ANTIBIOTICS

Dr. Mushtak T. S.
Al-Ouqaili
Antibiotics-Lecture topics
-Nomenclature
-Developmental history
-Screening for antibiotics
-Recovery and isolation
-Antibiotic production
-Fermentation
-The manufacturing process
-Isolation and purification
-Refining
-Quality control
Penicillin production
Nomenclature
ANTIMICROBIAL AGENT

• any chemical or drug used to treat an

infectious disease, either by inhibiting or
killing the pathogens in vivo
ANTIMICROBIAL AGENT

Ideal Qualities:
1. kill or inhibit the growth of pathogens
2. cause no damage to the host
3. cause no allergic reaction to the host
4. stable when stored in solid or liquid form
5. remain in specific tissues in the body long
enough to be effective
6. kill the pathogens before they mutate and
become resistant to it
ANTIBIOTICS

 Substances derived from a microorganism

or produced synthetically, that destroys or
limits the growth of a living organism
ANTIBIOTICS – Sources

1. Natural
a.Fungi – penicillin, griseofulvin
b.Bacteria – Bacillus sp. (polymixin,
bacitracin) ; Actinomycetes
(tetracycline, chloramphenicol,
streptomycin)
2. Synthetic
ANTIBIOTICS – Classification

I. Accdg to antimicrobial activity

1. Bactericidal
2. Bacteriostatic

II. Accdg to bacterial spectrum of activity

1. Narrow spectrum
2. Broad spectrum
ANTIBIOTICS – Classification

III.According to absorbability from the

site of administration to attain
significant concentration for the
treatment of systemic infection
1. Locally acting
2. Systemic
Selective Toxicity

 Drugs that specifically target microbial

processes, and not the human host
cellular processes.
Antibiotics
 Naturally occurring antimicrobials
– Metabolic products of bacteria and
fungi
– Reduce competition for nutrients and
space
 Bacteria that produce them:
– Streptomyces, Bacillus,
 Molds
– Penicillium, Cephalosporium
History

 Ancient remedies

 Ehrlich

 Domagk

 Fleming
Neem Plant

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Neem Plant
 Uses: Arthritis, blood purifier and detoxifier, convalescence
after fever, cough, diabetes, eczema, fever (used with black
pepper and gentian), inflammation of muscles and joints,
jaundice, leukorrhea, malaria, mucus membrane
ulcerations, nausea, obesity, parasites, rheumatism, skin
diseases/inflammations, cleanses liver, syphilis, thirst,
tissue excess, tumors, vomiting, worms, drowsiness, loss
of appetite. Leaves—heal ulcers in urinary passage,
emmenagogue, skin diseases. Fruit—skin diseases,
bronchitis. Kernel powder —washing hair. Effective as a
pesticide.

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Propolis

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Propolis
 Propolis is plant resin compound, different fabric
compositions, wax, essential oils, iron,
microelements – copper, zinc, manganese,
cobalt, plus pollen, flavonoids, salivary gland
secretions of bees. Propolis is used as a bio-
stimulator which enhances endurance and
eliminate fatigue. Because its antiviral properties,
antitoxic and anti-inflammatory propolis finds
more and more uses. Recovery is a good
stimulator of affected tissue injuries, cuts...

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Ehrlich’s Magic Bullets
Ehrlich formulated the principles of selective
toxicity which means that the compound inhibits
or kills the microorganisms without having a
similar effect on the host organism (e.g., human).
He was recognized the specific chemical
relationships between microbial pathogens and
drugs, the development of drug resistance, and
the role of combined therapy.

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Gerhard Domagk - Prontosil
Fleming and Penicillin
IN 1928, ALEXANDER FLEMING MADE ONE OF THE MOST
IMPORTANT CONTRIBUTIONS TO THE FIELD OF
ANTIBIOTICS. IN AN EXPERIMENT, HE FOUND THAT A
STRAIN OF GREEN PENICILLIUM MOLD INHIBITED THE
GROWTH OF BACTERIA ON AN AGAR PLATE. THIS LED TO
THE DEVELOPMENT OF THE FIRST MODERN ERA
ANTIBIOTIC, PENICILLIN. A FEW YEARS LATER IN 1932, A
PAPER WAS PUBLISHED WHICH SUGGESTED A METHOD
FOR TREATING INFECTED WOUNDS USING A PENICILLIN
PREPARATION.

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Selman Waksman

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After the discovery of penicillin, other antibiotics were
sought. In 1939, work began on the isolation of potential
antibiotic products from the soil bacteria Streptomyces.
It was around this time that the term antibiotic was
introduced. Selman Waxman and associates discovered
streptomycin in 1944. Subsequent studies resulted in the
discovery of a host of new, different antibiotics including
actinomycin, streptothricin, and neomycin all produced
by Streptomyces.

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Between 1962 and 2000, no major classes
of antibiotics were introduced

Fischbach MA and Walsh CT Science 2009

A Changing Landscape for
Numbers of Approved Antibacterial Agents
18

16
Number of agents approved

14

12

Resistance
10

2
0
0
1983-87 1988-92 1993-97 1998-02 2003-05 2008
Bars represent number of new antimicrobial agents approved by the FDA during the period listed.

Infectious Diseases Society of America. Bad Bugs, No Drugs. July 2004; Spellberg B et al. Clin Infect Dis. 2004;38:1279-1286;
New antimicrobial agents. Antimicrob Agents Chemother. 2006;50:1912
Microbial
Sources
of
Antibiotics
Administration of Antibiotics

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-Screening for antibiotics
-Recovery and isolation

The large-scale production of an antibiotic depends on

a fermentation process. During fermentation, large
amounts of the antibiotic-producing organism are
grown. During fermentation, the organisms produce
the antibiotic material, which can then be isolated for
use as a drug. For a new antibiotic to be economically
feasible, manufacturers must be able to get a high
yield of drug from the fermentation process, and be
able to easily isolate it. Extensive research is usually
required before a new antibiotic can be commercially
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scaled up.
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Factors affecting antibiotic production:
1. Medium Composition:
-Carbon source
-Nitrogen source
-Inorganic phosphates
-Inorganic salts
-Trace metals
-Precursors
-Inhibitors
-Inducers
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 2.Fermentation conditions

-pH
-Temperature
-Oxygen

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The Manufacturing Process:-

Although most antibiotics occur in nature, they

are not normally available in the quantities
necessary for large-scale production. For this
reason, a fermentation process was developed. It
involves isolating a desired microorganism,
fueling growth of the culture and refining and
isolating the final antibiotic product. It is
important that sterile conditions be maintained
throughout the manufacturing process, because
contamination by foreign microbes will ruin the
fermentation.
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Starting the culture
1-Before fermentation can begin, the desired
antibiotic-producing organism must be isolated and
its numbers must be increased by many times. To do
this, a starter culture from a sample of previously
isolated, cold-stored organisms is created in the lab.
In order to grow the initial culture, a sample of the
organism is transferred to an agar-containing plate.
The initial culture is then put into shake flasks along
with food and other nutrients necessary for growth.
This creates a suspension, which can be transferred to
seed tanks for further growth.
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2-The seed tanks are steel tanks designed to provide an
ideal environment for growing microorganisms. They
are filled with the all the things the specific
microorganism would need to survive and thrive,
including warm water and carbohydrate foods like
lactose or glucose sugars. Additionally, they contain
other necessary carbon sources, such as acetic acid,
alcohols, or hydrocarbons, and nitrogen sources like
ammonia salts. Growth factors like vitamins, amino
acids, and minor nutrients round out the composition of
the seed tank contents. The seed tanks are equipped
with mixers, which keep the growth medium moving,
and a pump to deliver sterilized, filtered air. After about
24-28 hours, the material in the seed tanks is
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transferred to the primary fermentation tanks.
Fermentation:-

3-The fermentation tank is essentially a

larger version of the steel, seed tank, which
is able to hold about 30,000 gallons. It is
filled with the same growth media

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Isolation and purification
4-After three to five days, the maximum amount of
antibiotic will have been produced and the isolation
process can begin. Depending on the specific antibiotic
produced, the fermentation broth is processed by various
purification methods. For example, for antibiotic
compounds that are water soluble, an ion-exchange
method may be used for purification. To isolate an oil-
soluble antibiotic such as penicillin, a solvent extraction
method is used. At the end of this step, the manufacturer
is typically left with a purified powdered form of the
antibiotic, which can be further refined into different
product types.

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5-Antibiotic products can take on many different forms.
They can be sold in solutions for intravenous bags or
syringes, in pill or gel capsule form, or they may be sold
as powders, which are incorporated into topical
ointments. Depending on the final form of the
antibiotic, various refining steps may be taken after the
initial isolation. For intravenous bags, the crystalline
antibiotic can be dissolved in a solution, put in the bag,
which is then hermetically sealed. For gel capsules, the
powdered antibiotic is physically filled into the bottom
half of a capsule then the top half is mechanically put in
place. When used in topical ointments, the antibiotic is
mixed into the ointment.

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6-From this point, the antibiotic product is
transported to the final packaging stations. Here,
the products are stacked and put in boxes. They
are loaded up on trucks and transported to various
distributors, hospitals, and pharmacies. The entire
process of fermentation, recovery, and processing
can take anywhere from five to eight days.

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Spectrum of Activity
Determining Microbial Sensitivities
 Disk Diffusion
Method

 Dilution Method

 Serum Killing
Power

 Automated
Methods
Drug Mechanisms of Action
Antibacterial Antibiotics
Inhibitors of Cell Wall Synthesis
 Penicillin (over 50 compounds)
– Share 4-sided ring (b lactam ring)
 Natural penicillins
• Narrow range of action
• Susceptible to penicillinase (b lactamase)
Prokaryotic Cell Walls
Cell wall synthesis
Bactericidal
– Penicillin and cephalosporins –
binds and blocks peptidases
involved in cross-linking the
glycan molecules
– Vancomycin – hinders
peptidoglycan elongation
– Cycloserine – inhibits the
formation of the basic
peptidoglycan subunits
Antibiotics weaken the cell wall, and cause the cell to lyse.
Penicillin
 Penicillin chrysogenum
 A diverse group (1st, 2nd , 3rd generations)
– Natural (penicillin G and V)
– Semisynthetic (Ampicillin, Carbenicillin)
 Structure
– Thiazolidine ring
– Beta-lactam ring
– Variable side chain (R group)
-4C – 28C - 38C water activity 0.98

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Penicillins

Figure 20.6
Semisynthetic Penicillins

 Penicilinase-resistant penicillins
• Carbapenems: very broad spectrum
• Monobactam: Gram negative
 Extended-spectrum penicillins
 Penicillins + b-lactamase inhibitors
Penicillinase (b Lactamase)
Other Inhibitors of Cell Wall
Synthesis
 Cephalosporins
– 2nd, 3rd, and 4th
generations more
effective against
gram-negatives

Figure 20.9
 Cephalosporin
 Cephalosporium acremonium (mold)
 Widely administered today
– Diverse group (natural and
semisynthetic)
 Structure
– similar to penicillin except
• Main ring is different
• Two sites for R groups
The different
R groups
allow for
versatility
and improved
effectiveness.
Other Inhibitors of Cell Wall
Synthesis
 Mycobacteria:
interfere with
mycolic acid
synthesis or
incorporation
– Isoniazid (INH)
– Ethambutol
Other Inhibitors of Cell Wall
Synthesis
 Polypeptide antibiotics
– Bacitracin
• Topical application
• Against gram-positives
– Vancomycin
• Glycopeptide
• Important "last line" against antibiotic resistant S.
aureus
Inhibitors of Protein Synthesis
 Broad spectrum, toxicity problems
 Examples

– Aminoglycosides: Streptomycin,
neomycin, gentamycin
– Tetracyclines
– Macrolides: Erythromycin
– Chloramphenicol
 Aminoglycosides
 From Streptomyces
 Inhibit protein synthesis

Streptomyces synthesizes many

different antibiotics such as
aminoglycosides, tetracycline,
chloramphenicol, and
erythromycin.
Tetracycline

 Inhibits proteins synthesis

 Broad spectrum and low cost
 Commonly used to treat sexually
transmitted diseases
 Minor side effect – gastrointestinal
disruption
Erythromycin

 Inhibits protein synthesis

 Broad-spectrum
 Commonly used as prophylactic drug
prior to surgery
 Side effects - low toxicity
Streptomycin - treat Plague
Chloramphenicol

 Broad-spectrum
 Treat typhoid fever, brain abscesses
 Rarely used now due to side effects –
aplastic anemia
Injury to the Plasma Membrane

 Polymyxin B (Gram negatives)

– Topical
– Combined with bacitracin and neomycin (broad
spectrum) in over-the-counter preparation
Injury to the Plasma Membrane
 Polymyxin B (Gram negatives)
– Topical
– Combined with bacitracin and
neomycin (broad spectrum) in over-
the-counter preparation
Inhibitors of Nucleic Acid
Synthesis
 Rifamycin
– Inhibits RNA synthesis
– Antituberculosis
 Quinolones and fluoroquinolones
– Ciprofloxacin
– Inhibits DNA gyrase
– Urinary tract infections
Inhibition of Nucleic Acid Synthesis

 Rifampin binds to
DNA-dependent
RNA polymerase
and inhibits
intiation of RNA
synthesis
Folic acid synthesis

Sulfonamides (sulfa drug) and

trimethoprim
– Analogs
– Competitive inhibition of enzymes
– Prevents the metabolism of DNA,
RNA, and amino acid
Summary of Targets
Antibiotic Resistance

Figure 20.20
Antimicrobial Resistance

 Relative or complete lack of

effect of antimicrobial against
a previously susceptible
microbe
 Increase in MIC
Mechanisms of Antibiotic
Resistance

• Enzymatic destruction of drug

• Prevention of penetration of drug
• Alteration of drug's target site
• Rapid ejection of the drug
Antibiotic Selection for Resistant
Bacteria
What Factors Promote Antimicrobial
Resistance?

 Exposure to sub-optimal
levels of antimicrobial
 Exposure to microbes
carrying resistance genes
Inappropriate Antimicrobial Use

 Prescription not taken correctly

 Antibiotics for viral infections
 Antibiotics sold without medical
supervision
 Spread of resistant microbes in
hospitals due to lack of hygiene
Inappropriate Antimicrobial Use

 Lack of quality control in manufacture or

outdated antimicrobial
 Inadequate surveillance or defective
susceptibility assays
 Poverty or war
 Use of antibiotics in foods
The Future of Chemotherapeutic
Agents
 Antimicrobial peptides
– Broad spectrum antibiotics from
plants and animals
• Squalamine (sharks)
• Protegrin (pigs)
• Magainin (frogs)
Resistance to Drugs

 Chromosomal

 Plasmid borne
Mechanisms of Drug Resistance

 Mutations in Target molecules

 Alterations in membrane permeability

 Enzyme development
Mechanisms of Drug Resistance

 Enzyme Activity Changes

 Alterations in Anabolic Pathways

Generations of Drugs

 First/Second/Third
Line Drugs

 Cross Resistance
Resistance Factors – R Factors

 Transferred through conjugation,

transformation or transduction
 Many bacteria also maintain transposable
drug resistance sequences – tansposons
that are duplicated and inserted from one
plasmid to another or from a plasmid to a
chromosome
Conjugation – plasmids and chromosomal
elements, conjugative transposons plasmids

 Conjugative plasmid – plasmids that

transfer themselves by conjugation must
carry a number of genes encoding
proteins needed for the conjugation
process itself (tra genes)
 Self-transmissable plasmids (STP) are
usually at least 25kb
 Mobilize plasmids – much smaller than
STP because they need only 1 or 2 genes
(mob genes)
Resistance Genes

 Acquire sequential transposon insertions

 Integrons are probably responsible for
evolution of many of the plasmids that
carry multiple resistance genes
Integrons

 Integrons like transposons are linear DNA

segments that insert into DNA
 Unlike transposons, integrons integrate at
a single site and do not encode a
transposase
 Conjugative transposons – located in the
bacterial chromosome, also integrate into
plasmids
Mechanism of Transfer

 Excise themselves from the donor

genome to form a covalently closed circle
that does not replicate
 The circular intermediate transfers
similarly to a plasmid
 In the recipient, the circular intermediate
integrates in the chromosome by a
mechanism that does not duplicate the
target site
Origin of Antibiotic Resistant Genes

 First it was assumed that antibiotic

resistance genes appeared only after
antibiotics began to be widely used in
medicine
 The genetic diversity within some classes
of resistance makes it clear that these
genes have been evolving for a much
longer time
Origin of resistance
 Hypothesis: resistance genes first evolved
in the antibiotic-producing bacteria such
as Streptomyces spp. As a mechanism for
protecting them from the antibiotics they
produce.
 Genes for antibiotic production are
frequently found in the same gene
clusters with genes encoding resistance
proteins
Specific mechanisms of drug
resistance
Bacteria lose its sensitivity
to a drug by expressing
genes that stop the action
of the drug.
Gene expression

 Synthesis of enzymes that inactivate the

drug
 Decrease in cell permeability and uptake
of the drug
 Change in the number or affinity of the
drug receptor sites, or
 Modification of an essential metabolic
pathway
Resistance

 Some bacteria can become resistant

indirectly by lapsing into dormancy, or, in
the case of penicillin, by converting to a
cell-wall-deficient form (L form) that
penicillin cannot affect.
Drug Inactivation Mechanisms

 Produce enzymes that

permanently alter drug
structure (beta lactamases)
Resistance

 Resistance to the aminoglycoside

antibiotics is a specific case in which
microbial cells have lost the capacity to
transport the drug intracellulary
Natural selection and drug resistance

 When a population of bacteria is exposed

to a drug, sensitive cells are inhibited or
destroyed and resistant forms survive and
proliferate
 In ecological terms, the environmental
factor has put selection pressure on the
population, allowing the more fit microbe
to survive, and the population has evolved
to a condition of drug resistance.