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 HealthCurator, Emory University HerbariumE-mail: cquave@emory.eduLab Website: http://etnobotanica.us/Twitter: @QuaveEthnobot

IntroductionPlants as a source of medicine

WillowAspirin

FoxgloveDigoxin/Digitoxin

MayapplePodophyllin/ Etoposide

PoppyCodeine/ Morphine

Plant natural products are notably absent among approved antibiotics.

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 medicineNature Medicine 17: 1217–1220

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

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/

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

drugCooper & Shlaes. 2011. Nature 472:32

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

Who can fill the antibiotics void?▫Academic scientists?▫Small biotech?▫A unique opportunity for phytochemists,

pharmacognosists and ethnobotanists?

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

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

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.)

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Diffusion AssayOther Assay

PubMed search of 2015 literature revealed that 17% of in vitro studies on antibacterial activity of plant extracts used outdated diffusion techniques.

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)

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:

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

Models for Biofilm Formation & Dispersal

microtiter plate

catheters in vivo (Imaging with IVIS)

flow cells

catheters in vitro

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

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)

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)

Castanea sativa Mill., Fagaceae

Quave et al. 2015 PLoS ONE 10(8): e0136486.

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

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

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

XX

X X

Not Bactericidal

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

X

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)

224C-F2 reduces dermatopathology & morbidity

Quave et al. 2015 PLoS One 10(8)

•Early-stage toxicity tests as a counter-screen to antimicrobial assays are critical!

?

“First, do no harm” - Hippocrates

“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

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.0136486http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0136486

Potential for off-target effects

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!

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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!!

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

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

AcknowledgementsQuave Lab:James T. Lyles, PhDKate NelsonRina LeeTracy LiJustin RobenyAdam MackieMatt MendelsohnXinyi Huang

Past lab members:Emily MapelliNami MottoghiAmelia MuhsAlex PijeauxPaula TylerEugenia Addie-NoyeMatt DorianParth Jarivala

Philanthropic DonorsR01 AT007052

Join us! Society for Economic Botany

http://www.econbot.org/

Questions? Traditional Medicine for infectious disease

Herbarium voucher Plant DNA

Botanical Extracts

Microbiome

Biofilm formation

Planktonic growth

Quorum sensing

In vivo toxicity & efficacy

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Cytotoxicity

Combo formulation testing

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Antibioticpotentiation

E-mail: cquave@emory.edu Website: http://etnobotanica.us/ Twitter: @QuaveEthnobot