Antibiotic Resistance: A Losing Battle?

By: Amy Botta & Gregory Conn

1.

Antibiotic Resistance: A Losing Battle?

2. What is Antibiotic Resistance?

3. History of Antibiotics
4. Mechanisms of Resistance
a. Mechanisms
b. Resistance Plasmids
c. Mutagenesis
5. The Spread of Resistance
6. Combatting Multi-drug Resistance Bacteria
7. Looking for new Antibiotics
a. Analogs of Existing Drugs
b. Computerized Drug Design
c. Other Antimicrobial Agents
8. Bacteriophage as an Alternative to Antibiotics
9. Summary
10. References

What is Antibiotic Resistance?

Antibiotics are chemical substances that kill or suppress the growth of microorganisms.
Generally, antibiotics function in one of the five following ways:
1. Inhibition of nucleic acid synthesis (e.g. Rifampicin; Chloroquine)
2. Inhibition of protein synthesis (e.g. Tetracyclines; Chloramphenicol)
3. Action on cell membrane (e.g. Polyenes; Polymyxin)
4. Interference with enzyme system (e.g. Sulphamethoxazole)
5. Action on cell wall (e.g. Penicillin; Vancomycin)

A bacterium is said to be resistant to an antibiotic if it has the ability to withstand antibiotic

treatment. Some bacteria are naturally resistant to specific antibiotics. In other cases, bacteria
develop resistance over time to an antibiotic to which they were previously susceptible (Yim,
2007).

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.
History of Antibiotics

Although antibiotic compounds were not isolated until around the 1930s, the
Egyptians 3000 years ago used molds to treat some infections and rashes. As well, the
Chinese and various Indian tribes had also used molds to treat infections
(http://utut.essortment.com/whatisthehist_rgic.htm).

In the 1890s, two German Scientists, Rudolf Emmerich and Oscar Low, began
experimenting with various compounds in an attempt to kill microbes. However, rather than
using chemical compounds, they used one type of microbe to kill other infectious bacteria.
They marketed the microbe Bacillus pycyoneus, which was isolated from the puss of infected
bandages, under the trade name pyocyanase, and although it was successful in killing cholera
and other diseases in some patients, it caused their conditions to worsen in others. Although
no longer used today, pyocyanase was the first antibiotic drug to be used in hospitals.

The first modern day antibiotic compound was isolated in 1928 by Alexander
Fleming. Although, the ability of the mold Penicillium notatum to kill bacteria had been
noticed before in the 1890s, it was not until 1928 that the significance of this discovery was
realized when the mold was rediscovered by Alexander Fleming.

Figure 1. Alexander Fleming (http://www.wienerzeitung.at/bilder/dossier/flemming.jpg)

Fleming, who had been trained as a surgeon, had been searching for chemical compounds
that could kill infections but would not harm the patient. One day while cleaning up his lab,
he noticed that one of his Petri plates, on which he was growing the bacterium
Staphylococcus aureus, had become infected with mold. However, not only was the plate
infected with mold but wherever the mold was present there was an area around it that no
bacteria where growing in. The mold which had infected the plate was known as Penicillium
notatum. Although he was able to prove that the mold was able to kill bacteria even with
significant dilution, he was unable to isolate the penicillin successfully from the mold. It
wasnt until 1939 that 2 scientists, Ernst Chain and Howard Florey, developed a way to
isolate penicillin and used it to treat bacterial infections during the Second World War. For
their work on discovering and isolating the first antibiotic, Fleming, Chain, and Florey, were
awarded the Nobel prize for Medicine in 1945
(http://library.thinkquest.org/25462/history.html).

Around the same time as Fleming, another doctor, Gerhard Domagk, was working on
the creation of a synthetic molecule with antibiotic properties named protonsil. Protonsil was
the first in a series of drugs now known today as sulfonamides. These synthetic drugs have

one major characteristic in common; they all contain a sulphonamide. Although generally not
as effective as the naturally occurring antibiotics, sulfonamides have been used to
successfully treat many types of infections such as urinary tract infections
(http://utut.essortment.com/whatisthehist_rgic.htm).

Figure 2. sulfonamide Group (http://en.wikipedia.org/wiki/Image:Sulfonamide-group.png)

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Mechanisms of Resistance

Mechanisms

In general, bacteria have developed several ways to counteract the introduction of foreign
antibiotics, which are encoded by resistance plasmids (see section below) or at a
chromosomal level (Madigan and Martinko, 2006):

1.

The bacteria do not have the proper cellular structure the drug targets. One
needs to be sure on what type of bacteria you are treating, as if you are using a drug
that is only functional against a structure only found in Gram-negative bacteria, it
would most likely be useless against Gram-positive bacteria

2.

The antibiotic may not be permeable through the bacterial membrane. Whether it
is the size and/or structure of the antibiotic or whether its because of the membrane
features of the bacteria, the drug cannot be actively transported into the cell. For
example, vancomycin, an antibiotic that acts by inhibiting bacterial cell wall
formation and used for the treatment of a wide variety of Gram-positive bacteria,
cannot be effectively used against Gram-negative bacteria as certain factors associated
with the outer membrane (such as porins, lipopolysaccharides, and vancomycin large
size) prevent vancomycin from entering (Caillet and Alix, 1996)

3.

The antibiotic may become inactive by modification. There are thousands of

enzymes at the disposal of any given bacteria that can simply cut a certain chemical
bond, phosphorylate a hydroxyl group, or some other chemical processing that will
disrupt the antibiotic and render it inactive. An example of this is the use of
chloramphenicol, an antibiotic that acts by preventing peptide bond formation in
bacteria, is deactivated when its hydroxyl groups are acetylated by an acetylating
enzyme (chloramphenicol acetyltransferase) (Okamoto et al, 1967)

Figure 3. chloramphenicol (photos: http://en.wikipedia.org/wiki/Chloramphenicol)

4.

The target of the antibiotic is modified. This will render the antibiotic useless as it
will not be able to recognize its active site. An example of this is the use of
aminoglycosides, a spectrum of antibiotics that all function by inhibiting the 30S
subunit of bacterial ribosomes. Bacteria can develop resistance by a simple mutation
to the site where the aminoglycosides bind, preventing aminoglycosides from
recognizing their binding site.
(http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/aminoglycosideresistance.ht
m)

5.

The bacteria may alter a biochemical pathway in a way which develops

resistance to an antibiotic. This means that the bacteria can find alternative routes to
making cellular structures, molecules or other biochemical products that are
specifically targeted by an antibiotic. An example of this is the resistance of
sulfonamides. Sulfonamides function by inhibiting folic acid synthesis in bacteria,
which is needed in all cells for growth. Bacteria can move around this problem by

taking up folic acid from its environment directly, abandoning the need to synthesize
its own folic acid (Madigan and Martinko, 2006)

6.

The bacteria have mechanisms to actively displace the antibiotic out of the cell
(efflux). Many cases of resistance share this mechanism, as it proves to be quite
effective. This can involve anything from specialized proteins that actively transport a
drug across the cell memebrane, to proton pumps located on the cell membrane that
actively pump out the antibiotic. One of the many examples of this resistance is in
tertacyclines, where specialized proton pumps called Tet proteins actively transport
tetracycline out of the cell, while bringing H+ protons in (Poole, 2005).

Figure 4. Diagram of Tet protein functionality

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Resistance Plasmids

There are many different ways in which a bacterium is able to obtain antibiotic
resistance. One of the most common ways is through the acquisition of resistance plasmids.
Resistance plasmids are small pieces of DNA that are not part of the bacterial genome; they
can either be present in a linear or a circular form. A bacteria can obtain a plasmid through
either horizontal or vertical gene transfer. In vertical gene transfer the plasmid is transfered to
the offspring during division. However, horizontal gene transfer involves the transfer of genes
from one bacterium to another bacterium that is not its offspring. As shown in figure 5 there
are 3 main ways that a bacteria can obtain a resistance plasmid through horizontal gene
transfer; transformation, conjugation, and transduction (Yim, 2007).

Figure 5. Diagram of the 3 main methods of Horizotal Gene

Transfer (Yim, 2007)

Certain types of bacteria are able to undergo natural transformation, which means that
they are capable of obtaining small pieces of free DNA from the environment and
incorporating it into either their genome or the cell. The DNA obtained is not necessarily a
plasmid but if another bacterium containing a resistance plasmid has lysed and that plasmid is
not left in the environment it is possible for another bacteria to uptake that plasmid and
incorporate it, hence that bacterium which was not previously resistant to a particular
antibiotic is now resistant. The next method of horizontal gene transfer is known as
conjugation. Conjugation occurs when there is direct cell to cell contact between any two
bacteria and a small piece of DNA, usually a plasmid, is transferred from one bacterium to
the other. In conjugation the bacteria need not be related, or even the same strain, the only
requirement is that both bacteria need to be able to undergo conjugation. The final method of
horizontal gene transfer is that of Transduction. Transduction occurs when viruses specific to

bacteria, known as bacteriophage, obtain host DNA and transport this DNA to the next
bacteria it infects. Usually this only occurs between bacteria which are related (Yim, 2007).

Once the plasmid has been incorporated into the bacterial cell, the plasmid codes for
resistance a certain antibiotic according to the mechanisms previously described above. Of
the mechanisms listed, as seen in figure 6, there are 3 common mechanisms usually used;
antibiotic degrading enzymes, antibiotic altering enzymes, and efflux pumps (Yim, 2007).

Figure 6. The common mechanisms of resistance encoded for by resistance plasmids (Yim, 2007).

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Mutagenesis

Originally it was hypothesized that all cases of antibiotic resistance originated from
random mutations in the DNA. When a bacterium is ready to divide it replicates its DNA.
Although replication of DNA is fairly accurate, a spontaneous mutation can occur with a

frequency of 1x10^-7. However, a single mutation does not usually cause the occurrence of
antibiotic resistance, therefore usually multiple mutations must occur in order for antibiotic
resistance to arise (Yim, 2007).

Recently, research has shown that when bacteria are treated with certain kinds of
antibiotics they appear to develop a resistance to these agents faster than by what random
mutations can explain (Criz et al, 2005). Two of these antibitics are Ciprofloxacin and
Rifampicin.

Ciprofloxacin, or 1-cyclopropyl-6-fluoro-4-oxo-7piperazin-1-yl-quinoline-3-carboxylic acid, functions by

blocking the enzyme DNA gyrase. DNA gyrase or
topoisomerase II, is responsible for the supercoiling and
uncoiling of DNA. Without the presence of DNA gyrase,
this causes double-stranded DNA breaks and no
replication of the DNA is able to occur. With the formation of double-stranded DNA breaks,
single-stranded DNA is formed. Ciprofloxacin is part of the large group of quinolone
antibiotics which are thought to be some of the most important antibiotics available today.
Ciprofloxacin was used specifically to treat anthrax during the outbreaks experienced several
years ago (Brian, 2001).

Figure 7. Ciprofloxacin (http://en.wikipedia.org/wiki/Image:Ciprofloxazin.svg)

Unlike Ciprofloxacin, Rifampicin targets the -subunit, of the RNA polymerase

present in bacteria. By binding to the -subunit, it prevents the transcription of messenger
RNA. However, the DNA is still unwound and this leads to an accumulation of singlestranded DNA at the replication forks (Cirz, 2005)

Figure 8. Rifampicin
(http://en.wikipedia.org/wiki/Image:Rifampicin.png)

Because Ciprofloxacin and Rifampicin lead to the formation of single-stranded (ss) DNA,
this causes the bacterium to activate a repair mechanism known as the SOS response (Criz et
al, 2005). The SOS response is activated when there is an accumulation of ss-DNA at the
replication forks where RNA polymerase is unable to bind and protect the ss-DNA. The SOS
reponse is a post replication DNA repair mechanism that allows DNA replication to skip over
errors and damage that have occurred.

Figure 9. SOS Response (

http://www.science.siu.edu/microbiology/micr460/PageMill%20Images/image36.gif)

Usually the SOS reponse is activated due to damage occurred when the bacteria is
exposed to UV light. As seen in figure 9, the UV light causes the formation of dimers in the
DNA and this causes RecA to be activated and to cleave the lexA protein. Once lexA protein
is cleaved, it activates a series of enzymes which allow replication to occur without the
correction of the errors. Hence, this causes an increase in the mutation rate. Although the
activation of the SOS-response has mostly been studied in correlation with exposure to UV
light, it has also been shown to be activated whenever there is an accumualation of ss-DNA in
the cell, as RecA has a high affinity for ss-DNA.

Based on the reasoning that the accumulation of ss-DNA would cause an activation of
the SOS-reponse and this in turn, would lead to an increase in the mutation rate. Therefore
Cirz et al (2005), set out to see if the increased mutation rate, and hence the increased
development of, that occurred upon the addition of Cciprofloxacin and rifampicin was a
result of the activation and use of the SOS-response. Using both in vivo and in vitro models,
they were able to show that the presence of a functional RNA polymerase and a functional
RecA protein is responsible for the rapid rates of antibiotic resistance observed in bacteria
treated with these antibiotics (Cirz et al, 2005).

In the in vivo method 6 week old pathogen-free female mice were made
immunocompromised through injection with cylcophosphamide. Once their white blood cell
count was low enough E. coli broth cultures containing either the wildtype or a mutant in
which the SOS-response system has been rendered inactive due to a mutation in LexA, where
injected into the thigh muscles of the mice. Following several hours of incubation the mice
were then either administered ciprofloxacin or rifampicin. After 24, 48 and 72 hours, 2 mice
were sacrificed and their thigh muscles were removed and tested for the presence of nonresistant E. coli and resistant E. coli. As shown in Figure 9 in the LexA mutant the amount of
antibiotic resistant bacteria is significantly less than the number of antibiotic resistant bacteria
found in the wild type. Therefore, this shows that the SOS-response system is most likely
involved in the higher mutation rate, and hence the higher observed antibiotic resistance rate
(Criz et al, 2005)
Criz et al, 2005

Figure 10. Adapted from Criz et al, 2005. Survival and Mutation of E. coli Mutants In Vivo after Starting
Antibiotic Therapy. Survival and mutation of DlacZ and lexA(S119A) mutants of E. coli ATCC 25922 in thighs
of neutropenic (low white blood cell count) mice at 24-h intervals after starting therapy with (A) ciprofoxacin or
(B) rifampicin. Open circles and triangles correspond to the total cfu/thigh of the DlacZ and lexA(S119A)
strains, respectively. Solid circles and triangles represent the number of drug-resistant DlacZ and lexA(S119A)
cfu/thigh, respectively.

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The Spread of Resistance

The emergence of antimicrobial resistance in our society is becoming a serious

problem. In North America, bacteria strains like Staphylococcus aureus have shown in the
past decade to become resistant to antibiotics that are usually prescribed to treat its infections,
such as vancomycin and methicillin. We already know that bacteria can be naturally resistant
to antibiotics through various mechanisms, R plasmids, mutagenesis, etc., but resistance can
be induced by humans, and their improper use of antibiotics. This can lead to widespread
resistance to any drug in several bacterial species and the development of super bugs.

One way this occurs is the improper prescription of antibiotics to a patient that a viral
infection, which may be due to misdiagnosis from the doctor. Statistics show that
approximately only 20% of reported clinical infectious diseases have shown that treatment
with antibiotics was necessary and 50% of diagonoses do not prescribe the correct amount of
drug (Madigan and Martinko, 2006). The antibiotics prescribed may or may not affect, but it
will most likely lead to a few bacteria in the population developing resistance to the drug. We
can also say that the misuse of the drug by the patient leads to resistance. For example, if a
patient is taking antibiotics for strep throat (colonization by Streptococcus pyogenes), the
patient may stop short of using the prescribed amount of drug when he feels better. This will

lead to a small percentage of surviving bacteria at the site of infection that have developed
resistance to the drug, hence making it harder to treat when it infects the patient again.
Another problem is the rampant overuse of one antibiotic in a population. Tons of common
antibiotics such as tetracycline, streptomycin and kanamycin have been used worldwide since
their discovery, and as such, the amounts of bacterial species resistant to those drugs have
increased proportionally (Madigan and Martinko, 2006). Examples of this include the use of
penicillin for Neisseria gonorrhoeae, responsible for the STD gonorrhea. Prior to 1980,
penicillin was the one of the leading treatments for gonorrhea, which led to the rampant use
of the drug. Today, almost all clinical isolations of gonorrhea contain beta-lactamase, the
protein that cuts beta-lactam rings and is associated with penicillin resistance. This led
doctors to halt the treatment of the STD with penicillin (Madigan and Martinko, 2006). More
drugs have been developed to combat gonorrhea, but medicine must come up with new drugs
each year, as more and more drugs are becoming resistant.

Figure 11. (from left to right) Streptococcus pyogenes

(http://en.wikipedia.org/wiki/Image:Streptococcus_pyogenes_01.jpg), Neisseria gonorrhoeae
(http://www.nature.com/emboj/journal/v21/n4/coverfig.gif)

This misuse of antibiotics is not exclusive to humans. Bacterial infections can be

easily transmitted though animal to humans in many of our food products that we eat; the
most common are Escherichia coli and Salmonella colonization of tainted meat products. For
livestock, it is common to use certain antibiotic for growth promoting purposes and for the
prevention of disease, not treatment. This has been common clinical veterinary practice for a
long time, and due to the tones of antibiotics that have been used in animal feeds, pathogenic
bacteria have become resistant to many of these antibiotics, and thus become hard to treat if
and when it infects humans. An example of this is the use of fluoroquinones in poultry
livestock. Fluoroquinones have been used extensively to prevent respiratory diseases in
poultry (Madigan and Martinko, 2006). As a unforeseen side effect of this Campylobacter
jejuni, one of the most common causes of food-borne pathogens, has become resistant to the
drug (as of 2002 in the US, about 13.5% of all chicken carcasses contain the resistant strain
(Lathers, 2002))

Figure 12. Campylobacter jejuni (http://www.pref.aichi.jp/eiseiken/67f/campylo.jpg)

There is also work currently underway to develop novel drugs that can bock or inhibit
some of the mechanisms of resistance. Some of these drugs by themselves dont actively
work to kill bacterial cells, but prevent the mechanics of resistance from functioning

properly; therefore potentiating antibiotics that it is taken with. One kind of drug that is being
developed is efflux inhibitors. It is believed that the inhibitor will directly block the efflux
pump by binding competitively or non-competitively with the pump, but the drug could also
act by preventing ATP-binding for ATP-dependent pumps or disrupt the protein gradient of
the cell membrane (Marquez, 2005). For example, some tetracycline efflux proteins (namely
TetB) have shown to be inhibited through rational drug design by various analogs of
tetracycline, such as doxycycline and 13-cyclopentylthio-tetracycline (see photos). Both of
these drugs have been proven to be effective against E. coli that have developed resistance to
tetracycline, but these drugs are far from universal for treating tetracycline resistance, as there
are many structural variations between the various Tet efflux proteins (Marquez, 2005).

Figure 13. (Top) Tetracycline (http://en.wikipedia.org/wiki/Image:Tetracycline_structure.svg), (Botton

left to right) TetB efflux protein inhibitors (Marquez, 20