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22
 
Louis Pasteur portrait inhis later years.Photomicrograph of 
 Bacillus anthracis
 (fuchsin-methylene bluespore stain).
Image of 
Vibrio cholerae
 
Prof.Sir 
Alexander Fleming
 
 Antibiotics
1
 
The word antibiotic is derived from the term
 Antibiosis
, which means against life. antibioticsare chemical compounds produced by micro organisms (M.O) which, in low concentrations,have the capacity to inhibit or kill, selectively, other micro organisms.The selectivity or selective toxicity, means that the antibiotic compound inhibits or kills theM.O without having a similar effect on the host organism (e.g. humans).Essentially all definitions limit antibiotics to compounds that exert their action in lowconcentrations.This definition excludes compounds such as: ethanol that are active at higher concentrations
.
 The term antimicrobial agent is a general term which includes:
1-
 
 Natural, antibiotics from M.O2-
 
Synthetic, chemotherapeutic agents ,or synthetic not found in nature, obtained by chemical synthesis3-
 
 Natural, obtained from non-microbial sources, from higher plants and animal...etc.
The first scientific recording of antibiotic activity was made by
 Louis Pasteur 
 who in 1877 reported that animals injected with an inoculation containing
 Bacillus
 
anthracis
and certain other common
 Bacilli
failed to develop
anthrax 
2
.
anthrax 
2
. Ten years after Pasteur's discovery, another scientist called
 Emmerich
( 1887 ) accidently discovered that a
 guinea-pig 
3
which had previously been injected with
Streptococcus
 
erysipelatis
failed to develop
Cholera
4
when injected with virulent cultures of 
Vibrio
 
cholerae
.The recognition of the phenomenon of antibiosis had now been established butin 1928, Sir 
 Alexander Fleming 
observed the inhibition of bacteria by a colonyof 
 Penicillium
 
notatum
that had developed as a contaminant on a Petri dish. Hereported in 1929 the possible clinical use of the substance formed by the
 penicillium
culture. These findings stimulated large scale production and testingof the substance which is now known as
 penicillin
, and the search for other antibiotics.This led to the discovery of 
 Streptomycin
,
Chloramphenicol 
, and many other antibiotics.
1
 
Chemical
 
substance
 
that
 
in
 
dilute
 
solutions
 
can
 
inhibit
 
the
 
growth
 
of 
 
microorganisms
 
or
 
destroy
 
them
 
with
 
little
 
or
 
no
 
harm
 
to
 
the
 
infected
 
host.
 
2
 
Fatal
 
disease
 
characterized
 
by
 
infections
 
of 
 
the
 
skin
 
lungs
 
and
 
digestive
 
system
 
that
 
is
 
caused
 
by
 
the
 
poisonous
 
bacteria
 
Bacillus
 
anthracis
 
(this
 
bacteria
 
is
 
used
 
in
 
biological
 
weapons).
 
3
 
Somebody
 
or
 
something
 
used
 
as
 
the
 
subject
 
of 
 
an
 
experiment,
 
test,
 
or
 
trial.
 
4
 
An
 
acute
 
and
 
often
 
fatal
 
intestinal
 
disease
 
that
 
produces
 
severe
 
gastrointestinal
 
symptoms
 
and
 
is
 
usually
 
caused
 
by
 
the
 
bacterium
 
Vibrio
 
cholera.
 
 
23
 
Screening for Antibiotic
:
 
In searching for new antibiotics, relatively simple and rapid methods have been developed for screening M.O for antibiotic producing ability.Soil samples are commonly employed in the screen programme because they are a rich sourceof antibiotic-producing organisms. Most of these organisms are members of a group called
 Actinomycetes
1
(have morphologic characteristics between fungi and bacteria). In general thescreening method involves
:
 1-
 
Treating the soil sample (or sample from another source) with chemicals that inhibit thegrowth of interfering bacteria and fungi but do not affect
 Actinomycetes
.
Cycloheximide
is used as antifungal agent for this purpose, and a 1:140 dilution of  phenol is used as an antibacterial agent.2-
 
Various dilutions of the treated samples are streaked on
agar 
2
plates containing mediumthat support the growth of 
 Actinomycetes
.
 3-
 
After 
incubation
3
for 3 to 7 days at 25 to 30
˚
C, the plates are examined for characteristic colonies of 
 Actinomycetes
. These colonies are then selectively transferredonto fresh medium.4-
 
Giant colonies of the selected organisms are grown, and plugs are cut from the coloniesthat include the organism and the underlying agar.5-
 
The plugs are then placed on an agar plates that has been seeded with a test organismthat gives an indication of the potential usefulness of the antibiotic
.
 6-
 
The test plates are incubated under conditions suitable for maximum growth of the testM.O, and if after incubation there is a clear zone (zone of inhibition) around the plug of 
 Actinomycetes
, it can be assumed that an antibiotic is produced by
 Actinomycetes
whichinhibited the growth of the test organism.7-
 
The next step in the screening procedure is to determine whether the chemical substance(antibiotic) that produced the inhibition is a new antibiotic or a known compound.This can be determined by chromatographic and spectroscopic methods, and the data(result) obtained are compared to previously identified antibiotic from which the produced compound cab be determined whether a new antibiotic or not.
1
 
Rod
‐
shaped
 
bacterium:
 
a
 
rod
‐
shaped
 
or
 
filamentous
 
bacterium
 
belonging
 
to
 
a
 
large
 
group
 
that
 
includes
 
some
 
that
 
cause
 
diseases
 
and
 
some
 
that
 
are
 
the
 
sources
 
of 
 
antibiotics.
 
2
 
A
 
culture
 
medium
 
based
 
on
 
a
 
seaweed
 
extract.
 
Use:
 
growing
 
microorganisms
 
in
 
laboratories.
 
3
 
The
 
maintenance
 
of 
 
cells
 
or
 
microorganisms
 
under
 
a
 
controlled
 
temperature
 
in
 
or
 
on
 
a
 
medium
 
so
 
that
 
they
 
can
 
multiply.
 
 
24
 
Commercial Production Of Antibiotic
When a new antibiotic has been discovered by the usual screening method, the next importantstep is investigation into the chemical, physical and biologic properties of the antibiotic beforethe commercial production is decided (or started).The important requirements for productionof antibiotics are
:
 1-
 
The organism must produce the antibiotic in submerged culture as opposed to surfaceculture
.
 2-
 
The organism must excrete the antibiotic into the culture medium
.
 These requirements are important considerations in production costs to determine whether theantibiotic can compete with other antibiotics for a portion of the market
.
 Other important considerations are:-
 
Chemical stability.-
 
The minimum inhibitory concentration (MIC) against strains of pathogenic organism.-
 
Toxic manifestations in mammals, and activity in VIVO.The commercial production of antibiotics by fermentation almost always involves growth of the producing organism in aerated tanks containing thousands of gallons of nutrient medium.Spores or vegetative growth from a stock culture of the organism are used to start thefermentation process.It is important to maintain stock cultures (e.g. by
lyophilization
1
) for repeated fermentative production. The several hundred gallons of vegetative growth that are necessary for 
inoculating 
2
the large fermentation tanks are obtained by successively transferring theorganism to increasingly larger volumes of nutrient.In the production of antibiotics by fermentation process, there are two distinct phases:1-
 
The growth phase of the organism, which also termed the
Trophophase
.2-
 
The antibiotic production phase, also termed the
 Idiophase
.The following diagram illustrates these phases during the production of a typical penicillin byfermentation process carried out in a culture medium containing:
 
1-
 
Glucose and lactose as the sources of carbon nutrition.2-
 
Corn steep liquor for nitrogen sources.3-
 
Phosphate buffer.
1
 
Dry
 
blood
 
plasma
 
or
 
other
 
biological
 
substances
 
by
 
freezing
 
in
 
a
 
vacuum
 
(also
 
lyophilisation).
 
2
 
To
 
introduce
 
microorganisms
 
into
 
a
 
culture
 
medium.
 
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