The Potential Use of Reactive Oxygen Species as Antibacterial Treatment
Joshua T. Bram
April 15, 2013
Microbiology 201H
Pennsylvania State University
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The development of safe antibiotics and the advancement of treatment of bacterial
infections could be considered the most revolutionary and life-changing development of
the 20th century. When Alexander Fleming left a Petri dish unattended and contaminated
with Penicillium mold over vacation, little did he know that mere years later his
subsequent discovery of the antibiotic penicillin would reshape the face of medicine.
Since that time, a myriad of different antibiotics with a broad array of applications have
been isolated and developed for use as treatments of naturally occurring bacterial
infections. From the discovery of penicillin in 1940 (still used to treat skin and chest
infections) to tetracyclines in 1953 (broad-spectrum antibiotics) to Carbapenems in 1976
(often a last-ditch, broad-spectrum antibiotic), a surge in the discovery of new and
effective antibiotics gripped the medical field (NHS Choices). Doctors could then treat a
wide variety of infections and diseases, drastically increasing life expectancy from 62.9
years at birth in 1940 to 78.7 years at birth in 2010 (Deaths: Final Data for 2010). The
drugs were and are a “miracle” to be lauded, and in the words of Winston Churchill,
“[We] should be bound to celebrate, among others, Saint Penicillin.”
However, as quickly as antibiotics have crept to every corner of the world and
drastically improved the quality of living for billions across globe, the threat of reduced
efficacy of the drugs has also spread in recent years. It is a well-known fact that drugresistant strains of pathogenic bacteria are appearing such as XDR-TB, an extremely drug
resistant form of Tuberculosis, and MRSA (Methicillin-Resistant Staphylococcus
aureus). Yet, many ordinary citizens do not understand the reasons that this resistance
has evolved in recent years. One of the principle reasons of the development antibiotic
resistance is the misuse of antibiotics (Kardar). Viral infections and other diseases in
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humans are often misinterpreted as bacterial infections, leading to a patient’s prescription
of antibiotics that will only serve produce drug-resistant strains of bacteria. In fact, a
recent study done by the Center for Disease Control and Prevention found that in the
United States, 258 million rounds of antibiotics were prescribed last year for a population
of roughly 309 million, an astonishing rate of 0.833 prescriptions per individual annually
(Stobbe). Combined with the overuse of “old” antibiotics when patients become sick,
patients not completing their prescribed drug regimens, and the use of antibiotics in many
animal feeds, bacteria are exposed to antibiotics at a much higher frequency than at any
point in history.
Some bacteria are naturally resistant to antibiotics through random mutations or
past selective pressures, but are often outcompeted by so-called “normal” bacteria whose
metabolic pathways have not yet been altered. When a strong selective pressure such as
an antibiotic is applied to those bacterial populations, the resistant bacteria are more fit
and their phenotype will be naturally selected for. On the other hand, when those
antibiotics are not present, the resistant bacteria are often less “fit” due to extra energy
and resources being spent on sidestepping the actions of the antibiotic. This fact has lead
scientists to the solution of cycling antibiotic usage to diminish resistance patterns.
Essentially, a drug would be removed from the medical community for a period of years
until all of the resistant bacteria have been outcompeted and replaced with so-called
“normal” bacteria that could then be hit again with the fresh wave of old antibiotics.
While this solution certainly seems viable in the long run, it does not change the fact that
during the period that certain antibiotics are removed from the system, more antibiotics
are needed to combat new strains and resistant patterns in the microbial world. This
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represents one of the largest problems facing drug manufacturers and researchers today:
where do they find new antibiotics? Many antibiotics are produced naturally and slightly
modified for maximum efficacy, while a few others are entirely synthetic, but there is
obviously a limit on the number of antibiotics that can be developed. While screening of
thousands of natural and synthetic chemicals for antimicrobial properties is certainly
feasible, although not entirely practical, doctors are now facing a shortage of effective
drugs to combat these new super bugs. Doctors are in need of new and innovative
“weapons” against microbes, something that Dr. James Collins of the Wyss Institute at
Harvard University may just have discovered.
Before his work can be fully appreciated, the functions of antibiotics must first be
understood. Generally, antibiotics can be divided into several main classes of drugs with
a variety of different actions against the bacterial cells. The most common antibiotics are
from a group known as the beta-lactam antibiotics, which includes Cephalosporins and
Penicillins (Brock Biology of Microorganisms). These antibiotics prevent proper cell
wall synthesis by inhibiting the action of transpeptidation, which forms the cross-links
between glycan-linked peptide chains (Brock Biology of Microorganisms). Cell wall
synthesis continues, but the synthesized wall is much weaker without the cross-linkages
and eventually lyses. Two other common classes of antibiotics are known as the
Aminoglycosides and Tetracyclines. Both inhibit efficient protein synthesis by targeting
the 30S ribosomal subunit (Brock Biology of Microorganisms). Aminoglycosides are
more efficient against gram-negative bacteria while Tetracyclines are a more broadspectrum class of antibiotics. The last two major classes of antibiotics are the Macrolides
and the Quinolines. Macrolides inhibit protein synthesis by irreversibly binding to the
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50S ribosomal subunit, and thus are similar in action to the Tetracyclines and
Aminoglycosides. Quinolines on the other hand are rather different and are actually
synthetic antibiotic agents. Quinolines interfere with DNA gyrase and prevent the super
coiling of bacterial DNA, a vital step for packaging DNA in the cell (Mechanisms of
Antibiotic Action and Resistance). This class of antibiotics is incredibly useful, not only
because DNA gyrase is only found in all bacteria (both gram positive and negative), but
also because DNA gyrase is only found in bacteria due to the fact that human DNA
contains histone proteins that super coil the DNA.
While these antibiotics have proven incredibly effective at combating pathogenic
bacterial infections, the misuse of antibiotics and natural resistance patterns have resulted
in extensive antibiotic resistance across many different strains. To resist the beta-lactam
group of antibiotics, bacteria evolve methods of preventing the antibiotic from entering
the cell and also the hydrolysis of the drug itself (Mechanisms of Antibiotic Action and
Resistance). For Aminoglycoides and Tetracyclines, mutations often arise and are
selected for that decrease the efficiency or prevent the binding of the drug to the
ribosomal binding site of the 30S ribosomal subunit. Other methods of resistance include
active efflux pumps that pump the antibiotic out of the cell constantly, preventing its
action. When looking at Macrolide resistance, another defense that bacteria have evolved
is the methylation of the 23S ribosomal subunit, a component of the 50S subunit, which
again prevents the binding of the antibiotic and thus its action. With quinoline resistance,
it is often found that gyrase subunit binding sites have been altered to prevent the drug
from binding (Mechanisms of Antibiotic Action and Resistance). These can generally be
classified into the major resistance categories of: altering the active binding site,
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damaging or altering the antibiotic, and preventing the antibiotic from entering the cell.
Because these are so varied and are so advantageous in the presence of antibiotics, it is
easy to see why these resistance patterns have spread, especially given that antibiotic
resistance genes can be passed on to other cells via conjugation of plasmids.
Even still, there remains hope. Several recent studies conducted by James Collins
have told the scientific community a great deal about the function of antibiotics and
might hold several keys for the future. One area that James Collins has looked at is the
natural production of reactive oxygen species such as superoxide O2-, H2O2 (hydrogen
peroxide), and OH radicals (Dwyer). O2- is mostly produced through various biological
pathways of cellular respiration along with significant levels of hydrogen peroxide and
OH radicals. However, the key for many bacteria species is that they produce superoxide
dismutases (SODs) that can deal with O2- and H2O2, ultimately producing water and
oxygen as byproducts, which are not harmful to those species. On the other hand, OH
radicals are destructive to a variety of macromolecules including lipids, proteins and
DNA (Dwyer). OH radicals are produced via a process known as the Fenton cycle, where
ferrous iron is oxidized by H2O2 to ferric iron and the OH radical (Prousek).
Understanding this natural pathway to Collins because he believes, and has demonstrated,
that it can be utilized to combat the spread of antibiotic-resistant bacteria.
Remarkably, in a study of Collins’ from 2009 titled “Role of Reactive Oxygen
Species in Antibiotic Action and Resistance”, he and his collaborators found that
quinolines, which bind DNA gyrase and prevent the super coiling of DNA leading to
double-stranded DNA breaks and a failure to replicate, also lead to an increase in the
production of reactive oxygen species (ROS). This resulted from the decreased regulation
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of the Fenton cycle and thus the production of harmful OH radicals due to the damage
caused to the DNA. The quinolines also resulted in the activation of another regulated
process that is mediated by the production of superoxide, with both processes
contributing to the efficacy of the quinolines. Beta-lactams and Aminoglycosides also
showed similar antimicrobial properties that promoted the production of harmful OH
radicals (Pinpointing How Antibiotics Work). The OH radicals specifically are dangerous
due to their interaction with the nucleotide guanine. When the guanine is damaged, the
bacteria via the SOS response pathway attempt to repair the nucleotide, but only serve to
hasten their own death. This is due to the fact that not only is the SOS response pathway
error-prone, but also because Collins and his colleague Walker found that when the SOS
regulon was activated, ROS production was increased, and thus the cells self-perpetuate
their own demise. The radicals induce this cycle, and when the pathway attempts to fix
the damage, double-stranded DNA breaks often result to the action of the repair enzymes
themselves and kill the cells (Pinpointing How Antibiotics Work). Because this system is
so broad in its action, Collins viewed these radicals as a viable research area and potential
antimicrobial agent.
Not only was Collins right to view these reactive oxygen species as a viable
option for antimicrobial research, he may have in fact discovered a possible future of the
antibiotics field. Because these ROS’ showed such promise, Collins and his team decided
to focus on ways of increasing the production of ROS or decreasing their regulation. This
is a rather novel approach because it does not focus on developing a new antibiotic, but
instead directly interferes with the bacterial metabolism (Kusek). Importantly, to cut
down on wasting time, the team utilized sophisticated computer modeling of the
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Escherichia coli genome and metabolic pathways to determine which genes played the
largest roles in the production of ROS. They deleted genes that seemed to be important
for ROS metabolism and determined their role in the bacteria, analyzing hundreds of
reactions (Kusek). When those genes were subsequently knocked out in the laboratory,
they actually found 80-90% verification of their predictions. More importantly, as
detailed earlier, they found that by combining gene deletions with antibiotic stressors, the
cells died at much higher rates than normal antibiotic-stressed cells. As a follow-up to the
gene deletions, the team employed the use of a chemical compound known as carboxin to
see if it increased the production of ROS (Brynildsen). The compound inhibits succinate
dehydrogenase, an integral part of both the citric acid cycle and electron transport chain
that is composed of a flavoprotein, a class of proteins that are known to have the ability to
produce OH radicals when inhibited ("Succinate Dehydrogenase”). The compound had
no effect on wild-type cells when applied alone, but when applied in addition to
ampicillin the cells showed increased sensitivity due to the additional oxidative attack
combined with the antibiotic stressor (Brynildsen). Unfortunately carboxin is toxic and
thus would not be suitable for use in humans ("Carboxin”). This does however provide
hope for the future that further chemicals could be used to increase ROS production in
combination with current antibiotics. Currently, Collins and his team are using molecular
screening techniques to identify other molecules that will increase ROS production in E.
coli as well as in other more dangerous bacterial strains such as Tuberculosis. In fact,
Collins has founded his own biotechnology company called EnBiotix to further the
screening process in a timely manner, although as of yet they have had no success
(Johnson).
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Going into the future, this technique of increasing ROS production could be
potentially revolutionary to the field of medicine. Although there have been no new
chemicals that have shown promise as an ROS boosting agent, there is certainly a lot of
promise. There are definitely concerns that must first be faced. The biggest concern, and
also a potential repercussion of any chemical that is found to increase ROS production is
what effect that drug or compound has on the human body. While Collins’ team has
clearly taken this into account with the compound carboxin, this is something that has to
be taken into consideration with future compounds that seem like concrete solutions. The
effects need to be studied at all levels, even if the drugs show no immediate apparent
effect on the patient. Important routes that need to be explored by Collins’ team are any
potential differences in the pathways that produce reactive oxygen species in humans
versus bacteria. Unfortunately as of now, the exact effects of carboxin on humans are not
well understood, but the fungicide has been shown to impact the kidneys and liver,
although it is not shown to be carcinogenic in nature (“Carboxin”). This demonstrates
that there is a potential difference in the pathways between humans and bacteria as the
drug should damage all parts of the body if it does in fact cause ROS production in the
human body, although as said before it is not well understood. If Collins’ team decides to
explore these differences they might be able to better find drugs that can more efficiently
and safely kill the bacteria.
Hypothetically, if a drug is someday found that is safe for humans but also boosts
ROS production, it has the potential to be a cure-all for many drug-resistant bacteria.
While current strategies are being employed to deal with such bacteria, this method of
combining antibiotics with another compound could serve as an effective multi-drug
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regimen to which it is harder for bacteria to evolve. Any such compound could then be
used in the public health arena to regain our advantage over the microbial world and
ensure the survival of the nearly 16 million people that die each year from infectious
diseases (“Microbiology by Numbers”).
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