1. Teixobactin and Ichip:
Novel Solutions for the
Treatment of Resistant
Bacterial Infections
ELIZABETH DAUGHERTY, PHARM.D. AND MPH CANDIDATE
VIRGINIA FLEMING, PHARM.D., BCPS, FACULTY ADVISOR
UNIVERSITY OF GEORGIA COLLEGE OF PHARMACY
OC TOBER 14, 2015

2. Objectives
• Discuss Antibiotic Resistance
• What is it?
• What are the costs?
• How does it happen?
• Who is at risk?
• What is the treatment?
• Introduce Ichip, a revolutionary method of in situ bacterial cultivation
• Introduce Teixobactin, a novel antibiotic popularly referred to as “resistance-proof”
• Examine the clinical relevance of these discoveries
2

3. Antibiotic Resistance
• “The ability of bacteria to resist the effects of an antibiotic – that is, the bacteria are not
killed, and their growth is not stopped. Resistant bacteria survive exposure to the antibiotic
and continue to multiply in the body, potentially causing more harm and spreading to other
animals or people.”
• Resistant infections cause severe illnesses that:
• Require lengthy hospitalizations and expensive medical treatment
• Involve increased recovery time
• May be be ultimately untreatable, resulting in death
• Resistant infections are generally treated with second- or third- line drugs of choice that are:
• Less effective, and possibly ineffective
• More toxic
• More expensive
3

4. Antibiotic-Resistant
Pathogens (Superbugs)
1. Clostridium Difficile (CDIFF)*
2. Carbapenem-Resistant
Enterobacteriaceae (CRE)
3. Neisseria gonorrhoeae
4. Multidrug-Resistant
Acinetobacter
5. Drug-Resistant Campylobacter
6. Fluconazole-Resistant Candida
7. Extended Spectrum
Enterobacteriaceae (ESBL)
8. Vancomycin-Resistant
Enterococcus (VRE)
9. Multidrug-Resistant
Pseudomonas aeruginosa
10. Drug-Resistant Non-Typhoidal
Salmonella
11. Drug-Resistant Salmonella
Serotype Typhi
12. Drug-Resistant Shigella
13. Methicillin-Resistant
Staphylococcus aureus (MRSA)
14. Drug Resistant Streptococcus
pneumoniae
15. Drug-Resistant Tuberculosis
(MDR/DXR TB)**
16. Vancomycin-Resistant
Staphyloccocus aureus (VRSA)
17. Etrythromycin Resistant Group
A Streptococcus
18. Clindamycin-Resistant Group B
Streptococcus
4

5. Antibiotic Resistance Costs
• Financial Costs
• $18588 - $29069 in medical costs per patient
• $20 Billion in health care system costs each year
• Societal Cost
• Hospital stays are extended by 6.4 – 12.7 days on average
• $35 Billion to U.S. households due to lost wages
• Cost of Death
• Patients with antibiotic-resistant infections are twice as likely to die as antibiotic-susceptible infections
• Premature deaths represent and emotional and financial burden on society
5

6. 6

7. Antibiotic Resistance Development and Spread
• The use of antibiotics is the single most
important factor leading to antibiotic
resistance around the world.
• Simply using antibiotics creates resistance.
• Using antibiotics inappropriately speeds
up the development and spread of
resistance
• Antibiotics are not properly prescribed up
to 50% of the time
• Not needed (both humans and food-animals)
• Inappropriate drug
• Inappropriate dosage
• Inappropriate duration
7

8. Antibiotic Resistance Risk Factors
• Patient Populations:
• Cancer chemotherapy
• Complex surgery
• Rheumatoid arthritis
• ESRD/Dialysis
• HIV/AIDS
• Organ and bone marrow
transplants
• Previous high-risk infection
• Environmental Factors:
• Geographical location
• Overcrowding (hospitals and farms)
• Poor hygiene/sterilization
8

9. Antibiotic Resistance “Treatment”
9

10. 10

11. 11

12. The Search for New Antibiotics
• Bacteria produce antibiotics as self-defense
mechanisms against other bacteria. They
compete with and try to out-evolve one
another
• Nearly all of our antibiotics are either
• Natural compounds produced by bacteria
• Synthetic derivatives of those compounds
• We look for new antibiotics in biologically dense
and competitive environments, but drug screens for
tend to re-discover the same compounds over and
over again
• Approximately 99% of all bacteria are “uncultivable”
in a laboratory, which makes them impossible to
screen for possible antibiotic targets
12
Antibiotic Producer organism Activity
Penicillin Penicillium chrysogenum Gram-positive bacteria
Cephalosporin
Cephalosporium
acremonium
Broad spectrum
Griseofulvin Penicillium griseofulvum Dermatophytic fungi
Bacitracin Bacillus subtilis Gram-positive bacteria
Polymyxin B Bacillus polymyxa Gram-negative bacteria
Amphotericin B Streptomyces nodosus Fungi
Erythromycin Streptomyces erythreus Gram-positive bacteria
Neomycin Streptomyces fradiae Broad spectrum
Streptomycin Streptomyces griseus Gram-negative bacteria
Tetracycline Streptomyces rimosus Broad spectrum
Vancomycin Streptomyces orientalis Gram-positive bacteria
Gentamicin Micromonospora purpurea Broad spectrum
Rifamycin Streptomyces mediterranei Tuberculosis

13. Literature Search
• PubMed
• Limits
 Publication date: 2010 – 2015
 Journal Article
• Search terms: “teixobactin, resistance”
• Returned 5 results
• Search terms: “ichip, bacteria”
• Returned 9 results
13

14. Study 1
Use of Ichip for High-Throughput In Situ
Cultivation of Uncultivable Microbial Species
D. Nichols, N. Cahoon, E. Trakhtenberg, L.
Pham, A. Mehta, A. Belanger, T. Kanigan, K.
Lewis, S. D. S. Epsetin
Applied and Environmental Microbiology, 76(8),
2445-2450. doi:10.1128/AEM.01754-09
February 19, 2010
14

15. Objectives
• To demonstrate that cultivation of environmental microorganisms inside the Ichip incubated
in situ leads to a significantly increased colony count over that observed on synthetic media
• To demonstrate that species grown in Ichips are different from those registered in standard
petri dishes and are highly novel.
• Rationale:
• Most of the bacterial diversity of nature is inaccessible for either basic or applied research. The
microbial species cultivable by conventional methods are widely considered over-mined for secondary
metabolites, and the probability of discovery of a novel bioactive compound is low.
• The “uncultivable” microbial majority arguably represents our planet’s largest unexplored pool of
biological and chemical novelty.
15

16. 16

17. Methods
• Colony Count
• Seawater and Soil samples were taken from the
local environment
• Samples were diluted to a concentration of 103
cells/ml in warm aqueous LB agar
• Three each of diffusion chambers, ichips and petri
dishes were inoculated with 500μL of cell-agar mix
(approximately 500 cells each)
• Cells were incubated for 2 weeks
• Seawater samples were suspended in seawater on water tables
of the flowthrough seawater system
• Soil samples were buried in waterlogged soil
• Petri dishes were incubated in the lab at room temperature
• After incubation, 5-10% of the agar from each
vessel was selected randomly and examined under
a microscope to determine microbial recovery
• Microbial diversity
• Microorganisms grown in ichips and petri dishes
were identified using 16S rRNA gene sequences
• Sequences were clustered into operational
taxonomic units (OTUs) based on 99, 97, 95 and
90% similarities
• The sequence least different from the others
within each group was compared to the NCBI
database to establish identity
17

18. Results: Colony Counts
• The colony counts were higher in the ichips
than in diffusion chambers or petri dishes
regardless of the environment studied.
• In ichips, growing cells constituted over 40%
(seawater) and 50% (soil) of the number of
cells inoculated but were not statistically
different from those obtained from the
diffusion chambers (P = 0.05).
• Colony counts in petri dishes were
approximately 5-fold lower, with statistically
significant difference from either the ichip-
or diffusion chamber-derived recoveries (P =
0.02).
18

19. Results: Microbial Diversity
• Four libraries of gene fragments were
established
• Ichip, seawater: 635 clones
• Ichip, soil: 525 clones
• Petri dish, seawater: 265 clones
• Petri dish, soil: 314 clones
• 173 additional clones were too short or
chimeric to sequence
19

20. Results: Microbial Diversity
• 10 different phyla were detected overall, 5
were unique to either the ichip or petri dish
• 129 species were detected in the ichips
• 6 species found in both habitats
• 85 species were detected in the petri dishes
• 17 species found in both habitats
• Species overlap between the ichip and petri
dish arms were minimal, even when
comparing OTUs that are 90% similar
• Only one species was shared between the ichip
and petri-dish cultures (Vibrio sp. ATC
EU655333)
20

21. Results: Phylogenetic Novelty
• Ichip- and petri dish-derived strains (OTUs
sharing 99% identity) were different in the
degrees of their phylogenetic novelty.
• The same is observed even with the 90% OTUs.
• ichip- and petri dish-based methods recover
not only entirely different microbial strains and
species but also different genera and families.
• In petri dishes, the most frequently
observed class of strains shares 97 to 100%
identity with previously cultivated species.
• In ichips, the most frequently observed class
of strains exhibits 94 to 97% identity with
known species.
21

22. Authors’ Conclusions
• The ichip is a practical device for massively parallel in situ cultivation of environmental
microorganisms.
• Ichip incubation leads to a significantly increased colony count compared to what can be
achieved with traditional laboratory-based methods
• Organisms growing in ichips are more likely to be novel than those grown by standard
approaches.
• The microbial species cultivated in ichips are different and more diverse than those grown
with traditional laboratory-based methods
• Discovery of a novel antibiotic from microorganisms cultivable by conventional means (petri
dishes in laboratories) is extremely unlikely, with a probability of 10-7 per isolate.
22

23. Limitations
• The ichip devices were allowed to incubate in the soil/seawater for only 2 weeks, which may
have limited the bacterial growth of some more slowly-growing species.
• Diffusion chambers were not utilized in the microbial diversity and novelty tests.
• After initial cultivation in the ichip, it is still difficult to domesticate the colonies in vitro
• 26% of chamber-reared colonies domesticating after the first round of in situ cultivation
• 40% after two rounds
• 60% still unable to be grown in lab
23

24. Seminarian’s Conclusions
• Ichip represents a significant improvement over previous cultivation techniques, but further
improvements to this system can still be made.
• Ichip is a promising new technology that can be used in different environments around the
world to cultivate previously unknown bacteria.
• Considering the wealth of antibiotics we have derived from the 1% of easily cultivable
bacteria, it is exciting to think of what new antibiotics will be derived from the isolates
discovered by ichip.
24

25. Study 2
A new antibiotic kills pathogens without
detectable resistance
Losee L. Ling, Tanja Schneider, Aaron J. Peoples,
Amy L. Spoering, Ina Engels, Brian P. Conlon,
Anna Mueller, Till F. Schäberle, Dallas E.
Hughes, Slava Epstein, Michael Jones, Linos
Lazarides, Victoria A. Steadman, Douglas R.
Cohen, Cintia R. Felix, K. Ashley Fetterman,
William P. Millett, Anthony G. Nitti, Ashley M.
Zullo, Chao Chen, & Kim Lewis
Nature, 517, 455-459.
doi:10.1038/nature14098
January 22, 2015
25

26. Objective
• Report the discovery of a new cell wall inhibitor, Teixobactin:
• Isolation and cultivation of producing strains
• Extract preparation and screening for
activity
• Sequencing of the strain
• Strain identification
• Biosynthetic cluster identification
• Strain fermentation and purification of
teixobactin
• Proposed Mechanism of Action
• Minimum inhibitory concentration (MIC)
• Spectrum
• Minimum bactericidal concentration (MBC)
• Time-dependent killing
• Resistance studies
• Mammalian cytotoxicity
• Hemolytic activity
• Macromolecular synthesis
• Intracellular accumulation of UDP-N-acetyl-
muramic acid pentapeptide
• Cloning, overexpression and purification of
S. aureus UppS and YbjG as His6-tag fusions.
• In vitro peptidoglycan synthesis reactions
• Synthesis and purification of lipid
intermediates
• Antagonization assays
• Complex formation of teixobactin
• hERG inhibition testing
• Cytochrome P450 inhibition
• In vitro genotoxicity
• DNA binding
• Plasma protein binding
• Microsomal stability
• Pharmacokinetic analysis
• Mouse sepsis protection model
• Mouse thigh infection model
• Mouse lung infection model
26

27. Eleftheria terrae
• Discovered by a team of scientists from
Northeastern University and NovoBiotic
Pharmaceuticals
• A Gram-Negative bacteria initially isolated from
a sample of soil taken from “a grassy field in
Maine.”
• Cultured in situ using ichip (isolation chip)
technology
• Produces Teixobactin as a defense mechanism
against other bacteria
• Teixobactin is transported outside E. terrae’s
outer membrane permeability barrier and
targets binding sites exposed on cell membrane
of nearby Gram-positive bacteria
27

28. Mechanism of Action
28

29. Minimum Inhibitory Concentration (MIC)
• MIC was determined by broth microdilution
• Test mediums:
• Mueller-Hinton broth (MHB) for most species
• MHB with 3% lysed horse blood for Streptococci
• Haemophilus Test Medium for H. influenza
• Middlebrook 7H9 broth for mycobacteria
• Schaedleranaerobe broth for C. difficile
• Incubation:
• 20 h at 37°C for most species
• 2 days at 37°C for M. smegmatis
• 7 days at 37°C for M. tuberculosis)
• MIC was defined as the lowest concentration
of antibiotic with no visible growth.
29

30. 30
MRSA →
VISA →
VRE →
TB → ← CDIFF
← Anthrax

31. Resistance Studies
• S. aureus was cultured in the presence of subinhibitory
(0.25, 0.5, 1, 2, and 4 X MIC) levels of Texiobactin and
Ofloxacin (control).
• Every 24 hours for 30 days, cultures that allowed growth were diluted
into fresh media containing 0.25, 0.5, 1, 2 and 4 X MIC Teixobactin.
• Any cultures that grew at higher than the MIC were passaged on drug-
free MHA plates and the MIC was determined by broth dilution.
• A change in the MIC greater than the parent S. aureus titer would
indicate that resistance had developed.
• The experiment was repeated a second time for another 30
days.
• The experiment was repeated a third time for 27 days with
smaller incremental increases in the drug concentration
during passaging (0.25, 0.5, 0.75, 1, 1.25, 1.5, and 2 X MIC).
• No S. aureus mutants resistant to Teixobactin were found.
31

32. Mechanism of Resistance?
• E. terrae does not employ an alternative
pathway for cell wall synthesis that other
bacteria could “steal.”
• The most common type of antibiotic resistance
(inactivating enzymes like β-lactamase) is
unknown for Vancomycin and therefore unlikely
to develop for Teixobactin.
• If there is a bacteria that already exists which is
resistant to Teixobactin, it is uncultivable in lab
(just like E. terrae) and unlikely to pass its
resistance to other bacteria in humans.
32

33. Toxicity and Adverse Effects
• Mammalian cytotoxicity
• Teixobactin showed no toxicity towards
exponentially growing NIH/3T3 mouse embryonic
fibroblasts and HepG2 cells.
• Teixobactin cannot target mammalian cells
• Hemolytic activity
• Teixobactin was added to resuspended human red
blood cell precipitants.
• After 1 hour the cells were centrifuged and the
supernatant was measured for lysed cells.
• No hemolytic activity
• Cardiotoxicity
• Teixobactin showed a lack of activity against hERG.
• Genotoxicity
• Teixobactin did not produce genotoxic metabolites
when tested in an in vitro micronucleus test.
• No evidence of genotoxicity was observed
• Cytochrome P450 Inhibition
• Teixobactin and control compounds were incubated
with human liver microsomes to determine their
effect on 5 major human cytochromes.
• 16% inhibition of 1A2, 2C9, 2C19, and 3A4.
• 33% inhibition of 2D6.
33

34. Animal Studies
• All animal studies conformed to institutional
animal care and use policies.
• Neither randomization nor blinding was
deemed necessary
• All animal studies were performed with female
CD-1 mice, 6–8-weeks old.
• Three independent studies:
• Protective Dose (PD50) determination in S. aureus
septic mice
• Localized S. aureus infection in neutropenic mice
• S. pneumoniae lung infection in
immunocompetent mice
34

35. Mouse sepsis protection model
• Mice were infected with 0.5ml MRSA suspension
(3.48x107 c.f.u. per mouse) to induce sepsis.
• Intraperitoneal injection
• This concentration and method are known to achieve ≥
90% mortality within 48 hours
• Mice were treated with single doses of IV
Teixobactin, Vancomycin or Saline at one hour post-
infection
• 6 Mice per treatment group
• Survival was observed at 48 hours post-infection
• Probability was determined by non-parametric log-
rank test
• * P < 0.05
• *** P < 0.001
• PD50 was determined to be 0.2 mg/kg
35

36. Mouse thigh infection model
• Mice were rendered neutropenic
(immunocompromised) with two doses of
cyclophosphamide delivered on days 4 and 1 before
infection.
• MRSA was injected into the right thighs of each
mouse.
• A single dose of IV Teixobactin, Vancomycin or
Saline was given two hours post-infection.
• 4 mice per treatment group
• One group of mice was euthanized to serve as the time-
of-treatment controls
• The remaining mice were euthanized at 26 hours
post-infection. The thighs were aseptically removed
and processed for c.f.u. titers.
• Teixobactin showed efficacy in concentrations
similar to Vancomycin.
36

37. Mouse lung infection model
• Mice were intranasally infected with
Streptococcus pneumoniae (1.5x106) to induce
pneumonia.
• IV Teixobactin was given twice at 24 and 36
hours post-infection.
• SC Amoxicillin was given once at 24 hours as a
control.
• Mice were euthanized at 48 hours post-
infection. The lungs were aseptically removed
and processed for c.f.u. titers.
• Teixobactin caused a 6 log10 reduction of c.f.u.
in mice lungs and demonstrated comparable
treatment to Amoxicillin.
37

38. Authors’ Conclusions
• Teixobactin is a promising therapeutic candidate; it is effective against drug-resistant
pathogens in a number of animal models of infection.
• Teixobactin has a novel mechanism of action that inhibits Pepdioglycan and Wall Teichoic Acid
(WTA) synthesis by binding to Lipid II and Lipid III.
• Bacteria will not develop resistance to Teixobactin in the same way they developed resistance
to Vancomycin.
• Vancomycin resistance (probably) originated in the bacteria that produces it, Amycolatopsis orientalis.
• Eleftheria terrae does not have a mechanism to protect itself against Teixobactin that other bacteria
can borrow/copy.
• It may take 30 years or longer for bacteria to develop resistance to Teixobactin, if at all.
38

39. Limitations
• Not a clinical study, not randomized, not human-based
• Synthetic semi-synthesis or de novo synthesis procedures, which will be necessary before entering
Clinical Trials, have yet to be developed
• Needs to be tested against more resistant strains of bacteria including VRSA, DRE and MDR/XDR
TB, though these are extremely rare strains with isolates that are not widely available.
• Ofloxacin was used as the control during resistance testing. Vancomycin or Methicillin/Oxacillin
would have been a better comparison than a fluoroquinolone.
• Further research still needed:
• In Vivo Efficacy in animal models, in which standard therapies have failed
• In Vivo Toxicology
• Full benchmark trials that compare Teixobactin to Vancomycin, Talavancin, Daptomycin, Linezolid and
standard INH/RI/RIPE treatments for Tuberculosis.
39

40. Seminarian’s Conclusions
• Even more exciting than the discovery of Teixobactin is the successful use of iChip. More
antibacterial agents could be discovered in the near future.
• The development of resistance to Teixobactin seems unlikely, but is not impossible. There is
no such thing as a “resistance-proof” antibiotic.
• Since Teixobactin has no appreciable toxicity to human mammalian, it will likely prove safe to
use in humans. Though, only human clinical trials can confirm this.
• There are several drugs that treat various resistant strains of S. aureus and Enterococci. While
there have been a few very rare cases of infections resistant to all known treatment that
Teixobactin could be used against, the most exciting use for Teixobactin may be for MDR or
XDR TB. More testing should be done comparing Teixobactin and current TB treatment.
40

41. Clinical Relevance
• The discovery, development and approval of
new antibiotics has slowed drastically since
the 1960’s and we must use the antibiotics
we have now with extreme caution.
• Antibiotic resistance is a real and present
threat to modern medicine, and there are
currently no antibiotics in use that are
“resistance-proof.”
• Whether or not Teixobactin is FDA-
approved, it is the first of a new class of
antibiotics that pharmacists can expect to
utilize at some point in their careers.
41

42. Clinical Relevance
• Bacteria will eventually develop resistance
to Teixobactin, but pharmacists can make it
last for decades by using this drug
responsibly and appropriately.
• In the war against antibiotic resistance:
• Offense: R&D of new antibiotics
• Defense: Antimicrobial stewardship
• We need BOTH to win!
42

43. References
• Arias, C. A., & Murray, B. E. (2015). A New Antibiotic and the Evolution of Resistance. The New
England Journal of Medicine, 372, 1168-1170. doi:10.1056/NEJMcibr1500292
• Brunning, A. (2015, January 08). Teixobactin: A New Antibiotic, and A New Way to Find More.
Retrieved August 19, 2015, from http://www.compoundchem.com/2015/01/08/teixobactin/
• Burch, D. (2009, July 28). The Rational Use of Antibiotics in Relation to Antibiotic Resistance.
Retrieved September 28, 2015, from http://www.thepigsite.com/articles/2832/the-rational-use-of-
antibiotics-in-relation-to-antibiotic-resistance/
• Centers for Disease Control and Prevention. (2013). Antibiotic Resistance Threats in the United
States, 2013. Retrieved August 29, 2015, from http://www.cdc.gov/drugresistance/threat-report-
2013/
• Cooper, M. A., & Shlaes, D. (2011). Fix the antibiotics pipeline. Nature, 472(7341), 32-32.
doi:10.1038/472032a
• Ledford, H. (2015). Promising antibiotic discovered in microbial ‘dark matter’. Nature.
doi:10.1038/nature.2015.16675
43

44. References
• Ling, L. L., Schneider, T., Peoples, A. J., Spoering, A. L., Engles, I., Conlon, B. P., . . . Lewis, K. (2015). A
new antibiotic kills pathogens without detectable resistance. Nature, 517, 455-459.
doi:10.1038/nature14098
• New Antibiotic Development: Barriers and Opportunities in 2012. (2012, May 23). Alliance for the
Prudent Use of Antibiotics Clinical Newsletter, 30:1, 8-10.
• Nichols, D., Cahoon, N., Trakhtenberg, E. M., Pham, L., Mehta, A., Belanger, A., . . . Epstein, S. S.
(2010). Use of Ichip for High-Throughput In Situ Cultivation of "Uncultivable" Microbial
Species. Applied and Environmental Microbiology, 76(8), 2445-2450. doi:10.1128/AEM.01754-09
• Plumer, B. (2013, December 14). The FDA is cracking down on antibiotics on farms. Here’s what you
should know. The Washington Post. Retrieved August 19, 2015, from
http://www.washingtonpost.com/news/wonkblog/wp/2013/12/14/the-fda-is-cracking-down-on-
antibiotics-at-farms-heres-what-you-should-know/
• Servick, K. (2015). The drug push. Science, 348(6237), 850-853. doi:10.1126/science.348.6237.850
• Wright, G. (2015). Antibiotics: An irresistible newcomer. Nature, 517, 442-444.
doi:doi:10.1038/nature14193
44

45. Questions?
THANK YOU FOR YOUR ATTENTION!
45

46. Supplemental Material
46

47. 47

48. Failures in Antibiotic Discovery
• Scientific
• All the “low hanging fruit” have already been
plucked
• Drug screens for new antibiotics tend to re-discover
the same compounds
• Finding new targets is complex and expensive
• Economic
• Poor (negative!) return on investment for drug
companies
• Antibiotics are used short term, rather than chronically
• New antibiotics are specifically reserved as last-resort drugs
• No financial incentive to create drugs that aren’t
used very often
• Patents are not issued for “naturally occurring”
molecules
• Regulatory
• Large number of indications that must be
independently tested
• Strict requirements due to high likelihood of
adverse events
• Unfeasible/Unreasonable limitations for clinical
trials
48

49. The Diffusion Chamber
• Precursor to the Ichip
• A metal washer sandwiched between two
semipermeable membranes
• Incubated on the surface of marine sediment
• Allows for the one-way passage of nutrients
and growth factors to promote bacterial growth
without contamination
• Successfully cultivated several new isolates
• Not mass-producible
49

50. 50

51. FDA Approval
51