Teixobactin and Ichip: Novel Solutions for the Treatment of Resistant Bacterial InfectionsEL IZABETH DAUGHERTY, PHARM.D . AND MPH CANDIDATEV IRGIN IA FLEMING, PHARM.D. , BCPS , FACULTY ADVISORUNIVERS ITY OF GEORGIA COLLEGE OF PHARMACYOCTOBER 14 , 2015

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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

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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

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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

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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

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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

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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

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Antibiotic Resistance “Treatment”

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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

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

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Literature Search

• PubMed• Limits

Publication date: 2010 – 2015 Journal Article

• Search terms: “teixobactin, resistance”• Returned 5 results

• Search terms: “ichip, bacteria”• Returned 9 results

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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

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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.

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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

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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).

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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

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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)

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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.

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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.

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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

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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.

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Study 2A 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

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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

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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

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Mechanism of Action

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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.

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MRSA →

VISA →

VRE →

TB → ← CDIFF

← Anthrax

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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.

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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.

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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.

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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

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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

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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.

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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.

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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.

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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.

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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.

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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.

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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!

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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

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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

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Questions?THANK YOU FOR YOUR ATTENTION!

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Supplemental Material

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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

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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

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FDA Approval