1. Saman Amirzadegan & Krista Jastrzembski BINF 704
A new antibiotic kills pathogens without detectable resistance Losee L. Ling, Tanja Schneider2, Aaron J. Peoples, Amy L. Spoering, Ina Engels, Brian P. Conlon, Anna Mueller, Till F. Scha¨berle, 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
Jasmine and I will be presenting a review of an article entitled “A new antibiotic kills pathogens without detectable resistance”. The lead authors on this study are Losee Ling from NovoBiotic Pharmaceuticals, iIn Massachusetts and Tanja Schneider from the university of Bonn in Bonn Germany. This article appeared in Nature in January of this year and we will provide a link so that you can go and read it if you like.
Saman Amirzadegan & Krista Jastrzembski
BINF 704

2. Antibiotic Resistance
So we are only a few weeks past Halloween and Jasmine and I are going to talk about something really terrifying…..Antibiotic resistance. Antibiotic resistance has received a lot of attention both in the scientific community and the mainstream media over the past several years. As more and more bacteria evolve to resist the bacteracidal efforts of even our most potent antibiotics we are struggling to keep up with new drug options. Jasmine and I are going to talk about a new method of antibiotic discovery as well as a promising early result of this technique

3. Overview A Short History of Antibiotics
Development of Antibiotic Resistance
Teixobactin
Cultivation
Identification
Mechanism of Action
Discussion
In this presentation I am going to cover a short history of antibiotics, just to give an idea of their overall importance to medical science. I will then jump into the unique ways in which bacteria are able to so quickly evolve to develop resistance to these antibiotics and the repercussions of this antibiotic resistance. Finally I will move on to discuss Teixobactin specifically including the cultivation of the microorganism responsible for it’s development and general identification of the compound. After than Jasmine will tell you more about texiobactin including how it works and why it is thought to be so promising.

4. 1909 – Paul Ehrlich et. al. discover a drug effective against syphilis
The Antibiotic Era
1909 – Paul Ehrlich et. al. discover a drug effective against syphilis
The modern antibiotic age is often thought to begin with Paul Ehrlich and the discovery of a compound to cure syphilis.
Starting in 1904 Paul Ehrlich, along with colleagues Alfred Bertheim and Sachario Hata began a systematic screening of drugs for to fight Syphilis, which at the time was endemic and nearly incurable.
In 1909, after screening 606 compounds, they discovered arsphanamine which cured syphilis infected rabbits and showed promise against the disease in humans.
For more than 30 years this drug, known commercially as Salvarsan and a later derivitave called Neosalvarsan, was the most frequently prescribed drug.

5. Discovery of Penicillin
September 3, 1928
Alexander Fleming returns to his lab after a lengthy vacation to discover Penicillin
On September 3, 1928 Alexander Flemming returned to his, rather messy, lab after a long vacation to discover that some of his plates had grown mold and that this mold seemed to be active against the bacteria growing on the plates.
Flemming was, in fact, not the first person to obvserve these antibacerial properties. He was, however, the first person to persist in resolving the issues surrounding purification and stability of the active substances.
Only a few years after an Oxford research team led by Howard Florey and Ernest Chain published a paper describing the purification of the compound, Penicillin proved invaluable during WWII where it was used to treat illness such as mengitis and syphilis as well as general battlefield injuries. The drug is credited with greatly reducing the death toll from gangrene, septicimia as well as the need for amputations.
From 1940, when penicillin gained popularity until

6. Antibiotic Mechanisms of Action
Bactericidal antimicrobials commonly fall into four categories. DNA gyrase inhibitors induce DNA double strand breaks, while rifamycins interfere with DNA dependant RNA synthesis. Cell-wall Synthesis inhibitors obviously damage the cellular envelope and protein synthesis inhibitors lead to protein mistranslation.
Additionaly, recent evidence has indicated that all classes of antibiotics induce a common stress response which eventually leads to their death
Despite this array of mechanisms leading to cellular death, microbials are evolving to resist these drugs on a daily basis.

7. Superbugs from Super-fast Evolution
1940 Penicillin introduced – Resistant bacteria uncommon
1950’s Penicillin resistant S. aureus common in hospital settings
1961 – Methicillin introduced
1962 – Methicillin resistant A. aureus strains begin to appear
2002 – Vancomycin resistant strain of S. aureus isolated from patient in Michigan
Bacteria evolve with alarming speed.
Let’s look at MRSA, or methicillin resistant Staph aureus, for example. When Penicillin was introduce din the early 1940’s resistant bacteria were essentially unheard of. By the 1950’s they were commonplace in hospitals, which is unfortunately where most of these resistant strains are initially discovered. In 1961 methicilin was introduced to combat these resistant strains, but only a year later methicillin resistant strains of S. aureus had begun to appear. Today, we have strains of S. Aureus that are resistant to an extensive list of antibiotics, including vancymycin which is often considered to be our end game antibiotic. So, unfortunatly, at this point it appears that bacteria are winning this biological battle and we need a better arsenal to fight them.
Superbugs from Super-fast Evolution

8. Bacteria’s evolutionary advantages
Vertical Transmission
Generation time
Population Size
Horizontal Transmission
No need to wait for random mutations
Multiple resistance genes
When it comes to evolutionary prowess, bacteria have several advantages that we, as humans, lack.
Generation time – For known, culturable bacteria generation time commonly ranges from 15 to 60 minutes. Meaning, that by the time this presentation is over a culture of S. aureus will have created a whole new generation of cells, any one of which is potentially more drug resistant than it’s parent cell.
Large population size – In addition to being highly prolific, bacteria develop enormous populations, once again resulting in in creased opportunities for beneficial mutations.
Horizontal transmission allows genes from one bacterium to be integrated into the genome of another, even non-related, bacterium. These bits of DNA, also known as plasmids, may be incorporated into the new bacteria’s dna and then passed to future generations through vertical transmission. This means that bacteria do not need to wait for random mutation to confer antibiotic resistance. As a result, even species that have never encountered a particular antibiotic may, through chance, contain a few individuals already containing resistant genes.

9. These plasmids may carry genes for resistance for multiple antibiotic agents. The plasmid on this slide is a good example of this. This single plasmid carries genes for resistance to four antibiotics as well as a disinfectant. When a bacterium carrying this plasmid is selected for due to pressure from one of these antibiotics it confers selection for all four of the antibiotics. This results in microorganisms that are resistant to multiple drugs.
----- Meeting Notes (11/3/15 23:08) -----
Even if they have never actually been exposed to those drugs.

10. Mechanisms of Antibiotic Resistance
Modification of the Antibiotic
Removal from the cell
Modification of the target site
There are several mechanisms by which these genes can cause a microorganism to become resistant to a particular antibiotic.
Most commonly the cell will enzymtically inactivate the antibiotic by modifying an existing cellular enzyme to react with the antibiotic in such a manner that the antibiotic no longer affects the cell. This is illustrated in red or orange on the left side of the cell here in the slide. An example of this would be penicillinases which are enzymes that degrade penicillin
Other, less common, be equally as effective methods include physically pumping the antibiotic out of the cell using efflux pumps. This is the method of resistance to tetracycline.
Finally, the cell may alter the antibiotics target site as show in green. This keeps streptomycin from recognizing and binding to the ribosome to block protein synthesis.
Cells may also over produce antibiotic target sites such as in trimethoprim resistance.

11. The costs of Antibiotic resistance
2 million yearly infections in the US
23,000 deaths directly related to antibiotic resistant infections
$20 billion lost to excessive healthcare costs
$35 billion due to lost productivity
A 2013 publication by the CDC characterizes antibiotic resistant bacteria as Nightmare organisms that pose a catastrophic hreat to people in every country of the world.
Every year in the united states 2 million people will contract a serious infection caused by a bacteria resistant to the antibiotic designed to treat it. As least 23,000 of those people will die as a direct result of the infection. And many more will die of complications related to their illness.
In addition to the human cost, antibiotic resistance have econimic costs. Estimates for the costs of excessive healthcare, such as extended hospital stays, costlier treatment and the need for additional doctor visits range as high as 20 billion dollars a year with an additional 35 billion dollars for lost productivity.
As you can see, antibiotic resistance poses very real challenges for society.

12. Antibiotic Discovery tapers after 1960
As you can see from the graph above, Flemming’s discovery lead to a surge in antibiotic discovery. In recent years, however, the introduction of novel antimicrobials has decreased dramatically, with only two . The difficulty of culturing microbial species, coupled with the relatively low profitability of antibiotics has resulted in the development of fewer and fewer antibiotics even as the demand, due to increased microbial resistance, increases.
Antibiotic Discovery tapers after 1960
Difficulty of new discovery combined with increasing antibiotic resistance results in potential public health crisis

13. iChip device for growing uncultured microorganisms
Most naturally occurring antibiotics have been developed by screening cultivatable soil microorgnisms. Unfortunatly the vast majority of these soil organisms are not able to be grown in culture. The authors of the article have used an ingenious method to isloate and grow unclutured organsms. A soil sample is diluted to the point where a single microorganism can be isloated and delivered into a single well. A semi-permiable membrane covers each side of the wells and the entire device is then returned to the soil. This allows a single organism to be grown in it’s natural environment. This method increases the growth recover from around 1% on nutrient plates to nearly 50%.
It has been speculated that the efficiency of this new iChip technology may lead to a return to the previously tedious task of screening for naturally occuring compounds with activity profiles of interest.

14. This is another view of the iChip device, it gives a better idea of the scale of the mechanism.
In this study the soil was obtained from a field in Maine, diluted to a concentration of 1 cell/ 20ul. 20ul of this dilutant was then innoculated onto the iChip. After one month incubation in native soil isolates were streaked onto SMS agar test for the ability to propagate outside of the natural environment as well as for colony purification. Extracts from each suitable isolate, 10,000 in all, were screened for anitmicrobial activity against plates overlaid with S. aureus. An extract from the isolate provisionally named Eleftheria terrae showed the greatest amount of activity.

15. Eleftheria terrae New species of b-proteobacteria
New genus related to the genus Aquabacteria
16S sequencing
DNA/DNA hybridization
This is Eleftheria terrae, a new species of b-proteobacteria.
Using GoTaqaGreen master mix from promega and universal primers E8F and U1510R the 16S sequence was amplified for and sequenced by Macrogen in Cambridge Massachusetts. This sequence was compared by BLAST to isolates in the ribosomal Database Project. The whole genome was submitted to for RAST analysis to produce a list of close relatives. Based upon 16s and genoic sequencing along with in situ DNA/DNA hybridization the unknown organism is was determined to belong to a new genus related to the genus Aquabacteria, a genus of gram-negative bacteria not known for their ability to produce antimicrobial agents. .

16. Teixobactin Molecular Mass: 1,242 Da
Depsipeptide containing enduracididine, methylphenylalanine and four D- amino acids.
The active fraction was partially purified and a previously unreported compound with a molecular mass of 1,242 Daltons was detected Via mass spectrometry.
The compound was then isolated and a complete stereochemical assignment was done using nuclear magnetic resonance spectroscopy and advanced Marfey’s analysis. The result was Teixobactin as you can see here.

17. Here we have teixobactin along with it’s predicted biosynthetic gene structure. This is an unusual depsipeptide in that it contains enduracididine, a non-proteinogenic amino acid, a methylated phenalylnine as well as 4 of the less common d-amino acids.

18. This final slide, before I pass things off to Jasmine, gives you an idea of the post translational modifications taking place to complete the structure of Teixobactin.
And now I’m going to pass things off to my partner jasmine who is going to tell you about how Teixobactin works and why it seems so promising as a new tool in our arsenal against microbial disease.

19. Image source: http://www.medscape.com/viewarticle/715971_2
Different cell membranes = differents ways of uptaking antibiotics/stuff outside of the membrane
Image source:

20. Resistance
Teixobactin is effective against many gram positive* pathogens, some of which are unresponsive to vancomycin (last resort)
Gram negative exception
M. tuberculosis = impermeable to gram stain; waxy coat; still has peptidoglycan tho
S aureus = gram pos
E coli = gram neg

21. S. Aureus treated w/various antibiotics
*Control was no antibiotic; Oxacillin and Vancomycin are other powerful antibiotics
Teixobactin = lysis of bacterial cells [degradation is what makes the liquid clear]
Image source:
Fig 2c

22. Resistance
No mutants (S.aureus or M.tuberculosis) observed when plated on teixobactin – NO RESISTANCE
No S.aureus mutants observed, even after 27 day repeated exposure to teixobactin – STILL NO RESISTANCE 
Indicates “non – specific” mechanism & toxicity
BUT no toxicity observed in mammalian cells (highest possible dose)
Mutants = antibiotic resistance genes; in other words, the bacteria did not adapt to over come the antibiotic, they were defeated by it
Why mammialian cells? [ask the class to interact here]
>>because eventually they will want to use it on humans!!! And we are mammalian

23. Mechanism of Action
Teixobactin is non – haemolytic* and doesn’t bind DNA
How did the research team figure out specifically where teixobactin is active?
Rate of label* incorporation into S.aureus’ main biosynthetic pathways
Teixobactin inhibits peptidoglycan* synthesis
Teixobactin has no observed effect on label incorporation into DNA, RNA, or protein
Switching gears slightly: from the observations that teixobactin doesn’t really seem to cause antibiotic resistance, to how it actually works:
Non – haemolytic: doesn’t rupture red blood cells
Rate of label: a process in which biomarkers are tagged, either with fluorescence or another visual tag, and observed as they go about normal processes
*peptidoglycan: cell wall component of many bacterial cells; both gram + and gram neg, AND teixobactin inhibits its synthesis

24. Fig 3a
Teixobactin has no observed effect on label incorporation into DNA, RNA, or protein <<what does this actually mean?
Cell used was gram pos S.aureus
Grey bars = teixobactin
White bars = other antibiotic controls – we know where and how they work, so they are good controls
So, in S.aureus, the presence of teixobactin [grey] does not affect synthesis of DNA, RNA, and Proteins, but it does effect the synthesis of peptidoglycan. Again, they used biomarker tags to observe this, specifically radioisotopic

25. Mechanism of Action
In vancomycin, the absence of resistance suggests that the mechanism target is not a protein. Could the same be true for teixobactin?
Vancomycin binds lipid II* – does teixobactin bind this also?
S.aureus built-up a peptidoglycan precursor (UDP-MurNAc-pentapeptide), when exposed to teixobactin in concentrations of 1x – 5x the MIC*
Vancomycin control does the same; indicates inhibition of a peptidoglycan biosynthesis step
In test tubes, teixobactin blocked peptidoglycan synthesis reactions: lipid I, lipid II, or undecaprenyl - pyrophosphate
Fig 3
*lipid I, lipid II, or undecaprenyl - pyrophosphate are a peptidoglycan precursors / targets for teixobactin activity
>>undecaprenyl – pyrophosphate, a peptidoglycan precursor carrier molecule
UDP-MurNAc-pentapeptide is an precursor of peptidoglycan biosynthesis
*minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation [Wikipedia def]

26. Mechanism of Action
What we know so far: Teixobactin is specifically involved with peptidoglycan precursors, as opposed to the other enzymes involved in its biosynthetic pathway
The follow – up question: What specific part of these precursors is the is teixobactin targeting?
How to investigate: Study direct involvement with peptidoglycan precursors coupled in pairs
Reinterate 3 main precursors: lipid 1, 2, undecaprenylpyrophosphate, but there were some others…
*undecaprenyl: lipid carrier molecule, transporting the hydrophilic peptidoglycan precursor molecules across the cytoplasmic membrane <
*thin layer chromatography: chromatography in which compounds are separated on a thin layer of adsorbent material, typically a coating of silica gel on a glass plate or plastic sheet < google def

27. Paired peptidoglycan precursors:
purified and incubated with teixobactin at various molar ratios
extracted and analyzed by thin – layer chromatography
Teixobactin bound if amount of lipid intermediates were reduced
Different substances travel at different speeds, thereby separating out
Image Source:

28. Mechanism of Action
What did teixobactin bind? All peptidoglycan precursors and wall teichoic acid*
Fun fact! Lipid I and lipid II form a stable complex impermeable to the effects of teixobactin
Teixobactin > Vancomycin??
*wall teichoic acids present in gram pos bacteria, covalently attached to peptidoglycan and extend through and beyond the cell wall
Teixobactin > vancomycin??
Teixobactin is effective against vacomycin resistant enterococci with modified lipid II, suggesting that teixobactin binds to modified lipid II, whereas vancomycin cannot

29. Mechanism of Action
WTA is not needed for the organism to survive, inhibition of wall teichoic acid biosynthesis has lethal toxic intermediates (late stage)
Teichoic acids bind autolysins*; complex prevents uncontrolled peptidoglycan break down.
Inhibition of teichoic acid synthesis by teixobactin releases autolysins; lytic/killing activity of the bacteria increases
Obviously WTA is not essential, since gram negative bacteria don’t have it
*Autolysins exist in all bacteria containing peptidoglycan, breaking it down in small pieces so that growth and division of cells can occur <Wikipedia def

30. In vivo efficacy Teixobactin stable in serum; low toxicity in mice
Up until now, everything we’ve discussed about teixobactin has been done in the lab; but is it as effective in the body?
[fig8a]: This graph shows how the antibiotic degrades/how long it is active; Pharmacokinetic parameters observed after intravenous injection of a single 20 mg/kg dose in mice. The outcome was flattering; the teixobactin serum level was retained above the minimal inhibitory concentration for 4 hours
Extended Data; Fig 8a

31. In vivo efficacy
Teixobactin also observed to be effective in mice with:
MRSA* septicemia* dose of 90% lethality, if administered with as little as 1mg/kg intravenously within an hour
In a follow up experiment the PD50* was observed to be 0.2 mg/kg; better than vancomycin* (PD50 of 2.75 mg/kg)
Thigh model of MRSA infection*
Streptococcus pneumonie infections
*septicemia: bacterial infection of the blood
*MRSA: methicillin resistant s. aureus
*PD50: protective dose of antibiotic in which 50% of those infected are protected
*Thigh model of infection: skin and soft tissue
Teixobactin > Vancoymcin
Vancomycin primarily used to treat MRSA

32. Summary
Antibiotics are an invaluable medical advancement that have greatly reduced suffering and death
Bacteria have evolutionary advantages when it comes to evading antibiotic activity
New techniques are necessary to advance the discovery of novel antibiotics
Teixobactin is a strong and promising antibiotic with efficacy better than that of the last resort antibiotic - Vancomycin
Further research must be done, in terms of clinical trials – in vitro human trials?
Except from Teixobactin Wikipedia page:
-”In early 2015, human clinical trials of teixobactin were predicted to be at least two years away.
-One co-discoverer estimated a drug development cost "in the low $100-millions" on a five to six year schedule.[13] 
-Pharmaceutical companies have been reluctant to make such investments in new antibiotics, because their wide prescription is likely to be discouraged in order to retard development of resistance, which has come to be considered almost inevitable.”

33. References
Clin Orthop Relat Res Oct;439:23-6. THE CLASSIC: penicillin as a chemotherapeutic agent Chain E, Florey HW, Gardner AD, Heatley NG, Jennings MA, Orr-Ewing J, Sanders AG.
Cell Sep 7;130(5): A common mechanism of cellular death induced by bactericidal antibiotics. Kohanski MA1, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ.
Lancet Infect Dis Dec;10(12): doi: /S (10) VRSA-doomsday superbug or damp squib? Gould IM.