This is a presentation delivered at the 16th Annual Conference on the Science of Botanicals and 5th Annual Interim American Society of Pharmacognosy Meeting from April 11-14, 2016 in Oxford, MS, USA. Abstract: Answering the Call to Arms: Tools for Assessing the Anti-infective Potential of Natural Products in a Time of Rising Antibiotic Resistance Quave CL1,2 1 Center for the Study of Human Health, Emory University, 550 Asbury Circle, Candler Library 107, Atlanta, GA 30322 USA. 2 Department of Dermatology, Emory University School of Medicine, 615 Michael Street, Whitehead 105L, Atlanta, GA 30322 USA. As antibiotic resistance continues to rise, the pool of viable anti-infective therapeutic options is becoming rapidly exhausted. New therapies are in high demand and natural products are a likely source of novel bioactive compounds to meet this need. In particular, botanical secondary metabolites represent a rich pool for antibiotic discovery efforts. Plants are often the primary ingredients used in traditional anti-infective therapies, and yet their activity and mechanisms of action are often poorly understood. Much of the antibacterial research on botanical extracts and essential oils has focused on growth inhibitory studies using outdated methods limited in their ability to obtain an accurate assessment of bioactivity. The emergence of new molecular and bioanalytical tools for drug discovery provides a unique opportunity for application to natural products research. Using Staphylococcus aureus as a model, tools for anti-infective testing of plant extracts will be reviewed, specifically focusing on the merits and limitations of each method. Examples include standardized methods for examining activity for the inhibition of growth (e.g., MIC, MBC), virulence (e.g., quorum sensing and toxin quantification) and pathogenesis (e.g., biofilms and antibiotic synergy). Data from our recent discoveries of novel biofilm [1] and quorum sensing [2,3] inhibitors isolated from medicinal plants (Rubus ulmifolius, Castanea sativa and Schinus terebinthifolius) will be presented in the review of these tools. Acknowledgements: This work was supported by a grant from the National Institutes of Health, National Center for Complementary and Integrative Health (R01 AT007052). The content is solely the responsibility of the authors and does not necessarily reflect the official views of NCCIH or NIH. References: [1] Quave CL, Estévez-Carmona M, et al. (2012) PLoS ONE, 7(1): e28737. [2] Quave CL, Lyles JT, et al. (2015) PLoS ONE, 10(8): e0136486. [3] Quave CL, Horswill AR (2014) Frontiers in Microbiology, 5: 706.

1. Answering the Call to Arms:
Tools for Assessing the Anti-infective
Potential of Natural Products in a Time
of Rising Antibiotic Resistance
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. Introduction
Plants as a source of medicine
Willow
Aspirin
Foxglove
Digoxin/Digitoxin
Mayapple
Podophyllin/ Etoposide
Poppy
Codeine/ Morphine
Plant natural products are notably absent
among approved antibiotics.

3. Natural products in the Nobel Prize spotlight
Dr. Youyou Tu
Ming dynasty version
(1574 CE) of the
handbook. “A handful of
qinghao immersed with
2 liters of water, wring
out the juice and drink it
all” is printed in the fifth
line from the right.
artemisinin
Artemisia annua L., Asteraceae
Tu, Y. 2011. The discovery of artemisinin
(qinghaosu) and gifts from Chinese medicine
Nature Medicine 17: 1217–1220

4. Common criticisms for natural
products as anti-infectives
• Chemically complex extracts
• Synergistic interactions
• Lack of mechanistic studies
• Breaks Lipinski’s rules for small molecules
• New patent ruling (Myriad) & interpretation on
natural products
• Lack of scientific rigor and reproducibility in
assessment of biological activity

5. On the precipice of the post-antibiotic era
• 2M serious
infections, 23k
fatalities linked to
MDR infection in
USA
http://www.cdc.gov/drugresistance/threat-report-2013/

6. Looking to the future: A perfect storm
• Big pharma has
shown lack of
interest/investment
in antimicrobial
development
▫ High risk, low return
▫ More lucrative drug
options
▫ “low hanging fruit”
already captured
▫ Inevitable resistance
▫ Limited lifespan of
drug
Cooper & Shlaes. 2011. Nature 472:32

7. What happens when we have nothing
left in the arsenal?
• Implications for entire
healthcare infrastructure
and for military as well:
▫ Would cripple fields of surgery,
dentistry, oncology, pediatrics, etc.
▫ Mexican War (1845-1848) and the
Spanish-American War (1898):
number of disease-related deaths
outnumbered battlefield related
deaths by seven to one
Murray et al. 2008. J Trauma Injury, Infection, and Critical Care 64: S221-S231

8. Who can fill the antibiotics void?
▫ Academic scientists?
▫ Small biotech?
▫ A unique opportunity for phytochemists,
pharmacognosists and ethnobotanists?

9. Ethnobotany as a tool for discovery
• >450,000 plant species on
Earth*
• Targeted approach necessary
*Pimm & Joppa 2015 Annals of the Missouri Botanical
Garden. 100: 170-176

10. Biological Assessment of Natural Products:
Are We Asking the Right Questions?
• Classic antibiotic discovery has focused on
bacteriostatic and bactericidal action
• What about other MOAs?
▫ Targeting resistance
 Anti-biofilm therapies
 Antibiotic adjuvants
▫ Bacterial disarmament (anti-virulence approach)
▫ Host-directed therapies

11. Every model has strengths & weaknesses
• The bioassay matters
▫ Disc/well diffusion are NOT
suitable:
 for establishing MICs; or
 for comparing bioactivity
between extracts or between
extracts & antibiotics
▫ Use Clinical & Laboratory
Standards Institute
methods for MIC & MBC
▫ Mechanistic driven assays
(reporters, biofilms, etc.)
88
515
Diffusion
Assay
Other Assay
PubMed search of 2015 literature
revealed that 17% of in vitro studies on
antibacterial activity of plant extracts
used outdated diffusion techniques.

12. Every model has strengths & weaknesses
• To establish MIC50 or
MIC90, a plate reader is
necessary ($$$), or:
▫ MIC can be established by
eye, and further MBC by
colony plate count ($)
• Biofilm can be assessed by
CSLM ($$$), or:
▫ MBIC & MBEC can be
assessed by crystal violet
stain assay ($)
Concentration (µg/mL)

13. Clinical relevance
• The dose matters
▫ Crudes with >512 µg/mL activity not relevant to clinic
• The species & strain matters
▫ Focus on greatest area of clinical need – MDR pathogens
(ESKAPE and Gram-negative bacteria)
▫ Use MDR clinical isolates
• Relevant controls matter
▫ Mutant strains for desired phenotype are key
• Planktonic growth does not fully represent the clinical
reality (most infections are in biofilm in host)
• Resources exist:

14. Acquired vs. intrinsic resistance
• 17M new biofilm
infections/year in US =
550k fatalities
• Uni- or Poly-microbial
• Heightened gene exchange
• Slow growth/metabolism
• Matrix presents a physical
barrier to host immune
response and antibiotic
therapy

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

16. Elmleaf Blackberry
• Traditional uses in S. Italy:
▫ Leaves: furuncles, abscesses, and
other skin inflammations
▫ Roots: hair loss
▫ Fruits: food use
Rubus ulmifolius Schott., Rosaceae: The
source of the bioactive composition
“220D-F2”.
Quave et al., J Ethnobiol and Ethnomed 2009. 4(5)
Quave et al., J Ethnopharmacol 2008. 118:418-428

17. 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)

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

19. Castanea sativa Mill., Fagaceae
Quave et al. 2015 PLoS ONE 10(8): e0136486.

20. 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

21. 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!
Quave & Horswill. (2014) Flipping the switch. Frontiers in Microbiology. 5(706):1-10

22. Quorum Quenching Approach
• Quorum quenching
▫ “Disarming” bacteria
▫ Protect the host
▫ Adjuvant to existing lines
of antibiotics
▫ Anti-virulence strategy
• Accessory gene regulator
(agr) system
▫ controls virulence
X
X
X
X X
Not
Bactericidal
Quave & Horswill. (2014) Flipping the switch. Frontiers in Microbiology. 5(706):1-10
X

23. 224C-F2 inhibits agr in a nonbiocide manner
X X
Not
Bactericidal
Quorum
Quenching
Growth (OD)
agr
(Fluorescence)
Quave et al. 2015
PLoS One 10(8)

24. 224C-F2 reduces dermatopathology & morbidity
Quave et al. 2015 PLoS One 10(8)

25. • Early-stage toxicity tests as a counter-screen to
antimicrobial assays are critical!
?
“First, do no harm” - Hippocrates

26. “First, do no harm” - Hippocrates
• Early-stage toxicity tests as a counter-screen to
antimicrobial assays are critical!
Galleria mellonella is useful for toxicity and antibacterial
efficacy tests BEFORE moving to vertebrate models

27. 224C-F2 has limited impact on growth of common skin microflora.
Quave CL, Lyles JT, Kavanaugh JS, Nelson K, Parlet CP, et al. (2015) Castanea sativa (European Chestnut) Leaf Extracts Rich in
Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance. PLoS
ONE 10(8): e0136486. doi:10.1371/journal.pone.0136486
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0136486
Potential for off-target effects

28. What is the functional relevance of
the human microbiome?
• Basic principles :
• Microbes and host have co-evolved
and have a complex relationship
influenced by our environment
• Dysbiosis as a result of anti-infective
therapy can lead to disease states
• Disease may be expressed as a
consequence of signals sent in both
directions (host-bacteria)
• Take-home message: counter
screens for off-target effects on
commensal bacteria are critical!
28

29. Predictions on resistance…
• Alexander Fleming’s 1945 Nobel speech:
▫ “The time may come when penicillin can be bought by
anyone in the shops. Then there is the danger that the
ignorant man may easily underdose himself and by
exposing his microbes to non-lethal quantities of the
drug make them resistant.”
▫ Is this also true for antimicrobial plant
extracts??
YES!!

30. Subtherapeutic use of natural products can
also lead to resistance: Tea Tree Oil example
• Sublethal treatment with TTO =
≥2-fold increase in MIC to
antibiotics
• Repeated exposure to sublethal
concentrations of TTO to MRSA
and MSSA = 4-fold increase in
MIC for TTO
• Repeated use of commercial
products with TTO may lead to
development of skin flora with
TTO resistance
McMahon et al. 2008. Lett Appl Microbiol 47(4): 263-8

31. Conclusions
• Natural products can play an important role in future
anti-infective/antibiotic discovery pipeline
• Scientific rigor in biological assessment of extracts is
critical
▫ Recognize strengths/limitations of models
▫ Use clinically relevant strains
▫ Use standard methods for core testing
▫ Consider off-target effects early in discovery process
• Consider alternate MOAs (host-directed, anti-virulence,
anti-pathogenesis, antibiotic potentiation, etc.)
• Balance (symbiosis vs. dysbiosis) is key to human health
and should be considered early in discovery process

32. Acknowledgements
Quave Lab:
James T. Lyles, PhD
Kate Nelson
Rina Lee
Tracy Li
Justin Robeny
Adam Mackie
Matt Mendelsohn
Xinyi Huang
Past lab members:
Emily Mapelli
Nami Mottoghi
Amelia Muhs
Alex Pijeaux
Paula Tyler
Eugenia Addie-Noye
Matt Dorian
Parth Jarivala
Philanthropic DonorsR01 AT007052

33. Join us!
Society for Economic Botany
http://www.econbot.org/

34. Questions? Traditional Medicine
for infectious disease
Herbarium
voucher
Plant DNA
Botanical
Extracts
Microbiome
Biofilm formation
Planktonic growth
Quorum sensing
In vivo toxicity & efficacy
ValidationofTM
Cytotoxicity
Combo formulation testing
ProductInnovation
Platform
Antibiotic
potentiation
E-mail: cquave@emory.edu Website: http://etnobotanica.us/
Twitter: @QuaveEthnobot