1. The discovery of new antibiotics, the Isolation Chip and Teixobactin
James Britton
Module: BIOC40160
Supervisor: Dr. Tadhg Cronin

2. Assessment Submission Form
Student Name James Britton
Student Number 14204224
Assessment title The discovery of new antibiotics, the Isolation Chip and
Teixobactin
Module title Drug Development & Clinical Trials (BIOC40160)
Module co-ordinator
Supervisor Dr. Tadhg Cronin
Date Submitted
Date received
Grade/ Mark
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Declaration of authorship:
I declare that all material in this assessment is my own work except where there is clear
acknowledgement and appropriate reference to the work of others.
Signed: James Britton Date: 22 Mar. 15

3. Table of Contents:
1. Summary………………………………………………………………………………………………………………..Page 1
2. Introduction……………………………………………………………………………………………………………Page 2
3. The Isolation Chip…………………………………………………………………………………………………..Page 5
4. Teixobactin…………………………………………………………………………………………………………..Page 10
5. Teixobactin Biosynthesis………………………………………………………………………………………Page 11
6. Teixobactin Efficacy & Resistance…………………………………………………………………………Page 13
7. Teixobactin – Mechanism of action………………………………………………………………………Page 16
8. In vivo studies………………………………………………………………………………………………………Page 18
9. Future challenges and Potential…….……..……………………………………………………..……...Page 19
10. Conclusions………………………………………………………………………………………………………….Page 20
11. References.……………………………………………………………………………….……………………….…Page 21

4. 1 | P a g e
Summary:
Bacterial infection is once again a serious threat to human health. Becoming almost
insignificant by the advent of antibiotics, in the last twenty years bacterial pathogens are
once again in the top 10 causes of death in the United States. The lack of profitability in
antibiotics along with the difficulty in discovering new compounds has pushed most large
pharmaceutical companies out of the antibiotics market. This and the continued growth of
antibiotic resistance has led to a scene where bacterial pathogens can cause serious
complications to anyone who needs treatments for other ailments or needs surgery.
However there are some who are continuing the search for new anti-infective compounds.
Researchers from the North-eastern University of Boston have developed a new assay
apparatus named the Isolation chip which allows for the isolated cultivation of up to 50% of
environmental bacteria. Classical methods only allow for the cultivation of under 1% of
bacterial species, as such the I-chip opens up a vast repertoire of new species potentially
harbouring useful new compounds. Recently newly described bacterium, Eleftheria terrae,
has been isolated and found to harbour a potent antibacterial compound, Teixobactin.
Teixobactin has been shown to have bactericidal activity against a wide variety of Gram
positive organisms including Methicillin & Vancomycin resistant Staphylococcus aureus.
Although it needs to be thoroughly examined and much development needs to take place
before Teixobactin or a derivative enters clinical trials, let alone the market at large, early
studies in murine models show high in vivo efficacy with low toxicity. Teixobactin may be
the first of a new wave of antibiotic compounds made possible by the advent of the I-chip.

5. 2 | P a g e
Introduction:
In 1942 the large scale production of Penicillin G by Florey and Chain ushered the world into
the antibiotic age (Florey, 1945). From then until the mid-1960s all of the major classes of
antibiotic used today were discovered (Lewis, 2012). Since then the only antibiotic of a new
class to reach clinical usage is the cyclic lipopeptide Daptomycin (Kirkpatrick et al., 2003).
This worrying innovation gap has allowed for the inevitable growth of resistance to the
majority of our antibiotic arsenal (Bush et al., 2011). According to a recent review of the
situation by So and Shah (2014) there are two major obstacles to the creation of new
antibiotic compounds; those being the barriers of economics and scientific innovation.
According to the review by So, antibiotics account for only 5% of the global pharmaceutical
market and have the lowest risk adjusted Net present value of all pharmaceuticals, more
than 10 times less valuable then musculoskeletal drugs, the most valued type of
pharmaceutical (So and Shah, 2014, Projan, 2003). Due to the inevitable growth of
resistance to antibacterials the time span of any given anti-infective is limited in comparison
with the essentially timeless usage of many other drug types (So and Shah, 2014). These
factors lead to the market returns on any successful antibiotic not covering the massive
costs (>$800 million) of development, driving potential investors away (Lewis, 2013).
The second, and more pressing issue that has hindered the creation of new antibiotic drugs
is the current stagnation in the detection of new drug leads (So and Shah, 2014). During the
golden age of antibiotic discovery the most commonly used method for the detection of
new compounds was the ‘Waksman platform’ (Lewis, 2013). This platform created by
Selman Waksman systematically screened Actinomycetes from soil and allowed Waksman
himself to discover over 20 antibiotics in his lifetime (Society, 2015). However the Waksman
platform declined in popularity and was discarded when it was realised that many seemingly
new products had been previously discovered and discarded (Lewis, 2013). After the failure
of the Waksman platform antibiotic development focused mainly on the modification of
existing compounds. From this time during the 1960’s onwards there was little progress
made towards the discovery and development of new classes of antibiotics. The only new
antibiotic classes discovered since then were the broad spectrum Fluroquinolones and the
Cyclic Lipopeptides (Lewis, 2013). During this time however resistance to the common
classes of antibiotics was growing and by the 1990’s it was obvious that the then current
practice of creating derivatives of existing drugs was futile (Lewis, 2013, Hughes and Karlén,

6. 3 | P a g e
2014). To combat this the pharmaceutical industry adopted new, more complex platforms
for compound discovery which encompassed many recent scientific advances such as High
throughput screening, Rational drug design, Combinatorial chemistry and Genomics (Lewis,
2013). While using this platform many companies tried to dictate their compounds to
Lipinski’s rule of five, a general rule which can predict how ‘Drug-like’ a substance is in
relation to pharmacokinetic and pharmacodynamics properties (Lewis, 2013, Lipinski et al.,
2001). This however failed as it was found difficult to identify compounds which were active
and able to reach their target sites in the target as well as those that obeyed Lipinski’s rule
(Lewis, 2013, Lewis, 2012, Hughes and Karlén, 2014). The failure of this advanced platform
has ultimately led us to the situation we find ourselves in today, the lack of success has
driven most large pharmaceutical companies away from first principles antibiotic discovery
and into more profitable areas.
Figure 1. Timeline of the introduction of antibiotic classes to clinical use. Information
taken from Lewis (2013).
A key factor to this failure as noted by Nichols et al. (2010) was the lack of correct
compound resources. Yet in spite of this there has been a steady, if slow pace of antibiotic
research coming from academic laboratories as well as some small biotechnology firms. It is
in one of these small research groups and their industry collaborators that a breakthrough
technology for new compound discovery has been developed (Nichols et al., 2010, Nichols
et al., 2008). During the era of renewed anti-infective research in the 1990’s the chemical
libraries used were lacking in a great resource, that of most microbial species. At this time,
until quite recently, one of the major challenges to antibiotic research and microbiology in
general was the inability to culture over 99% of the microbiome of the world, the Isolation
chip developed by Nichols et al. (2010) has changed this drastically.

7. 4 | P a g e
This pioneering work carried out by Nichols et al. (2010) has led to the creation of the
Isolation Chip (I-chip), a device which allows for the isolated culturing of previously
uncultivable microbes from various environments (Nichols et al., 2010, Nichols et al., 2008).
Once the isolated culture has been created it allows for the subsequent analysis of the
microbe in question, leading to the identification of the organism and the search for any
potentially useful compounds it may produce (Nichols et al., 2010). Since the inception of
the I-chip in 2010 it has been used to screen a large number of previously uncultivable
organisms taken from environmental samples around the globe, recently this has led to the
discovery of a novel antibiotic compound, Teixobactin (Ling et al., 2015).

8. 5 | P a g e
The Isolation Chip:
It had been hypothesised by Nichols et al. (2008) that the reason many microbial species
were uncultivable under classical culture conditions was due to the lack of environmental
cues caused by other species in their immediate environment and that separation from
these stimuli inhibits the growth of the organism in vitro. Nichols arrived at this hypothesis
after his groups work in 2008 during which they built upon the earlier work carried out by
Kaeberlein et al. (2002) (Nichols et al.). Kaeberlein devised a method which was to isolate
‘uncultivable’ microbes in a diffusion chamber and then place said chamber in a simulation
of their natural environment (Kaeberlein et al., 2002). Kaeberlein carried out this by first
taking a marine sediment sample and removing the sediment particles, leaving only the
microbial fauna behind. Once separated the microbial fraction was serially diluted in agar.
The diluted microbial fraction was then separated into diffusion chambers created from a
metal washer sandwiched between two 0.03μm pore polycarbonate filters (Kaeberlein et
al., 2002). It is important to note that there was a thin layer of air kept between the agar
and the top membrane, this allowed for the top membrane to be removed without
disturbing any colonies which may have formed on the agar surface.
Figure 2. Diffusion chambers as made by Kaeberlein et al. (2002). Note the
polycarbonate filters above and below the metal washers. Adapted from
Kaeberlein et al. (2002).

9. 6 | P a g e
Once the diffusion chambers were created and contained the diluted agar fractions the
sealed chambers were placed on a surface of their native marine sediment and submerged
in seawater (Kaeberlein et al., 2002).
Figure 3. Kaeberleins diffusion chambers on a marine
sediment submerged in seawater. Adapted from
Kaeberlein et al. (2002).
Using this method Kaeberleins team were able to obtain
colony recovery rates of over 40% of the estimated number
of cells inoculated (Kaeberlein et al., 2002). From this
Kaeberleins team isolated two previously undescribed
bacterial species, MSC1 and MSC2. It was observed that
both MSC1 and MSC2 were only cultivable either in diffusion chambers or when co-cultured
with the other organism (Kaeberlein et al., 2002).
From this Kaeberlein reasoned that the growth of previously unculturable microorganisms
shown in his experiments to be due to growth promoting signals (Kaeberlein et al., 2002,
Nichols et al., 2008). In the more recent paper by Nichols et al. (2008) explored this. Using
the same diffusion chamber isolation method they identified six uncultivable bacterial
species and a multitude of helper strains for each of the uncultivable species. The helper
species were so named due to their ability to aid in the creation of cultures of the
uncultivable bacteria. It was found that many of the helper strains were able to promote
growth of multiple uncultivable isolates. To determine if a helper promoted growth of one
of the uncultivable species the isolated helper species was placed in a diffusion chamber
which was subsequently placed on an agar plate inoculated with the organism in question
(Nichols et al., 2008).
It was found that upon repeated co-cultivation of uncultivable species with a helper
domesticated variants were created which were cultivable under classical conditions
(Nichols et al., 2008). Intriguingly co-culturing of the domesticated strain with its ‘wild’
ancestor led to the creation of cultivable strains from the ancestor, which in turn could be
used to repeat the process. From these results Nichols et al. (2008) reasoned that the use of
the diffusion chamber acts as a transitional stage between growth in native and laboratory

10. 7 | P a g e
conditions. The fact that cultivability could be selected for also suggested that it was caused
by a regulatory constraint which could be bypassed via selection (Nichols et al., 2008). Upon
further investigation it was found that a 24 amino acid long peptide isolated from the helper
species, β-caesin, acted as to signal colony growth in the uncultivable species, they deduced
that in the domesticated strains a similar peptide must be made which allows cultivation
also (Nichols et al., 2008). This work highlighted the importance of both microbial inter and
intra-species relationships. Similarly a study conducted by D'Onofrio et al. (2010) provided
further examples of this as they were able to promote the growth of uncultivable species by
growing them in the presence of cultivable organisms from similar environments. They
identified a range of Iron chelating compounds known as siderophores created by the
cultivable helper species as promoters for the previously uncultured strains (D'Onofrio et al.,
2010).
Building on this knowledge and expanding on the diffusion chamber growth method
developed by Kaeberlein et al. (2002) the Isolation chip was created (Nichols et al., 2010).
The Isolation chip (I-chip) created by Nichols et al. (2010) is a multi-layered chip with over
200 small wells which act as miniature diffusion chambers, these chip is covered on either
side by a porous membrane. Once covered with the membrane the chip is placed between
two plates which have holes to match those on the chip, the plates are then screwed
together to secure the device (Nichols et al., 2010). To use the chip the naked chip must be
placed in a dilute solution of any given environment, the dilution of the environmental
sample combined with the size of each individual well in the chip allows for, on average,
only one microbial cell to be placed in each well (Nichols et al., 2010).
Once sealed and seeded the I-chip will be placed in an agar solution made up from the same
environmental sample the I-chip was seeded from (Nichols et al., 2010). Placing the finished
chip in an environmental sample allows for any environmental stimuli created from native
microorganisms to reach those isolated in the I-chip wells (Figure 4). The I-chip is then
incubated for two weeks to allow for microbial growth, then each well was individually
examined for microcolonies using light microscopy (Nichols et al., 2010). If microbial growth
was found in the I-chip it was then identified using the gene sequencing of the 16S rRNA.
Using the I-chip in comparison with the diffusion chamber method and the standard petri
dish it was found that the I-chip gave a significantly higher rate of microbial recovery than
either of the other methods (Figure 5) (Nichols et al., 2010). Interestingly the percentage of

11. 8 | P a g e
microbial recovery from soil samples using the petri dish method was markedly higher than
the petri dish recovery from marine samples, possibly due to the nutrient state of an agar
plate resembling the soil more so than the sea.
Figure 4. Using the I-chip for cultivating microbial species. (A) Placing the I-chip in a dilute
environmental sample. (B) The I-chip captures a single cell per well. (C) Assembly of the I-
chip, the central chip is covered on both sides with a porous membrane which is sealed in
place by metal plates. Adapted from Nichols et al. (2010).
Figure 5. Rates of microbial recovery using the I-chip, diffusion chamber and petri dish
method. Samples are taken from (A) Seawater, (B) Soil. Adapted from (Nichols et al.,
2010).

12. 9 | P a g e
As well as this it was shown that the same sample, when recovered in a different way led to
the recovery of a different set of organisms (Figure 6) (Nichols et al., 2010).
Taxa I-chip Petri dish
Seawater Soil Seawater Soil
Alphaproteobacteria 1 1 2 6
Betaproteobacteria 3 0 33 33
Deltaproteobacteria 16 54 0 0
Epsilonbacteria 2 1 0 0
Gammaproteobacteria 9 12 8 17
Actinobacteria 0 0 4 4
Bacteroidetes 0 1 6 3
Table 1. The occurrence of taxa recovered from Seawater or Soil samples when grown
using the I-chip or a standard petri dish. Data from Nichols et al. (2010).
Nichols et al. (2010) estimated that due to its relatively simple method of use the I-chip
could potentially lead to the cultivation of novel microbial species at a rate of 100 per day
per researcher.

13. 10 | P a g e
Teixobactin
Recently, a compound with seemingly potent antibacterial activity has been discovered due
to the I-chip. The study carried out by Ling et al. (2015) screened 10,000 isolates captured
from I-chip procedures and tested them for antimicrobial activity by plating them on agar
plates overlaid with S. aureus. From this screening process Lings team identified a previously
unnamed gram negative bacteria they titled Eleftheria terrae which had potent antibacterial
activity against S. aureus. E. terrae was found to be a β-proteobacteria which upon 16S
ribosomal DNA sequencing was identified as a member of a previously unknown genus
closely related to the genus Aquabacteria (Ling et al., 2015). From E. terrae Ling et al. (2015)
isolated a 1242 Dalton compound which was the source of the antibacterial activity of E.
terrae, they named this compound Teixobactin. Teixobactin is an 11 amino acid long peptide
made through the action of two non-ribosomal peptide synthase (NRPS) proteins named
Txo1 and Txo2, encoded by txo1 and txo2 respectively.
Figure 6. The chemical structure of Teixobactin. Adapted from Ling et al. (2015).

14. 11 | P a g e
Teixobactin biosynthesis
Although not confirmed Ling et al. (2015) suggested a biosynthetic pathway for Teixobactin
formation. As mentioned Teixobactin is made through the action of the non-ribosomal
peptide synthases Txo1 and Txo2, in a similar manner to the cyclic lipopeptide Daptomycin.
In the production of non-ribosomal peptides the synthase production machine is made of
enzymes with multiple components which are responsible for the addition of individual
amino acids in the correct order to create short peptides (Fischbach and Walsh, 2006).
Usually NRPS peptide production lines contain three major domains, the thiolation domain
(peptidyl carrier protein) which binds the growing peptide chain to the NRPS, the
adenylation domain which selects and activates the amino acid monomers and the
condensation domain which forms peptide bonds allowing for the elongation of the
synthesised peptide (Fischbach and Walsh, 2006). NRPS peptide production starts with the
selection of the correct amino acid by an adenylation domain which then activates it with
ATP to make aminoacyl-AMP which is then bonded to the thiolate of a neighbouring
thiolation domain. The condensation domain then bonds the adjacent thiolate bonded
aminoacyl-AMPs creating a peptide bond (Fischbach and Walsh, 2006). This process
continues until the entire peptide is synthesised.
However this proposed pathway (as seen in Figure 7) is as of yet unproven. Also there are
many other factors which need to be considered which Ling et al. (2015) did not discuss.
Most importantly how are the genes behind Teixobactin production, txo1 and txo2
regulated? As a general rule antibiotics are products of secondary metabolism of a cell and
are usually only produced in reaction to specific signals. For example, Daptomycin, another
NRPS encoded antibiotic produced through the dpt gene cluster has been found to be
regulated indirectly by the global regulator AdpA and the specific regulator AtrA which is
only active when the bacteria experiences stress (Mao et al., 2015). In E. terrae we have no
idea how txo1 or txo2 are regulated, as well as little knowledge of the bacteria itself as it is
from a previously undescribed family of β-proteobacteria, as determined by Ling et al.
(2015). Another important factor which has not yet been discussed in the literature is how
Teixobactin exits the E. terrae cell after its production. There a variety of ways in which
bacteria export protein products such as secretion through type III secretion systems or by
the production of outer membrane vesicles (Bonnington and Kuehn, 2014, Lee and Rietsch,
2015). In many cases of protein export the protein in question will have a defining motif

15. 12 | P a g e
which signifies its mechanism of cell exit. In a typical protein the presence of this tag is easily
detectable through its gene sequence, however since Teixobactin is produced through a
NRPS system this tag may be more difficult to identify.
Figure 7. The biosynthetic pathway of Teixobactin as proposed by Ling et al. (2015). Txo1
and Txo2 contain 11 modules which each add a single amino acid residue to the peptide
through Condensation, Acetylation and Thiolation reactions. Adapted from Ling et al.
(2015).

16. 13 | P a g e
Teixobactin efficacy and resistance
After their initial discovery of E. terrae having an inhibitory effect on S. aureus and the
subsequent isolation of Teixobactin it was tested against a wide range of bacteria, both
gram positive and negative.
Organism Teixobactin MIC (μg ml-1)
S. aureus (MSSA) 0.25
S. aureus (MRSA) 0.25
Enterococcus faecalis (VRE) 0.5
B. anthracis ≤0.06
C. difficile 0.005
M. tuberculosis 0.125
K. pneumoniae >32
E. coli 25
Table 2. Minimum Inhibitory Concentration of Teixobactin with a selection of common
pathogens tested by Ling et al. (2015). Note that strong Teixobactin inhibitory activity is
limited to gram positive organisms, K. pneumonia and E. coli MICs being quite high.
Adapted from Ling et al. (2015).
As well as determining the activity of Teixobactin against a variety of organisms such as
those in table 2 above, Ling et al. (2015) also compared Teixobactin activity in S. aureus to
the hard-line Staphylococcal antibiotic Vancomycin. They found that at the same
concentration Teixobactin had a higher level of Staphylococcal clearance in exponential
phase cultures than Vancomycin but both were equally effective in early stage cultures
(Figure 8) (Ling et al., 2015). Additionally it was found that Teixobactin has high levels of
activity against Vancomycin intermediate S. aureus (VISA) (Figure 9).

17. 14 | P a g e
Figure 8. The Inhibitory effects of Teixobactin, Vancomycin and Oxacillin on (A)
Exponential phase S. aureus cultures and (B) Early phase S. aureus cultures. Adapted from
Ling et al. (2015).
Figure 9. The action of Teixobactin and
Vancomycin on Vancomycin intermediate S.
aureus cultures. Adapted from Ling et al. (2015)
Furthermore exposure of S. aureus cultures to sub inhibitory concentrations of Teixobactin
over a period of 27 days did not result in the formation of resistant strains (Ling et al., 2015).
This method of passaging pathogenic bacteria through media containing any given antibiotic
has been explored in a series of papers written out by a Spanish group led by José Martínez
(Martinez et al., 2011, Martinez and Baquero, 2000, Martinez et al., 2007). There a large
number of factors which affect the development of resistance to any antimicrobial. There
are three main categories of genes which must be considered when attempting to
understand bacterial resistance mutations. These are (A) genes which control the
production and cellular location of the antibiotics target; (B) genes which are able to
prevent the antibiotic reaching its target, either through altering the antibiotic itself through

18. 15 | P a g e
enzymatic action or removing it by efflux pump mechanisms; (C) Genes which code for
products which are involved in the antibiotic gaining access to its target (Martinez and
Baquero, 2000). Due to the fact that there is considerable amounts of cross talk between
different pathways and protein actions the list of genes in which a mutation could lead to
resistance extends greatly from genes whose products directly interact with the antibiotic
(Martinez and Baquero, 2000). The mutation rate of any implicated genes is also governed
by a variety of factors such as gene length, gene copy number, multiple easily mutable
nucleotide sequences in the gene and the distance of the gene from the origin of
replication (Martinez and Baquero, 2000). However as noted in their 2011 review the use of
a serial passaging experiment is insufficient to predict the true rise of resistance to a
particular antibiotic as it doesn’t take into account many factors such as horizontal gene
transfer and the selective pressures any bacteria will feel when in an environment
containing other organisms, either competitive or symbiotic (Martinez et al., 2011). Yet
serial passages still have use in their ability to identify genes which may be prone to
mutations enabling resistance (Martinez et al., 2011). Therefore to fully understand the
probability of resistance to teixobactin becoming prevalent it is crucial to have a thorough
understanding of its mechanism of action.

19. 16 | P a g e
Teixobactin – Mechanism of action
To elucidate the target of teixobactin, Ling et al. (2015) first sought to determine which of
the major biosynthetic pathways in S. aureus teixobactin acted on, either, the synthesis of
DNA, RNA, protein and peptidoglycan. This was carried out by measuring how Teixobactin
affected the level of incorporation of radiolabelled Thymidine (DNA), Uridine (RNA), Leucine
(protein) and glucosamine (peptidoglycan) in comparison with antibiotics known to act on
each of these. From this it was found that Teixobactin had little or no effect on DNA, RNA or
protein synthesis but had an extreme effect on the incorporation of glucosamine into
mature peptidoglycan (Ling et al., 2015).
Figure 10. Percentage incorporation of radiolabelled
DNA, RNA, Protein & Peptidoglycan precursors in S.
aureus when treated with Teixobactin (grey bars) or a
known antibiotic which effects the biosynthesis of the
appropriate molecule. Adapted from Ling et al. (2015).
The case for Teixobactin acting on some area of the
peptidoglycan synthesis was further strengthened by
the lack of resistance found in the serial passaging
experiment, this suggested that Teixobactins target was
not proteinaceous. Further study revealed that
Teixobactin treatment of S. aureus leads to a build-up of
the peptidoglycan precursor UDP-MurNAc-pentapeptide (Ling et al., 2015). In an effort to
determine which of the peptidoglycan precursors Teixobactin targets cultures of S. aureus
treated with Teixobactin were supplemented with each precursor compound. In cultures
supplemented with excess lipid II Teixobactin did not have an inhibitory effect (Ling et al.,
2015). Building upon this Teixobactin was added to purified cell wall precursor compounds,
it was found that along with lipid II Teixobactin interacts with lipid III, the precursor
molecule of wall teichoic acid (WTA). Using this knowledge a mechanism of Teixobactins
action was devised. Lipid II is also the main target of Vancomycin (Rubinstein and Keynan,
2014). Vancomycin binds to the D-ala-D-ala moiety of the Lipid II monomers prior to their
incorporation into the peptidoglycan wall (Rubinstein and Keynan, 2014). However if there

20. 17 | P a g e
is an alteration in this moiety Vancomycin fails to bind lipid II and loses its antibacterial
activity, as can be seen with Vancomycin Intermediate / resistant S. aureus (VISA/VRSA). The
activity of Teixobactin against VISA as shown by Ling et al. (2015) indicates that it does not
bind to the same area of lipid II as Vancomycin but seems to have a similar inhibitory effect.
Ling theorised that Teixobactin acts by binding to the lipid II precursor prior to its
incorporation into the peptidoglycan layer preventing said incorporation. However this
mechanism should yield similar levels of antibacterial activity as Vancomycin, yet as has
been shown Teixobactin MIC levels are considerably lower than those of Vancomycin
(Figures 8 & 9) (Ling et al., 2015). Teixobactins ability to bind lipid III was suggested to be the
cause of this differential activity. Ling brought two separate studies to back up his argument
which show the dangers to S. aureus of inhibiting WTA synthesis. The first showed that
inhibiting late stage WTA causes a build-up of toxic compounds leading to cell death (D'Elia
et al., 2006). The second details the activity of WTA in anchoring autolysins, preventing
them from hydrolysing the peptidoglycan (Bierbaum and Sahl, 1985).

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In vivo studies
After identifying Teixobactins mechanism of action Ling et al. (2015) tested its activity in
both Serum and Mice models. They found that in Serum Teixobactin showed low toxicity
with high stability and efficacy. Similarly Teixobactin had positive results in the three mouse
studies carried out. Teixobactin remained in the blood serum of the mice at levels over the
MIC for 4hrs. In subsequent experiments mice were inoculated with a dose of MRSA which if
untreated causes 90% mortality, these mice were treated with a range of Teixobactin doses
as low as 1mg per kg body weight, all mice survived when treated. From this Ling et al.
(2015) calculated the protective dose (PD50) of Teixobactin was 0.2mg/kg, proving its higher
in vivo efficacy then Vancomycin (PD50 = 2.75mg/kg).

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Future Challenges and potential:
The I-chip has the potential to entirely revolutionise the pace of discovery of antibiotics and
other useful natural products in a way never seen before. The classical methods for the
culturing of microbes gave at best a recovery rate of less than 1% of the total microbial
fauna. The I-chip has the ability to give a recovery rate of environmental microbes of up to
50%. This increases the pool for new useful compounds by over fifty times, I fully expect
many new compounds to be uncovered in the coming years due to the I-chip.
However there are drawbacks to the I-chip. As mentioned earlier the I-chip works on the
principle of allowing for the interaction of an isolated microbe with its environment. This
may cause an inherent problem in the scaling up a culture of any microbe of interest
discovered using the I-chip. It is not yet clear whether it is possible to create domesticated
strains of all microbes isolated using the I-chip in a similar manner to the work of Nichols et
al. (2008) with their isolates MSC1 and MSC2. It may be that many species identified with
the I-chip will be bonded to their microbiome in a stronger fashion, resulting in the need to
either co-culture the desired microbe with its required growth factor producers or find a
way to produce the product in question in a more easily cultivable organism like E. coli.
These problems may yet arise in Teixobactin. There are numerous other challenges which
Teixobactin must face if it is to reach clinical development. If it is indeed possible to create
industrial scale cultures of E. terrae it will be crucial to identify the exact biosynthetic
pathway to Teixobactin production. As outlined earlier there are numerous gaps in our
knowledge such as to the regulation of the Teixobactin NRPS genes txo1 and txo2,
Teixobactins export or the exact pathway which Txo1 and Txo2 synthesise it. If all of these
details are uncovered we will be able to experiment with ways in which to optimise
Teixobactin production like altering growth media components or causing site directed
mutagenesis. These methods have been used in the past to increase the production of other
antibiotics such as Daptomycin (Yu et al., 2011a, Yu et al., 2011b, Baltz et al., 2005).
Ling et al. (2015) have showed that Teixobactin has in vivo efficacy in mouse models as well
as desirable pharmacokinetic parameters. However no tests against human cell culture have
yet been carried out, while its non-toxicity in the murine model is promising prior to clinical
trials commencing toxicity and immunogenicity testing should be carried with human cell
culture and antibody assays.

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Conclusions:
 In the past 40 years there has been a significant lack of new antibiotic compounds
reaching clinical practice.
 This is thought to be due in large to the inability to culture over 99% of the worlds
microbial fauna using classical cultivation methods.
 The Isolation Chip as developed by Nichols et al. (2010) is a breakthrough platform for
microbial cultivation.
 It is estimated that the I-Chip will allow for the cultivation of up to 50% of the worlds
microbial fauna.
 The I-Chip works upon the principle that many microbial species cannot grow in
complete isolation and require the input of a variety of signals from their natural
environment in order to grow and replicate.
 The I-Chip allows for the Isolation of cells from an environmental sample while keeping
them in constant contact with their environment which allows for small cultures to
grow.
 Past studies have shown that some specific species need each other in culture to allow
for growth, but, upon repeated co-cultivation ‘domesticated’ strains were created which
could grow independently on classical media.
 Species ‘domestication’ could allow for the large scale cultivation of many species which
will be newly discovered with the use of the I-Chip. However, as this has only been
shown in one instance, domestication may not take place in all species.
 Using the I-Chip a previously undescribed bacterium Eleftheria terrae was isolated. E.
terrae was found to have strong inhibitory action against Gram positive organisms.
 On investigation a compound named Teixobactin was isolated from E. terrae. This was
the source of its antibacterial action.
 Teixobactin works by inhibiting the synthesis of Gram positive peptidoglycan while
simultaneously preventing the formation of Wall Teichoic Acid. This leads to the release
of autolysins into the peptidoglycan causing hydrolysis while also causing a build-up of
toxic precursors in the cell.
 Teixobactin has high activity against a range of Gram positive organisms and against M.
tuberculosis. However it is relatively ineffective against Gram negatives.
 In serial passaging experiments S. aureus cultures were unable to develop resistance to
sub-lethal concentrations of Teixobactin.
 In vivo Teixobactin showed high efficacy, good pharmacokinetic and pharmacodynamics
properties as well as efficient clearing of infection in mice models.
 The biosynthesis of Teixobactin is still unclear, whether E. terrae can be grown on a large
scale is yet to be seen.
 Teixobactin is the first potentially clinically useful compound to be discovered through
the use of the I-Chip. Hopefully this will encourage others to take on the I-Chip as the
discovery platform of choice for natural compounds.

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