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Antimicrobial Chemotherapy
A. History:
1. Antibiotics known for long time= chemicals produced
by certain organisms that killed other organism.
2. Ehrlich's
"magic
bullet":
1909,
discovered
"Salvarsan", chemical used to treat syphillis. Ehrlich
stressed Selective toxicity as key factor in success.
B. Structural analogues as drugs

1935. Domagk discovered sulfa drugs.
this drug prevented Staph aureus infections in vivo,
but not on petri plates.
C. Antibiotics:

After Sulfa drug success, more search for drugs.

Penicillin was discovered in 1929, not commercially
developed until WWII.

Since then major search for antibiotics. Found in 3
major groups of microorganisms:
1. Certain molds (Penicillium, Cephalosporium).
e.g. penicillin,cephalosporin
2. Certain strains of Bacillus e.g. bacitracin
3. Many strains of Actinomycetes (soil bacteria
that
grow
in
long
filamentous
masses).
Especially from Genus Streptomycetes. e.g.
streptomycin. Majority of antibiotics come from
these organisms.
Antibiotics may have:
1. a cidal (killing) effect or
2. a static (inhibitory) effect on a range of microbes.

The range of bacteria or other microorganisms that is affected by a
certain antibiotic is expressed as its spectrum of action.

Antibiotics effective against procaryotes which kill or inhibit a
wide range of Gram-Negative and Gram-Positive bacteria are said
to be broad spectrum.

If effective mainly against Gram-Negative or Gram-Positive
bacteria, they are narrow spectrum.

If effective against a single organism or disease, they are referred
to as limited spectrum.
A clinically-useful antibiotic should have as many of these
characteristics as possible.
1. It should have a wide spectrum of activity with the ability to
destroy or inhibit many different species of pathogenic organisms.
2. It should be nontoxic to the host and without undesirable side
effects.
3. It should be nonallergenic to the host.
4. It should not eliminate the normal flora of the host.
5. It should be able to reach the part of the human body where the
infection is occurring.
6. It should be inexpensive and easy to produce.
7. It should be chemically-stable (have a long shelf-life).
Types of Antimicrobial Agents and their Primary Modes of Action
1. Cell Wall antibiotics

Penicillins. First widely available drug, introduced in
1945.
Contains
ß-lactam
ring.
(Penicillin G) was first natural isolate
Benzylpenicillin

Activity:
binds
to
enzymes
that
carry
out
transpeptidation linkage in bacterial cells. Unique
target in procaryotes.

Note beta-lactam ring -- this is critical for activity.

INITIAL PRODUCTION: 1-10 ug/ ml. Gradual strain
improvement over years, today 85,000 ug/ml!!!

BenzylPenicillin G -- low activity vs Gram-Negative,
ß-lactamase sensitive

Modifications: side chain can be chemically modified.
e.g.
1. Methicillin, Oxacillin -- acid stable, ßlactamase
resistant
Methicillin
Oxacillin
2. Ampicillin -- broader spectrum (esp. vs GramNegative), acid stable, ß-lactamase sensitive.
3. Carbenicillin -- broader spectrum (esp. vs
Pseudomonads), acid stable but not effective
orally,

ß-lactamase
sensitive
Other antibiotics active against growth of cell wall:
Cephalosporins, Cycloserine, Bacitracin.
1. Cephalosporins (e.g. cefoxitin, cephalothin)
are ß-lactam antibiotics, dihydrothiazine ring
instead of thiazolidine ring. Broader spectrum
than
penicillins,
low
toxicity.
Relatively
resistant to pencillinase.
2. Bacitracin does not block transpeptidation, but
previous step in wall synthesis. Limited to
topical application, because of severe toxic
reactions.
2. Inhibitors of protein synthesis

Aminoglycosides: (have amino sugars linked by
glycosidic
bonds).
streptomycin,
gentamicin,
kanamycin, tobramycin, amikacin. Used mainly for
Gram-Negative infections. Not very effective against
anaerobes or Gram-Positive. Bind to 30s ribosomal
unit, block protein synthesis. Also cause misreading of
mRNA. Useful for a number of diseases. Different
degrees of toxicity (e.g. Gentamicin is very toxic, only
used for severe infections) .
Amikacin is best antibiotic for Gram-Negative rod
hospital
infections,
because
such
infections
(nosocomial) are often caused by strains with Rfactors, resistant to many common antibiotics.
Generally group is used as reserve antibiotics, when
others fail.

Tetracyclines. (4 ring system) Also bind to 30S
subunit,block protein synthesis. Effective against
variety of pathogenic bacteria- broad spectrum.
Together with ß-lactam antibiotics, the most important
group commercially.

Glycopeptide antibiotics. E.g. Vancomycin.
o
Two
antibiotics
commercially
available:
vancomycin & teicoplanin.
o
Vancomycin (Vancocin) -- worldwide use.
Discovered
1956,
produced
by
fungus
Amycolatopsis orientalis, from Indonesia. Drug
has relatively high toxicity and requires IV
administration. Currently the drug of last resort
for
treating
methicillin-resistant
staph
infections.
o
Teicoplanin (Targocid) -- another glycopeptide,
has longer half-life than vancomycin, can be
administered only once a day by intramuscular
or IV. Is effective against some vancomycinresistant strains.

Macrolide antibiotics. E.g. Erythromycin. Large
lactone rings connected to sugar groups. Binds to 50S
ribosome, blocks protein synthesis. Most active vs.
Gram-Positive organisms, eg. Strep. pyogenes. Now
routinely applied to eyes of newborns to prevent
gonnorhaea and chlamydia from infecting eye.
3. Effects on nucleic acid

affect the synthesis of DNA or RNA, or can bind to DNA or
RNA so that their messages cannot be read can block the
growth of cells

Two nucleic acid synthesis inhibitors which have selective
activity against procaryotes and some medical utility are the
quinolones and rifamycins.

Nalidixic acid is a synthetic chemotherapeutic agent which
has
activity
mainly
against
Gram-negative
bacteria.
Nalidixic acid belongs to a group of compounds called
quinolones. Nalidixic acid is a bactericidal agent. the main
use of nalidixic acid is in treatment of lower urinary tract
infections (UTI).

Fluoroquinolone
(ciprofloxacin):
have
a
broadened
spectrum against Gram-positive bacteria and was recently
touted as the drug of choice for treatment and prophylaxis of
anthrax, which is caused by a Gram-positive bacillus.

Rifampicin is a semisynthetic derivative of rifamycin that
is
active
against
Gram-positive
bacteria
(including
Mycobacterium tuberculosis) and some Gram-negative
bacteria.
The table below is a summary of the classes of antibiotics and their
properties including their biological source and mode of action.
Table 1. Classes of antibiotics and their properties
Chemical class Examples
Biological
source
Spectrum
(effective
against)
Mode of
action
Penicillium
notatum and
Gram-positive
Cephalosporium bacteria
species
Inhibits steps in
cell wall
(peptidoglycan)
synthesis and
murein
assembly
Gram-positive
and Gramnegative
bacteria
Inhibits steps in
cell wall
(peptidoglycan)
synthesis and
murein
assembly
Streptomyces
clavuligerus
Gram-positive
and Gramnegative
bacteria
"Suicide"
inhibitor of
betalactamases
Chromobacter
violaceum
Gram-positive
and Gramnegative
bacteria
Inhibits steps in
cell wall
(peptidoglycan)
synthesis and
murein
assembly
Carboxypenems Imipenem
Streptomyces
cattleya
Gram-positive
and Gramnegative
bacteria
Inhibits steps in
cell wall
(peptidoglycan)
synthesis and
murein
assembly
Aminoglycosides Streptomycin
Streptomyces
griseus
Gram-positive
and Gramnegative
bacteria
Inhibit
translation
(protein
synthesis)
Gram-positive
and GramMicromonospora
negative
species
bacteria esp.
Pseudomonas
Inhibit
translation
(protein
synthesis)
Beta-lactams
(penicillins and
cephalosporins)
Penicillin G,
Cephalothin
Semisynthetic
penicillin
Ampicillin,
Amoxycillin
Clavulanic Acid
Clavamox is
clavulanic acid
plus amoxycillin
Monobactams
Aztreonam
Gentamicin
Glycopeptides
Lincomycins
Vancomycin
Clindamycin
Streptomyces
orientales
Streptomyces
lincolnensis
Gram-positive
and Gramnegative
bacteria esp.
anaerobic
Bacteroides
Inhibits
translation
(protein
synthesis)
Gram-positive
bacteria, Gramnegative
bacteria not
enterics,
Neisseria,
Legionella,
Mycoplasma
Inhibits
translation
(protein
synthesis)
Damages
Gram-negative
cytoplasmic
bacteria
membranes
Macrolides
Erythromycin
Streptomyces
erythreus
Polypeptides
Polymyxin
Bacillus
polymyxa
Bacitracin
Polyenes
Amphotericin
Nystatin
Rifamycins
Tetracyclines
Rifampicin
Tetracycline
Inhibits steps in
murein
Gram-positive
(peptidoglycan)
bacteria, esp.
biosynthesis
Staphylococcus
and assembly
aureus
Gram-positive
bacteria
Inhibits steps in
murein
(peptidoglycan)
biosynthesis
and assembly
Streptomyces
nodosus
Fungi
Inactivate
membranes
containing
sterols
Streptomyces
noursei
Inactivate
membranes
Fungi (Candida)
containing
sterols
Streptomyces
mediterranei
Gram-positive
and Gramnegative
bacteria,
Mycobacterium
tuberculosis
Streptomyces
species
Gram-positive
and Gramnegative
bacteria,
Rickettsias
Bacillus subtilis
Inhibits
transcription
(bacterial RNA
polymerase)
Inhibit
translation
(protein
synthesis)
Semisynthetic
tetracycline
Doxycycline
Streptomyces
Chloramphenicol Chloramphenicol
venezuelae
Gram-positive
and Gramnegative
bacteria,
Rickettsias
Ehrlichia,
Borrelia
Inhibit
translation
(protein
synthesis)
Gram-positive
and Gramnegative
bacteria
Inhibits
translation
(protein
synthesis)
D. Assessment of bacterial sensitivity
Anti-microbial activity in-vitro

Disc diffusion tests
1. add test bacteria to small amount of melted agar
2. pour over surface of nutrient agar plate, let gel
3. add paper disks with known dose of antibiotic
to surface
4. incubate: antibiotic will diffuse into medium as
cells grow
5. examine plate: look for clear zones around disk
where growth is inhibited
6. measure diameter of clear zones: consult table
to find if this is clinically useful

Minimum inhibitory concentration (MIC) assay:
It give a more detailed assessment of bacterial susceptibility.
Suspension of the test bacteria are inoculated overnight with
doubling dilutions of antibiotics; the lowest concentration that
inhibits growth is MIC.

Epsilomenter or ‘E’ test:
New techniques for direct determination of MIC
1. A gradual increasing concentration of antibiotic is
fixed along plastic strip which applied to the surface
of inoculated agar plate.
2. after overnight incubation  tear drop shaped
inhibition zone is seen .
3. the zone edge intersects the graded test strip at the
MIC of the antibiotics
Example of organisms and antimicrobial agents for which
susceptibility is usually predictable:
Organism
Anti-microbial agents normally active
Streptococci
Penicillin, erythromycin, vancomycin
Enterococci
Ampicillin , vancomycin
Anaerobic cocci
Penicillin, erythromycin
Staphylococci
Oxacillin,
clindamycin,
gentamicin,
vancomycin
Haemophilus influenzae
Amoxiclav, tetracycline, erythromycin
E.
Gentamicin, ciprofloxacin, co-amoxclav
coli
&
Proteus
mirabilis
Gentamicin, ciprofloxacin, imipenem,
Pseudomonas
aeruginosa
Cefoxitin, co-amoxiclav
Bacteroides fragilis
Rickettsiae
&
Tetracycline, macrolides, rifampicin
Chlamydia
mycoplasmas
Tetracycline, erythromycin
Candida albicans
Nystatin, amphotericin B, azoles
Herpes simplex virus
Aciclovir
Examples of organisms and anti-microbial agents for which
resistance is usually predicted.
Organism
Streptococci
Antimicrobial agent not normally active
&
Aminoglycosides,
Enterococci
aztreonam
Staphylococci
Most
penicillin,
nalidixic
acid,
nalidixic
acid,
aztreonam
E. Coli
Penicillin, vancomycin, erythromycin
Klebsiella spp
Most penicillin, erythromycin
Pseudomonas
Most penicillin and cephalosporins,
aeruginosa
erythromycin
Anaerobes
Aminoglycosides,
nalidixic acid
azetreonam,
Drug Resistance:

Drug resistance first noted in Japanese hospitals;
serious increase in bacterial strains resistant to variety
of standard antibiotics.

Since then, many examples of drug resistance
developing. Ex: gonorrhea initially treated by
pencillin. But pencillin-resistant strains now account
for more than 25% of isolates, must use different
antibiotic.

Note: antibiotic resistance has always been present;
frozen bacterial cultures from before WW II have
been shown to include drug resistant individuals even
though antibiotics weren't yet used by humans.
Conclude that antibiotics are natural part of biological
activity, not surprising that some resistance should
have developed in course of evolution.

What is new, and different, is rate of development of
resistance. Some disesase, like TB, never easy to treat
even with the few antibiotics that were effective. Now
drug resistant strains appearing, TB becoming much
harder to treat.
Different ways for bacteria to develop drug resistance
1. mutations affecting cell surface can affect entry of
drug
o
prevents entry of drug into cell
2. receptor normally used by drug altered- no binding.
o
example: mutations can affect drug target in cell
(e.g. slight change in ribosomal RNA can
change affinity of ribosomes for erythromycin)
3. bacteria or plasmids can produce enzymes which
inactivate drug; e.g. pencillinases hydrolyze ß-lactam
ring.
o
Plasmids = small, circular DNA elements that
reside in bacterial cells, duplicate separately
from bacterial chromosome.
o
Some plasmids carry genes for antibiotic
resistance (enzymes that degrade antibiotic).
Called R-plasmids. Have been found for most
classes of antibiotics.
o
When antibiotics are in use, most bacteria are
killed. If R-plasmid exists, can be transferred to
other
cells,
resistance
spreads
through
population. Result: new population is resistant
to drug.
o
Note: possible for a single plasmid to carry
multiple drug resistance genes, spread all of
these as a single unit!
4. plasmid encoded drug pump
o
production of protein "pumps" to pump drug
out of cell
Ways to deal with antibiotic resistance
1. higher dose, different antibiotic, more than one drug
simultaneously
2. also restraint by physicians and control (no over the
counter use)
3. CORRECT use of drug. Most people take drugs
improperly, miss doses, allow conditions that favor
selection of drug resistant mutants.
Other anti-microbial agent:Antifungal Drugs
1. Polyenes, such as nystatin and amphotericin B,
combine with plasma membrane sterols and are fungicidal.
2. Azoles interfere with sterol synthesis and are used to
treat cutaneous and systemic mycoses.
3. Griseofulvin interferes with eukaryotic cell division and
is used primarily to treat skin infections caused by fungi.
Antiviral Drugs
1. Amantadine blocks penetration or uncoating of
influenza A virus.
2. Nucleoside and nucleotide analogs such as acyclovir,
AZT, ddI, and ddC inhibit DNA or RNA synthesis.
3. Protease inhibitors, such as indinavir and saquinavir,
block activity of an HIV enzyme essential for assembly of a
new viral coat.