Conference Session B5
Paper 27
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COMBATTING DRUG RESISTANT BACTERIA WITH CRISPR-CAS9
TECHNOLOGY AND ANTBIOTIC SCAFFOLDING
Emily Nutter, [email protected], Mahboobin 10:00, Meggan Rusiewicz, [email protected], Mena 1:00
Abstract — In today’s world, drug-resistant bacteria have
become a major public health and safety problem and the
rate at which bacteria are developing resistance is higher
than the rate at which new antibiotics are being developed.
Chemical engineers are devising a way to create new
antibiotics to overpower the two main types of drug resistant
super-bacteria: Gram-positive and Gram-negative. A current
approach to new antibiotics is a process that uses artificial
amendments made to the DNA of the selected microorganism
using the CRISPR-Cas9 (CRISPR) genome editor. The
CRISPR can be programmed to attack and replace the drugresistant genes. This serves two main purposes; to weaken the
bacteria and to allow the engineers to better understand the
genetic structure of the bacteria. Thus, they will be more
successful in modifying existing antibiotics and creating new
antibiotics to approach the drug resistant bacteria.
Currently, antibiotics are sorted by class based on their
structural scaffolds. Different classes are designed
specifically to attack a bacterium in one of several ways.
Engineers are looking at creating new synthetic scaffolds
inspired from existing classes to use as the basis for new
drugs that are tailored to beat the drug resistant bacteria.
Key Words —Antibiotics, CRISPR-Cas9, Drug Resistant
Bacteria, Gram-negative, Gram-positive, Scaffolds
THE RELATIONSHIP BETWEEN
ANTIBIOTICS AND BACTERIA
Drug resistant pathogenic bacteria has become an
imminent threat with the increased and sometimes
unnecessary reliance on antibiotics. Whether they are used to
treat bacterial infections in humans or to reduce and prevent
the spread of disease in animals farmed for consumption,
crops and other food products, this rate of use has a major
downside. Overexposure to antibiotics can cause bacteria to
mutate and become resistant to antimicrobial medication as it
feels pressured to survive. This creates a deficit of antibiotics
that are to win the fight against mutated resistant bacteria.
Bacteria are either categorized as Gram-positive or Gramnegative, both of which come with their own obstacles.
Before beginning to alter the antibiotics to overcome the
resistant bacteria, it is important for engineers to understand
University of Pittsburgh Swanson School of Engineering 1
2.10.2017
and control the mutations in the bacteria that create the
resistance. Engineers use CRISPR-Cas9 technology to study
and change targeted pieces of a bacteria’s DNA. The two
elements involved, the Cas9 enzyme and a guide RNA strand,
travel along the bacteria’s DNA strand, find a pre-specified
piece of strand, and can remove or change this targeted
portion. It is possible to use this technology to deliver a
bacteriophage, a virus that attacks bacteria, straight to the
core of bacteria. An improved understanding of the genetic
structure of the bacteria can help engineers to develop new
antibiotics. Antibiotics are designed with mechanisms that
target specific weaknesses in the bacteria: in turn the bacteria
develop mechanisms that work to resist. The base of every
antibiotic starts with a scaffold which allows for
modifications that improve the overall strength and
effectiveness of the antibiotic. Natural scaffolds are
developed from organisms found in nature, including fungi,
other bacteria, and other organisms. Engineers have created
completely synthetic scaffolds as well, which allow for a
larger variety of modifications. Bacterial resistance is a
human created problem and without immediate attention the
misuse of antibiotics will bring us back to a pre-antibiotic era
unless we can control the excessive use of antibiotics and
educate society.
WHY ARE BACTERIA BECOMING
RESISTANT TO ANTIBIOTICS?
There are several factors that can cause bacteria to develop
resistance to antibiotics. Prescribing antibiotics for viral
infections inappropriately happens more often than it should
with “about 44 percent of outpatient antibiotic prescriptions
written to treat patients with acute respiratory conditions,
such as sinus infections…An estimated half of these
outpatient prescriptions are unnecessary” [1]. Society is most
familiar with the concept of antibiotics yet does not
understand that antibiotics cannot function against virial
infections, therefore a prescription for antibiotics is expected
whenever they are feeling ill and visit their doctor. There is a
direct correlation between use of antibiotics and bacteria
developing resistance proven by “…countries with a higher
consumption of antibiotics show higher rates of resistance”
[2]. Medical professionals should focus on prescribing
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antibiotics only when necessary- making sure they are careful
to prescribe the correct antibiotic, in the correct dose, for the
correct duration. An estimated $1.1 billion is spent each year
on antibiotics that are unnecessarily prescribed [3]. Other
precautions include avoiding the use of broad-spectrum
drugs, which are able to treat both Gram-negative and Grampositive bacteria, and cycling through antibiotic classes. This
avoids overexposure to the same mechanisms of attack.
Antibiotics generally do not bring in much revenue due
to short-term prescriptions, so big pharmaceutical companies
do not focus on the development of new antibiotics; “in 2004,
only 1.6% of drugs in clinical development by the world’s 15
largest drug companies were antibiotics” [3]. While drugresistant bacteria are presenting a problem that is more and
more prevalent, the large majority of antibiotics prescribed
are already established, therefore discouraging companies
from investing time and money into new antibiotic research
for little financial return.
The food and livestock industry are major culprits of
unnecessary exposure and abuse of antibiotics. The livestock
industry, “…[has] been reported to receive over 13 million
kilograms, or approximately 80% of all antibiotics, in the
USA annually. Much of this is not in veterinary medicine, but
in the form of continuous sub-therapeutic application of
antibiotics for growth promotion and disease prevention in
intensively farmed animals” [4]. For the same reasons
bacteria can develop resistance to antibiotics misused in
humans, they develop resistance in the animals. Resistant
bacteria can be unintentionally transferred to humans when
they eat tainted meat. Misuse is not limited to just livestock
and antibiotics are sprayed prophylactically on fruit and other
food products grown industrially [3]. Sprayed antibiotics can
become runoff and create drug resistance in soil-based
bacteria or seep into ground water which can lead to
accidental ingestion of resistant bacteria when we drink from
the tap.
antibiotics [6]. Gram-positive bacteria have a thick layer of
easily penetrable peptidoglycan as the structure of their cell
wall making them easily defeated by antibiotics [7]. Gramnegative bacteria have a double membrane; a thin layer of
peptidoglycan, covered by a layer of lipopolysaccharides
(LPS) [8]. The endotoxicity of LPS produces virulent results,
making Gram-negative bacteria difficult to kill.
FIGURE 1 [9]
Displays the difference between the cell membrane of
Gram-negative and Gram-positive bacteria
Figure 1 demonstrates a visual approach to the difference
between the cell wall structures. The base plasma membrane
with proteins, shown in green, working as architectural
supports inserted throughout is shown in gray appears with
the same size and structure in both Gram positive and Gram
negative bacteria. The next layer, shown in blue, is the
peptidoglycan. Gram positive bacteria have double the
amount of peptidoglycan serving as their primary outer wall.
The peptidoglycan is thin and weak, with large holes in the
structure, making Gram-positive bacteria more susceptible to
antibiotics because they can’t stop foreign bodies from
entering through its cell wall [10]. Gram-negative bacteria’s
layer of structurally secure LPS, pictured in red, sits on top of
the layer of peptidoglycan with more proteins layered
throughout. The LPS layer gives more structural support to
the cell wall, fortifying it further. Their tougher, multilayered membrane makes them more dangerous and naturally
more resistant. Antibiotics are designed to target specific
weaknesses of both types of bacteria.
TWO MAIN TYPES OF BACTERIA
Bacteria are a group of microscopic living organisms
generally classified in two types: Gram-positive or Gramnegative. They are prokaryotic, meaning they have no
nucleus and are singled celled. While both types of bacteria
have a phosphorus-containing fatty lipid or phospholipid and
protein base called the plasma membrane, the rest of their cell
membrane structure differs. Gram-negative or Gram-positive
describes the structure of their peptidoglycan-based cell
membrane. Peptidoglycan is composed of sugar-based glycan
polymers and short peptide chains of amino acids cross
linked to form a mesh network. It also includes penicillinbinding proteins, called DD-transpeptidase, which are
structurally similar to the antibiotic penicillin. When a cell
wall breaks apart to duplicate, DD-transpeptidases serve to
cross-link and rebuild the peptidoglycan in the cell wall [5]. It
is the most basic component of the bacteria’s cell wall and its
structure is unique to bacteria, making it a target for
ANTIBIOTIC MECHANISMS
Bacteria are careful about what enters their single celled
system, so there are several different mechanisms employed
by the antibiotics that target different aspects of the bacteria.
Antibiotics can be bactericidal, meaning they kill bacteria. or
bacteriostatic, meaning they inhibit growth or duplication;
however, most antibiotics are not exclusively one or the other
and can be influenced by outside conditions to be more
bacteriostatic or bactericidal [11]. There are three modes of
action frequently seen in antibiotics used today: inhibition of
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cell wall synthesis, inhibition of protein synthesis, and
inhibition of DNA synthesis. The mechanisms are not limited
to the three examples discussed however, these are the most
prevalent and effective techniques.
The most common mechanism of attack is the inhibition
of cell wall synthesis. β-lactam antibiotics, including
penicillins and cephalosporins, use this method. β-lactams
target the bacteria when the cell is attempting to reproduce.
During this time, there are holes in the cell wall as it splits,
normally to be filled by DD-transpeptidases, penicillinbinding proteins. DD-transpeptidases are meant as a
placeholder to prevent the cell from exploding and to create a
bridge for the peptidoglycan to repair. Because DDtranspeptidases share a shape similar to the β-lactam ring,
penicillin will enter the holes in the bacteria instead of the
DD-transpeptidase and prevent the peptidoglycan from filling
the holes. This causes the cell to rupture. The easiest bacteria
to destroy by inhibition of cell wall synthesis are Grampositive bacteria because of their thick yet weak layer of
peptidoglycan that has no defense to the penicillin [12].
Figure 2 below shows the β-lactam ring in penicillin, the
square of carbons, nitrogen, sulfur, and a doubly-bonded
oxygen in the center of the scaffold.
cannot properly duplicate [15]. Without being able to
duplicate, the resistant bacterium remains stagnant in the
body and does not cause infection.
Protein synthesis inhibitors (PSIs) are another antibiotic
mechanism that attacks the bacteria during translation. The
commonly distributed classes of antibiotics that use this
mechanism are tetracyclines, aminoglycosides, and
macrolides. Both aminoglycosides and tetracylines bond to
the same site during the same step of translation, but
aminoglycosides bond irreversibly, killing the bacteria
defining them as bactericidal. Since proteins are essential
building blocks of any cell and are found everywhere, all
antibiotics could be considered PSIs; however, the classes of
antibiotic using this mechanism target protein assembly at the
ribosomal level [16]. Different classes typically have
different specific targets. Prokaryote protein synthesis has
several steps and PSI antibiotics can be manipulated to attack
the process at a different step if the bacteria develops
resistance [16].
If bacteria were easily and permanently defeated by the
attacks, there would be no need to develop new antibiotics,
but because bacteria are living they are able to evolve and
develop their own resistant mechanisms to fight back against
the drugs.
BACTERIA’S RESISTANT MECHANISMS
Bacteria develops resistance in two main ways: intrinsic
resistance or an acquired resistance from a genetic change or
a DNA transfer [17]. While both prove difficult to attack with
antibiotics, acquired resistance is entirely new, forcing
engineers to understand the mutation the bacterium has
undergone before attempting to design an antibiotic for them.
Intrinsic resistance is a mechanism built into the original
structure of the bacteria. There is no alteration or mutation
present and their ability to resist antibiotics in a naturally
occurring mechanism. As discussed previously, the difference
between Gram-negative and Gram-positive bacteria is the
tougher, multilayered cell membrane of Gram-negative
bacteria. Although there is a greater level of familiarity with
this type of resistance, it is still a challenge to develop
antibiotics to break through the membrane of the Gramnegative bacteria [10].
Acquired resistance is the main culprit of the current
dilemma with drug resistant bacteria. The bacterium acquires
resistance in one of two ways: through a genetic mutation or
through a DNA transfer. For a genetic mutation, a bacterium
changes its genetic structure to prevent the antibiotic from
attacking. Depending on which antibiotics are used against a
bacterium and the mechanisms of the antibiotic, the bacteria
will mutate in three main ways. The first is target resistance.
The original target of the antibiotic changes shape to prevent
the antibiotic from accessing it [18]. The second method is
enzymatic degradation [18]. A β-lactam based antibiotic such
as a penicillin or cephalosporin will become ineffective
against a bacterium that has developed β-lactamase, a type of
FIGURE 2 [13]
The basic structural scaffold of a Penicillin antibiotic
Antibiotics can be developed as broad spectrum, meaning
they are effective against both Gram-negative and Grampositive. Penicillin G is primarily effective against Grampositive bacteria, but Ciprofloxacin, a fluoroquinolone
antibiotic, who also have a β-lactam ring, are considered
broad spectrum due to its effectiveness against both Gramnegative and Gram-positive bacteria [14].
Inhibiting DNA synthesis is another technique used by the
classes quinolones and fluoroquinolones. Fluoroquinolones
are quinolone antibiotics with the inclusion of a fluorine
atom. These antibiotics target the production of DNA. An
enzyme called topoisomerase allows the double helix of DNA
to break apart and duplicate by relieving tension.
Topoisomerase II is found in bacteria and is the main target
of the fluoroquinolones. By attacking and disabling
topoisomerase, the DNA is under too much tension and
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enzymatic degradation that destroys the β-lactam ring by
breaking it open, rendering it incapable of inhibiting the cell
wall synthesis. For this specific example, clavulanic acid, a βlactamase inhibitor, is combined with amoxicillin, of the
penicillin class so it still has the β-lactam ring, to create
Augmentin, an antibiotic that counters mutated mechanism of
the bacteria [19]. For any bacteria that mutates to produce an
enzyme for enzymatic degradation, the new enzyme is
developed from an existing enzyme to react with the
antibiotics so that it can no longer complete its attack as
intended.
The final form of defense is an efflux pump. While
bacteria have natural proteins that pump out toxins as a form
of intrinsic resistance, they can develop efflux pumps that are
made specifically to remove the presence of antibiotics it
recognizes. This mechanism of resistance allows the
antibiotic into the cell but quickly removes via proteins that
act as pumps that regulate what enters and exits the cell.
There are drug specific efflux pumps, such as one against
tetracycline, but more dangerous still are multidrug efflux
pumps [20]. A multidrug resistant efflux pump is a single
efflux pump that functions in the same way as a regular
efflux pump, however it has the ability to work against
several type of drugs, therefore allowing the bacteria to
develop multidrug resistance. Oftentimes, the multidrug
resistant efflux pumps work against several drugs in the same
family, but can pump out drug of varying families as well
[20].
Bacteria do not have to develop the resistance by mutating
on their own and they can acquire it by sharing DNA with
other bacteria. Bacterial gene transfers can happen between
different bacterial species and a bacterium can acquire several
forms of resistance. This sharing of genes is called a
horizontal gene transfer (HGT). HGT can occur between
bacteria of the same species or between different species.
Three main processes by which HGT occurs are transduction,
transformation, and conjugation. Conjugation is the main
form of HGT and occurs when there is direct contact between
two bacteria and the resistant bacteria will transfer the
mutated DNA. Transformation is when a bacterium absorbs
mutated DNA present in its environment as a result of the
death of another bacterium. Transduction occurs when
bacteriophages, viruses that attack bacteria, transfer DNA
during their natural course of action [21].
To make the counter-fight against the bacteria, engineers
take advantage of the technology of the CRIPSR-Cas9
genome editor to understand the mutation the bacterium has
developed.
Regularly Interspaced Short Palindromic Repeats (CRIPSR)
and the Cas9 enzyme to explore and edit the drug resistant
bacteria. Prior approaches “…relied upon customizable
DNA-binding protein nucleases that required scientists to
design and generate a new nuclease-pair for every genomic
target” [22]. CRISPR technology can now replace this
practice due to its simplicity and adaptability. The CRISPR is
a genome editor that gives the power to engineers to edit
specifically targeted pieces of a bacteria’s DNA.
Components of CRISPR
CRISPR-Cas9 is made up of two key elements, the
enzyme Cas9 and the guide RNA (gRNA), which is a short
synthetic RNA composed of a scaffold sequence [22]. The
Cas9 follows the gRNA to the part in the DNA that needs
modification. The Cas9 acts as scissors to cut the DNA
strands and the cell then realizes the strand has been damaged
and will attempt to repair it. At this point, researchers can
introduce the changes they want to make to the genes [23].
The weakened cell, attempting to fix itself, will grab from
anywhere it can in order to fill the hole created by the Cas9
enzyme. Engineers can choose where they want to send the
CRIPSR, so they specifically target the mutated portion of the
bacteria to weaken the drug resistance that has developed and
re-sensitize it to the antibiotics [24]. They can also repress the
targeted genes so they do not try to re-replace the gene
amendment that has been artificially introduced. In doing so,
the drug resistant genes are removed completely or altered
and engineers have gained an improved understanding of the
genetic structure [25]. The edit they have introduced is
duplicated when the bacteria splits, eliminating the mutation
from the future generations of the bacteria as well. This
allows them to create new antibiotic generations or attempt to
reintroduce old version of antibiotics that were at one point
no longer effective.
USING CRIPSR-CAS9 AND
BACTERIOPHAGES TO MODIFY THE
BACTERIA’S GENES
FIGURE 3 [26]
A basic model of a CRISPR genome editor
Engineers attempt to understand the genetic structure of
bacteria using a method of DNA splicing with Clustered
In Figure 3, the process of the CRIPSR is demonstrated
with only the simplest elements necessary. The Cas9 enzyme,
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depicted as the blue background has opened the DNA strand
at the target sequence where it was instructed. The gRNA
brings the new DNA to the target sequence so that when the
strand begins its reparations it will grab at this first.
The CRISPR isn’t just used to return bacteria to its
antibiotic sensitive state, but to allow engineers to study the
effects of a DNA change on a bacterium. This means that an
unknown mutation can be identified if it is removed and help
us to better understand the functionality of the bacteria as a
whole. Because the technology of the CRISPR-Cas9 is
relatively new, it is still focused on animal models or isolated
human cells, but there is great potential for it to be used
routinely in humans [23].
same way they do the static antibiotics. Lastly, endolysins
target specific bacteria species, so beneficial bacteria are not
accidentally killed in the process, a practice which reduces
the chance of infection and side-effects [29].
Since
endolysins remove the threat of resistance, it is the goal of
engineers to extract and purify the endolysin for antibiotic
development [30]. Endolysins have a working mechanism
unrelated to that of antibiotics, so the resistant bacteria will
not be able to use their acquired method of resistance to fight
the endolysin.
Bacteriophages are unpredictable and can accidently share
mutated DNA between bacteria, a form of HGT. This
possibility deters engineers from looking solely at
bacteriophages. Looking for sources of new antibiotic
scaffolds can provide a safer, more reliable, and more
controlled approach to defeating antibiotics.
Bacteriophages
Bacteriophages (phages) are a bacteria attacking virus.
They were once at the forefront of antimicrobial research, but
upon the discovery of penicillin by, phages were largely
abandoned [27]. Because phages are a virus, there is
hesitation in using them as antibacterial treatments; however,
“…bacteriophages attack only host bacteria, not human cells,
so they are potentially good candidates to treat bacterial
diseases in humans” [28]. There are two lifecycles that a
bacteriophage can lead called: the lytic cycle or the lysogenic
cycle. The lytic cycle begins with the proteins in the tail of
the phage binding to the bacteria cell, where it injects its
DNA genome into the cell. The DNA is then copied and
resources in the bacteria’s cell are used to create more phages
within. Once there are enough phages inside, proteins, called
endolysin, released by the phages tear the peptidoglycan of
the cell wall and allow water inside, causing the bacteria to
explode. Afterwards, the phages are freed to find other host
cells nearby to infect. In the lysogenic cycle, the attachment
and DNA injection steps are the same as the lytic cycle,
except the DNA is not immediately copied or instructed to
make proteins. Instead it integrates itself into the bacterial
chromosome, now called the prophage. It lives dormant in the
cell and transfers with the DNA when the cell divides.
Eventually the prophage can become active again and
continue the rest of the cycle until the bacteria explodes.
Phages will either reproduce solely by the lytic cycle or they
can alternate between the lytic and lysogenic cycles since
they are copied along with the host DNA each time the cell
divides instead of destroying their host bacteria’s cell
immediately [28].
The protein produced by the phages, endolysin, is a
peptidoglycan degrading protein that damages the membrane
of the bacteria, especially in the thick, weak peptidoglycanbased cell membrane of Gram-positive bacteria. Endolysins
have characteristics that make them a candidate for fighting
resistant bacteria. Because phages are living organisms like
bacteria, they “…have co-evolved with bacteria over billions
of years, so endolysins target areas of the bacterial cell wall
that the bacterium cannot change during reproduction” [29].
The bacteria do not develop a resistance to the phages in the
ANTIBIOTIC SCAFFOLDS: THE BASIC
STRUCTURE OF AN ANTIBIOTIC
Antibiotics were originally derived from fungi, mold and
other living organisms. The organism is fermented by adding
sugars to increase the production. The molecules used are
then harvested separated from the organism and purified into
the final antibiotic [31]. These molecules harvested from the
fermented fungi, mold, or other organism are known as the
scaffold for designing antibiotics. They are the basic
chemical structure that remains fixed in all generations of an
antibiotic in a particular class. Engineers can add chemical
modification and design antibiotics to destroy Gram-negative
and Gram-positive bacteria based on their weaknesses.
Antibiotics are sorted into classes based on their mechanism
of attack. Their class also describes the molecular scaffold
used to build the antibiotic. Figure 4 demonstrates examples
of the scaffold for classes in five of the most commonly used
antibiotic classes; penicillin, cephalosporins, quinolones,
macrolides, and tetracyclines. These classes of drugs make up
the majority of drugs distributed for consumer use [32]. For
each generation built using the scaffold, the change is
indicated by the red group added to the scaffold, which is
kept in black to show the constant presence of the molecules.
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[35]. This will broaden the range of accessible antibiotics in
the same class. Of the five antibiotics in Figure 4, only the
quinolones have a completely synthetic scaffold. Completely
synthetic scaffolds can be more easily amended than natural
scaffolds because engineers have more control however
synthetic scaffolds are hard to create and many were
discovered by accident [32]. Innovations like the CRISPR
genome editor allows insight into the genetic structure of the
bacteria. It can help identify enzymes essential to bacteria
growth and these can be used as a structural compound in the
synthetic scaffolds of the antibiotics, possibly developing
new classes of antibiotics.
Looking for sources of new scaffolds
While it is innovative to develop synthetic scaffolds, it is a
complex chemical process and difficult to get the final
antibiotics approved because they are potentially unstable.
Looking in unexplored locations can reveal new molecules
and sources of scaffolds. The scaffolds of existing antibiotics
have come primarily from soil bacteria because they are easy
to collect and ferment into the scaffold for antibiotic
development. Looking at deep-sea sediment and other marine
sources has proved promising for the discovery of bacteria
and fungi that can be turned into molecular scaffolds [35].
Anthracimycin, discovered in 2013, is a new antibiotic
developed from marine derived bacteria. It was initially
found effective against the Gram-positive bacteria Bacillus
anthracis, which causes an anthrax infection and is being
tested against other bacteria that are structurally similar to
Bacillus anthracis. This includes the multidrug resistant
Gram-positive Staphylococcus aureus. Anthracimycin proved
effective against all strains of S. aureus, including the drug
resistant strain [36].
New natural scaffold locations are not limited to marine
soil. Bacteria living symbiotically in insects, ascidians (sea
squirts), and fungi are being investigated as sources of new
molecular scaffolds for antibiotic production [36]. Engineers
can’t take these organisms from anywhere: they must make
sure the new natural scaffolds are actually useful against the
pathogenic bacteria, be it Gram-negative, Gram-positive, or
both. The new molecular scaffolds shouldn’t be overlap with
existing antibiotic scaffolds to prevent an overlap in
resistance. The scaffold should also be able to be amendable
to the inevitable addition of synthetic variations included
when engineers attempt to improve the effectiveness of the
antibiotic.
FIGURE 4 [32]
Example of how an antibiotic scaffold is used to build new
antibiotics
In β-lactam antibiotics, like those in the penicillin class or
the cephalosporin class, Figure 4 shows the familiar β-lactam
ring built into the scaffold of the drug, making it vital to the
structure of each antibiotic in this class. During the period of
antibiotic production from 1950-1960 known as the golden
age of antibiotic discovery, most antibiotics scaffolds came
from natural sources [33]. Later generations created, which
are considered new antibiotics, are synthetically developed
from the base scaffold of the class. More than two-thirds of
clinically used antibiotics are from natural products or their
semi-synthetic derivatives [34]. These derivatives work
similarly to the first generation antibiotic, however they are
edited to avoid the development of bacterial resistance and to
improve their strength as an antibacterial, perhaps making it
into a broad spectrum antibiotic. “Just four…scaffoldscephalosporins, penicillins, quinolones, and macrolidesaccount for 73% of antibacterial chemical entities…”,
showing an obvious need for entirely new scaffolds [34]. One
way to go about developing new scaffolds is to create
synthetic scaffolds of pre-existing natural scaffolds. A
synthetic scaffold that is identical to a natural scaffold allows
engineers to manipulate the structure of the scaffold without
destroying it and its functionality. Bacteria will have less
intrinsic resistance to antibiotics with a synthetic scaffold
because the bacteria have not been previously exposed to the
structure and mechanism. As an example, bacteria that has
developed efflux pumps against tetracycline limits the
effectiveness of the antibiotic; however, fully synthetic
tetracyclines will allow for edits that are difficult to make to
scaffolds that are developed semi-synthetically. A similar
procedure is being applied to macrolides. Scientists are
generating a completely synthetic version of the macrolides
scaffold, giving more room for precisely controlled variations
ETHICAL DILEMMAS
When antibiotics were first discovered, no one knew that
the bacteria would eventually become resistant. Once the
resistance began, medical professionals and engineers started
working on new antibiotics to target this mutated bacterium.
Bacteria will continue to evolve and become resistant to new
and old classes of antibiotic. This raises an ethical dilemma to
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consider. Is it worth it to continue dedicating time, effort, and
funding into research and development of new antibiotics,
knowing they are trapped in a never-ending cycle where
bacteria will eventually become resistant to every new
antibiotic made? Or should professionals shift their focus on
to trying to find another way to treat bacterial infections?
The issue of drug resistant bacteria has spurred interest in
the discovery and creation of new antibiotics, but it is not
time efficient or cost effective and the process of producing a
new antibiotic can require upwards of “…ten years and $300
million to bring a new antibiotic to the market” [37].
Statistics like these contribute to a pharmaceutical company’s
decisions to contribute to the fight against resistance bacteria,
therefore a deficit exists in the development of new
antibiotics that are needed to keep pace with the evolution of
drug resistant bacteria.
Professionals have continued to strengthen antibiotics by
making semi-synthetic derivatives and broad spectrum
antibiotics that work against both Gram-positive and Gramnegative bacteria, but bacteria still become resistant. Bacteria
are not limited to one type of resistance, so the more
antibiotics it is introduced to, the more likely it is to develop
several forms of resistance. In addition to strengthening the
antibiotics by changing the chemical structure, engineers are
looking for sources of novel antibiotics, but “…many efforts
to find novel drugs in fungi and soil result in compounds that
are the same or very similar to previously discovered
antibiotics” [37]. Most new antibiotics are not shown to have
benefits outweighing the potentially dangerous side effects or
have not been able to demonstrate satisfactory efficacy. The
only way to preserve the potency of existing classes of
antibiotics is by decreasing the overall use [37].
Since there are many roadblocks in the process of
developing new antibiotics and no known way to overcome
drug resistant bacteria permanently, why aren’t the resources
spent on finding a different way to treat bacterial infections?
The problem with this is that it’s easier said than done. By
looking at the past, it is apparent that there were no options
on treating bacterial infections until antibiotics were
discovered. Antibiotics were the best option then, and still are
now. There are only a few alterative options, such as the
bacteriophages, but antibiotics are the most pre-established
and recognized option.
billions of years, so bacteriophages can target portions of the
bacteria that cannot change during reproduction [29].
Proteins produced by the bacteriophage only target bacterial
species and thus cannot kill healthy, living cells in the
process. This proves how sustainable of an option
bacteriophages are. They cut out the problem of antibiotic
resistant bacteria since bacteriophages are not an antibiotic,
and, as far as we know, no chance of growing resistance.
The sustainability of any solution to antibiotic resistant
bacteria is important to maintain quality of life. Antibiotics
have a low chance of being able to keep up with drug
resistant bacteria forever, so without a solution to combat
bacterial infections, the fate of the human race is at stake. If
we cannot come up with a solution to drug resistant bacteria,
this will become a problem that affects the whole world.
Many people die in third world countries due to lack of
antibiotics but now people are dying due to being infected
with bacteria that is resistant to all available antibiotics. With
this said, whichever solution found most appropriate and safe
to combat drug resistant bacteria must be sustainable in order
to keep the human species alive.
Not only do we need to have sustainable solutions, we
need to sustain good prescription practices. Doctors often
cater to patients request for antibiotics. “Patients often do not
understand the possible harm of taking antibiotics without
restraint, and doctors may feel too pressed for time to explain
why an antibiotic is unnecessary – so a prescription is written
instead” [38]. Because of this, twenty to fifty percent of
antibiotic use is deemed inappropriate [37]. Over-prescription
of antibiotics is a problem that affects everyone, even if that
individual does not partake in the use of antibiotics. Overuse
can be found in animal farming and agriculture since they
dispense large amounts in the form of animal feed or spray on
plants for disease prevention. To keep up the sustainability of
antibiotics, their use must be withheld unless it is a dire
situation in which it is the only course of treatment.
IMPACT IN THE FUTURE
The future of the public’s health is at stake and depends on
the engineers working towards antibiotics that can overcome
drug resistant bacteria. Without progression, we will have no
ways of treating bacterial infections or preventing the spread
of disease in animals and food products. Thankfully, such
progress has been made, but we must keep up with the rate of
change in resistant bacteria. The bacteria will continue to
become ever resistant, and it is in the hands of the engineers
to step up and continue improving. Whether the progression
be through modifying old antibiotics to overcome the drug
resistant bacteria, figuring out how to use the protein in
bacteriophages, or even discovering a new way to solve this
problem, all progress is good progress.
SUSTAINABILITY
In the case of antibiotics, sustainability is important for
quality of life. Illness is inevitable and we need a solution that
will stand up to bacteria with unceasing effort. At the current
rate of production of new antibiotics, continuing this practice
is not a sustainable option. We need a solution that hold up
the ever-changing drug resistant bacteria. That is why the
current research in bacteriophages may prove feasible as the
sustainable option. Bacteria cannot develop a resistance to
bacteriophages since they have co-evolved together over
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ACKNOWLEDGEMENTS
We would like to acknowledge the freshman engineering
department at Pitt, our chair Mark Shearer, and our co-chair
Kelly Donovan for their input and guidance.
9