet
Philanthropic Donors

56. Questions?
Traditional Medicine
for SSTI
Herbarium
voucher
Plant DNA
Botanical
Extracts
δ-hemolysin
RBC lysis
Biofilm formation
Planktonic growth
Quorum sensing
In vivo abscess
ValidationofTM
Cytotoxicity
Antibiotic Combos