ANTIBIOTICS Lecture 05

Bio 319: Antibiotics
Lecture Five

Topics:
•Mechanisms of Antibiotic Resistance
•Production of antibiotics
•Commercial production of penicillinsis

Dr. G. Kattam Maiyoh

Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 1

Antimicrobial Resistance
• Relative or complete lack of effect of
antimicrobial against a previously
susceptible microbe

Antibiotic resistance
• Relative or complete lack of effect of
antibiotic against a previously
susceptible bacreria


Antibiotic Resistance

Figure 20.20

Principal resistance strategies
for bacterial survival.
 Drugs such as tetracyclines or erythromycins are pumped back out
of bacterial cells through efflux pump proteins to
keep intracellular drug concentrations below therapeutic level.
 The antibiotic is destroyed by chemical modification by an enzyme
that is elaborated by the resistant bacteria. This is exemplified here
by the beta-lactamase secreted into the periplasmic space to
hydrolyse penicillin molecules before they reach their targets in the
cytoplasmic membrane of Gram-negative bacterium.
 The aminoglycoside antibiotic kanamycin can be enzymatically
modified at three sites by three kinds of enzymatic processing — N-
acetylation, O-phosphorylation or O-adenylylation — to block
recognition by its target on the ribosome.
 The target structure in the bacterium can be reprogrammed to have a
low affinity for antibiotic recognition. Here the switch from the
amide linkage in the D-Ala-D-Ala peptidoglycan termini to the ester
linkage in the D-Ala-D-Lac termini is accompanied by a 1,000-fold
drop in drug-binding affinity.


A. Efflux pumps


B. Enzymatic destruction of drug


Enzymatic modification

Aminoglycosides such as Kanamycin


Target modification

target structure in the bacterium can be reprogrammed to have a low affinity for
antibiotic recognition


Antibiotic Selection for Resistant Bacteria


What Factors Promote Antimicrobial
Resistance?
• Exposure to sub-optimal levels of
antimicrobial – innapropriate antibiotic use
(see next page)
• Exposure to microbes carrying resistance
genes


Inappropriate Antimicrobial Use
• Prescribing practices of providers. The use of
antibiotics for viral infections, use of broad
spectrum antibiotics and prescribing without a
laboratory request or doctor visit.
• Prescription not taken correctly
• Antibiotics for viral infections – common cold
• Antibiotics sold without medical supervision
• Spread of resistant microbes in hospitals due to
lack of hygiene
• Concerns of daycare providers (need to restrict
access).

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


Uses of antibiotics in agriculture?
– Growth promotion
– Disease prevention
– Sick animal treatment/plants – very large amounts
– Poultry
– Fish farms
– Fruit, potatoes, tobacco and others
– Ornamental plants


Should antibiotics for growth promotion and
disease prevention be banned?
• Adverse effect on animal industry
• reduced food supply
• increased cost of production
• increased disease incidence
 economic loss by farmers
• May not be totally necessary
• Might only require ban of specific antimicrobial drugs
that could select for resistance to drugs in human
medicine.


Consequences of Antimicrobial Resistance

• Infections resistant to
available antibiotics

• Increased cost of
treatment


Current problems of Resistance/MDR bacteria
Hospital Community
Gram Negative Gram Negative
Acinetobactor sp. E. Coli
Citrobacter sp. Neisseria gonorrhoeae
Enterobacter sp. S. typhi
Klebsiella sp. S. tythimurium
P. aeruginosa
Serratia marcescens
Gram Positive Gram Positive
Enterococcus sp.: vancomycin resistant Enterococcus sp.: vancomycin resistant
enterococci (VRE) enterococci (VRE)
Coagulase negative staphylococcus Mycobacterium turberculosis
MRSA MRSA
MRSA heterogenously resistant to Streptococcus pneumoniae
vancomycin
Streptococcus pyogenes

Multi-Drug Resistant TB


MRSA “mer-sah”
• Methicillin-Resistant
Staphylococcus aureus
• Most frequent nosocomial
(hospital-acquired)
pathogen
• Usually resistant to several
other antibiotics


Proposals to Combat Antimicrobial
Resistance
• Speed development of new
antibiotics
• Track resistance data nationwide
• Restrict antimicrobial use
• Direct observed dosing (TB)


Proposals to combat antimicrobial
resistance
• Use more narrow spectrum antibiotics
• Use antimicrobial cocktails
• Tx only the sick or at risk
• Producer education
• Further research before imposing bans


The Future of Chemotherapeutic Agents
• Antimicrobial peptides
– Antibiotics from plants and animals
• Squalamine (sharks)
• Protegrin (pigs)
• Magainin (frogs)
• DNA technology
• Antisense agents
– Complementary DNA or peptide nucleic acids that binds
to a pathogen's virulence gene(s) and prevents
transcription
– Phage therapy - use of bacteriophages to treat
pathogenic bacterial infections

Production of
Antibiotics


Production of Antibiotics
• The mass production of antibiotics
began during World War II with
streptomycin and penicillin.
• Now most antibiotics are produced
by staged fermentations in which
strains of microorganisms producing
high yields are grown under optimum
conditions
– nutrient media
– fermentation tanks
– holding several thousand gallons.


Production of Antibiotics
• The mold is strained out of the fermentation broth, and
then the antibiotic is removed from the broth by;
– filtration,

– precipitation, and

– other separation methods.

• In some cases new antibiotics are laboratory
synthesized, while many antibiotics are produced by
chemically modifying natural substances;
• Many such derivative penicillins are effective against
bacteria resistant to the parent substance.es are more
effective than the natural substances against infecting
organisms or are better absorbed by the body.


Raw Materials
• The compounds that make the fermentation
broth are the primary raw materials required
for antibiotic production.
• This broth is an aqueous solution made up
of all of the ingredients necessary for the
proliferation of the microorganisms.
• Typically, it contains;
– a carbon source like molasses, or soy meal,
both of which are made up of lactose and
glucose sugars.
– Other carbon sources include; acetic acid,
alcohols, or hydrocarbons

• These materials are needed as a food source for
the organisms.
• Nitrogen is another necessary compound in the
metabolic cycles of the organisms.
• For this reason, an ammonia salt is typically used.

Other elements


For E.g. Scheme for Penicillin Production


Steps in Production
• The production of a new antibiotic - lengthy and costly.
– First, the organism that makes the antibiotic must be identified
– desired microorganism must then be isolated
– Then the organism must be grown on a scale large enough to
allow the purification and chemical analysis of the antibiotic
– the antibiotic tested against a wide variety of bacterial species.
– This is a complex procedure because there are several thousand
compounds with antibiotic activity that have already been
discovered, and these compounds are repeatedly rediscovered.
– It is important that sterile conditions be maintained throughout the
manufacturing process, because contamination by foreign
microbes will ruin the fermentation.


Commercial Production over view
• After the antibiotic has been shown to
be useful in the treatment of infections
in animals, larger-scale preparation
can be undertaken.
• Commercial development requires a
high yield and an economic method of
purification.
• Extensive research may be needed to
increase the yield by selecting
improved strains of the organism or by
changing the growth medium.
• The organism is then grown in large
Large scale antibiotics steel vats, in submerged cultures with
production forced aeration.
• The naturally fermented product may
be modified chemically to produce a
semisynthetic antibiotic.


Steel Vats
•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 transferred to the
primary fermentation
tanks.


Fermentation
• Microorganisms are allowed to grow and multiply.
• During this process, they excrete large quantities of the
desired antibiotic.
• The tanks are cooled to keep the temperature between
73-81° F (23-27.2 ° C).
• It is constantly agitated, and a continuous stream of
sterilized air is pumped into it. For this reason, anti-
foaming agents are periodically added.
• Since pH control is vital for optimal growth, acids or
bases are added to the tank as necessary.


Isolation and Purification
• 3-5days, the maximum amount of antibiotic will
have been produced
• The isolation process can begin.
• Depending on the specific antibiotic produced,
the fermentation broth is processed by various
purification methods.


Water soluble Antibiotic
• For example, for antibiotic compounds that
are water soluble, an ion-exchange method
may be used for purification.
• In this method, the compound is first
separated from the waste organic materials in
the broth
• Then sent through equipment, which
separates the other water-soluble compounds
from the desired one.


Organic Antibiotics
• To isolate an oil-soluble antibiotic such
as penicillin, a solvent extraction method
is used.
• In this method, the broth is treated with
organic solvents such as butyl acetate or
methyl isobutyl ketone, which can
specifically dissolve the antibiotic.
• The dissolved antibiotic is then
recovered using various organic
chemical means.
• 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.

Refining/Packaging
• 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.

Antibiotics packaging


Pharmacology and Toxicity

• After purification, the effect of the
antibiotic on the normal function of
host tissues and organs (its
pharmacology), as well as its
possible toxic actions (toxicology),
must be tested on a large number
of animals of several species.

• In addition, the effective forms of
administration must be
determined..


Production
• Once these steps have been completed,
the manufacturer may file an
Investigational New Drug Application with
the Pharmacy and Poisions Board.
• If approved, the antibiotic can be tested
on volunteers for toxicity, tolerance,
absorption, and excretion.
• If subsequent tests on small numbers of
patients are successful, the drug can be
used on a larger group, usually in the
hundreds. If all goes well the drug can be
used in clinical medicine.
• These procedures, from the time the
antibiotic is discovered in the laboratory
until it undergoes clinical trial, usually
extend over several years.


Quality Control
• Quality control is of utmost importance in the production of
antibiotics.
• Since it involves a fermentation process, steps must be taken to
ensure that absolutely no contamination is introduced at any point
during production.
• To this end, the medium and all of the processing equipment are
thoroughly steam sterilized.
• During manufacturing, the quality of all the compounds is checked
on a regular basis.
• Of particular importance are frequent checks of the condition of the
microorganism culture during fermentation.
• These are accomplished using various chromatography techniques.
• Also, various physical and chemical properties of the finished
product are checked such as pH, melting point, and moisture
content

Penicillin – Industrial production
 The antibiotic substance,
named penicillin, was not
purified until the 1940s
(by Florey and Chain),
just in time to be used at
the end of the second
world war.
 Penicillin was the first
important commercial
product produced by an
aerobic, submerged
fermentation


 When penicillin was first
made at the end of the
Penicilium notatum second world war using the
fungus Penicilium notatum,
the process made 1 mg dm-
3.

 Today, using a different
species (P. chrysogenum)
and a better extraction
procedures the yield is 50 g
P. chrysogenum dm-3.
 There is a constant search
to improve the yield.


Antibiotic Production Methods
 Penicillin is produced by the fungus
Penicillium chrysogenum which
requires lactose, other sugars, and a
source of nitrogen (in this case a
yeast extract) in the medium to grow
well.
 Like all antibiotics, penicillin is a
secondary metabolite, so is only
produced in the stationary phase.

What sort of fermenter does it require?
 It requires a batch fermenter.
Fed-Batch: based on feeding  A fed batch process is normally used
of a growth limiting nutrient
to prolong the stationary period and
substrate to a culture.
so increase production.


Colony growth and penicillin Production


Purification
 Downstream processing is relatively easy since
penicillin is secreted into the medium
 So there is no need to break open the fungal cells.

 However, the product needs to be very pure, since
it being used as a therapeutic medical drug.

 It is dissolved and then precipitated as a potassium
salt to separate it from other substances in the
medium.


Purification

GKM/ANTIBIOTIC/2013
Tuesday, March 26, 2013 48

Batch-fed fermenter


Products
 The resulting penicillin (called penicillin G) can be chemically
and enzymatically modified to make a variety of penicillins
with slightly different properties.

 These semi-synthetic penicillins include penicillin V,
penicillin O, ampicillin and amoxycillin.


1. What is the Carbon source? lactose
2. What is the nitrogen source? yeast
3. What is the energy source? glucose
4. Is the fermentation aerobic or anaerobic? aerobic
5. What is the optimum temperature? 25 - 27ºC
6. Is penicillin a primary or secondary metabolite?secondary
7. What volume fermenter is used? 40 – 200 dm3
8. Why isn't a larger fermenter used? Too difficult to aerate
9. When is penicillin produced? 40 hours – after main increase in fungal mass
10.How long can it be produced for? 140 hours (180 – 40 hours)
11.What was the first fungus known to produce penicillin? Penicillin notatum
12.What species produces about 60mg/dm3 of penicillin? Penicillin chrysogenum
13.How did scientists improve the yield still further? Genetic modification
14.What is the substrate?Corn steep liquor
15.Why is batch culture used? Secondary metabolite
16.What are the processes involved in down-stream processing?
a) Filtration of liquid
b) Extraction from filtrate by counter current of butylacetate
c) Precipitation by potassium salts
17.Why can't penicillin be taken orally? Destroyed by stomach acid
18.Name the form of penicillin which can be taken orally. Penicillin V, ampicillin
19.How does Penicillin kill bacteria? Stops production of cell wall
20.Why are Gram negative bacteria not killed by penicillin?Different cell wall


Biosynthesis of Penicillin
•Three main and important steps to the
biosynthesis of penicillin G (benzylpenicillin)

1.Condensation of three amino acids L-α-
aminoadipic acid, L-cysteine, L-valine into a
δ-(L-α-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS)
tripeptide

2.ACV will undergoes oxidation which then
δ-(L-α-aminoadipyl)-L-cysteine-D-valine allows a ring closure so that a bicyclic ring is
isopenicillin N synthase formed

3.Exchange the side chain group so that
isopenicillin N will become penicillin G
isopenicillin N acyltransferase (IAT)
•The alpha-aminoadipyl side chain of
isopenicillin N is removed and exchanged for a
phenylacetyl side chain


ANTIBIOTICS Lecture 05

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