24. Antibiotics

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24. Antibiotics

• Now most of the antibiotics were discovered from
  soil microorganisms especially in microorganism
  Streptomyces spp.
• Engineered genes into production strains.
• Modification of existing antibiotics.

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• Antibiotics are chemicals produced by
  microorganisms and which in low concentrations
  are capable of inhibiting the growth of, or
  killing, other microorganisms.
• Broadened by some authors to include materials
  produced by living things plants, animals or
  microorganisms which inhibit any cell activity.
• Antibiotics may be wholly produced by
  fermentation.
• Semi-synthetic processes, in which a product
  obtained by fermentation is modified by the
  chemical introduction of side chains.

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• Some wholly chemically synthesized compounds are
  also used for the chemotherapy of infectious
  diseases e.g. sulfonamides and quinolones.
• Some antibiotics e.g. chloramphenicol were
  originally produced by fermentation, but are now
  more cheaply produced by chemical means.
• Only a small proportion of known antibiotics is
  used clinically, because the rest are too toxic.

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Classification of Antibiotics

• The classification to be adopted here is based on
  the chemical structure of the antibiotics and
  classifies antibiotics into 13 groups.
• This enables the accommodation of new groups as
  they are discovered.

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Grouping of antibiotics based on their chemical
structures
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The nomenclature of antibiotics

• The same antibiotic may have as many as 13
  different trade names depending on the
  manufacturers.
• Antibiotics are therefore identified by at least
  three names
• The chemical name, which prove long and is rarely
  used except in scientific or medical literature
• The group, generic, or common name, usually a
  shorter from of the chemical name or the one
  given by the discoverer
• The trade or brand name given by the manufacturer
  to distinguish it from the product of other
  companies.

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Some Antibiotics Produced By Microorganisms
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BETA-LACTAM ANTIBIOTICS

• The Beta-lactam antibiotics are so-called because
  they have in their structure the four membered
  lactam ring.
• A lactam is a cyclic amide. It is named as such,
  because the nitrogen atom is attached to the
  ß-carbon relative to the carbonyl.
• The Beta-lactam antibiotics inhibit the formation
  of the structure-conferring peptidoglycan of the
  bacterial cell wall.
• As this component is absent in mammalian cells,
  Beta-lactam antibiotics have very low toxicity
  towards mammal

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• The Beta-lactam antibiotics include the
  well-established and clinically important
  penicillins and cephalosphorins as well as some
  relatively newer members cephamycins,
  nocardicins, thienamycins, and clavulanic acid.
• Except in the case of nocardicins these
  antibiotics are derivatives of bicyclic ring
  systems in which the lactam ring is fused through
  a nitrogen atom and a carbon atom to ring
  compound.
• This ring compound is five-membered in
  penicillins (thiazolidine), thienamycins
  (pyrroline) and clavulanic acid (oxazolidine)
• It is six-membered (dihydrothiazolidine) in
  cephalosporins and cephamycins.

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The commercial productionof penicillin
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• First discovered by Fleming in 1932
• 19 of worldwide antibiotic market.
• Superior inhibitory action on bacterial cell wall
  synthesis
• Broad spectrum of antibacterial activity
• Low toxicity
• Outstanding efficacy against various bacterial
  strains
• Excessive use has led to development of resistant
  pathogens

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Secondary metabolites (idiolites) are produced
from Substrates provided by primary metabolism.

• Characteristics of secondary metabolites
• They are not essential for growth and
  reproduction.
• Their formation is extremely dependent on growth
  conditions.
• It is possible to get dramatic overproduction of
  secondary metabolites.

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COMMERCIAL PRODUCTION OF PENICILLIN
Originally used Penicillium notatum , now use
Penicillium chrysogenum. Initially produced via
surface mat culture. Problems! Inefficient,
slow penicillin synthesis and contamination.
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• DEEP LIQUID CULTURE
•  
• Inoculum prepared until it represents 5-10 of
  the volume of the fermentor. About 3-5 tonnes of
  wet mycelial mass will be used to inoculate a
  50,000 litre fermentor.
•  The fermentors vary from 38,000-380,000 litres.
• Three distinct phases
•  1. Trophophase Rapid mycelial growth (30-40
  hrs)
•  
• Idiophase Penicillin production via fed batch
  fermentation (5-7 days).
•  

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• 3. Carbon and nitrogen sources are depleted,
  antibiotic production ceases, the mycelia lyse
  releasing ammonia and the pH rises.
• Fermentor cooled by internal coils or external
  jackets (25-27oC).
• The pH is maintained between 6.8-7.4 by the
  automatic addition of H2SO4 or NaOH as
  necessary. 
• Oxygen added and mixed with mycelium.

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CULTURE MEDIUM   Composition of early
media corn steep liquor (cotton seeds,
peanut, Linseed or soybean meals) 2-4 lactose,
glucose or beet molasses 2-4 CaCO3 or phosphates
(buffer) 0.5-1 precursor 0.1-0.5   Cataboli
te repression of the enzymes responsible for
penicillin biosynthesis occurs in high
concentrations of glucose.   Use of precursors to
increase penicillin yield. 
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• Precursors of the appropriate side-chain are
  added to the fermentation.
• Thus if benzyl penicillin (penicillin G) is
  desired, phenylacetic acid is added.
• Phenyl acetic acid is nowadays added continuously
  as too high an amount inhibits the development of
  the fungus.
• High yielding strains of P. chrysogenum resistant
  to the precursors have therefore been developed.

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Use of precursors
 
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Extraction of penicillin after fermentation

• The broth is transferred to a settling tank.
• Penicillin is highly reactive and is easily
  destroyed by alkali conditions (pH 7.5-8.0) or by
  enzymes.
• It is therefore cooled rapidly to 5-10C.
• The separation of penicillin is based on the
  solubility, adsorption and ionic properties of
  penicillin.
• Since penicillins are monobasic carboxylic acids
  they are easily separated by solvent extraction.

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• The fermentation broth is filtered with a rotary
  vacuum filter to remove mycelia and other solids
  and the resulting broth is adjusted to about pH 2
  using a mineral acid.
• It is then extracted with a smaller volume of an
  organic solvent such as amyl acetate or butyl
  acetate, keeping it at this very low pH for as
  short a time as possible.
• The aqueous phase is separated from the organic
  solvent usually by centrifugation.

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• The organic solvent containing the penicillin is
  then typically passed through charcoal to remove
  impurities, after which it is back extracted with
  a 2 phosphate buffer at pH 7.5.
• The penicillin is then acidified once again with
  mineral acid (phosphoric acid) and the penicillin
  is again extracted into an organic solvent (e.g.
  amyl acetate).
• The product is transferred into smaller and
  smaller volumes, the penicillin becomes
  concentrated several times over, up to 80-100
  times.

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• The penicillin may be converted to a stable salt
  form in one of several ways which employ the fact
  that penicillin is an acid
• (a) it can be reacted with a calcium carbonate
  slurry to give the calcium salt which may be
  filtered, lyophilized or spray dried.
• (b) it may be reacted with sodium or potassium
  buffers to give the salts of these metals which
  can also be freeze or spray dried
• (c) it may be precipitated with an organic base
  such as triethylamine.

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NATURAL PENICILLINS

1. They are destroyed by acid in the stomach.
2. Sensitive to the enzyme penicillinase
3. Effective against Gram ve bacteria only.

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SEMISYNTHETIC PENICILLINS
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Chemical and Enzymatic Deacylation of Penicillins
to 6-APA
H
R
C
N
S
S
CH3
CH3
NH2
Penicillin acylase
CH3
CH3
O
Alkaline
N
N
COOH
COOH
Enzymatic
O
O
Penicillin V or G
(6-APA)
RPh or PhO
PCl5 ROH H2O
Chemical
Pyridine Me3SiCl
H
R
C
N
S
CH3
CH3
O
N
COOSiMe3
O
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• 6-Aminopenicillanic Acid (6-APA)
• Penicillin
• 6-APA Raw material for production of new
  semisynthetic penicillins (amoxycillin and
  ampicillin)
• Fewer side effects
• Diminished toxicity
• Greater selectivity against pathogens
• Broader antimicrobial range including
  G- -ve
• Improved pharmacological properties
• Gastric acid stability oral
  absorbability
• Resistance to beta-lactamases
• 
  

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6-Aminopenicillanic Acid (6-APA)
Chemical method Use of hazardous chemicals -
pyridine, phosphorous pentachloride, nitrosyl
chloride Enzymatic method Regio- and
stereo-specific Mild reaction conditions (pH
7.5, 37 oC) Enzymatatic process is cheaper by
10 Enzymes Penicillin G acylase (PGA)-
Escherichia coli, Bacillus megaterium,

Streptomyces lavendulae Penicillin V acylases
(PVA)- Beijerinckia indica var. Penicillium,

Fusarium sp., Pseudomonas acidovorans
Immobilized Enzyme Life, 500-2880
hours


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Penicillin G
Penicillinase (E.coli)
6 - APA
Side Chain Modification
Amoxycillin
AUGMENTIN
b-lactamase resistant
Clavulanic acid
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The Need for New Antibiotics

• The problem of multiple resistance to existing
  antibiotics
• The development of previously non-pathogenic
  microorganisms into pathogens
• Need to develop anti-fungal antibiotics
• Need to develop antibiotics specifically for
  agricultural purposes
• Need for anti-tumor and anti-parasitic drugs

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Antibiotic Production

• Development of pilot plant production methods
• Submission of licence for clinical trials
• Testing of purified antibiotic
• Development of plant scale production methods
• Obtaining a product licence for clinical use
• Other considerations
• Development of methods to control production of
  antibiotic
• Development of new applications
• Development of marketing and distribution system
• Financing of business

• Isolation or collection of cultures
• Screening of cultures to detect those with
  antimicrobial activity
• Development of methods for submerged-culture
  production
• Development of methods for isolation and
  purification of antibiotic
• Determination of antibiotic properties (physical
  adsorption and absorption, chemical reactions,
  solubility in solvents, stability to acids,
  alkalis, heat etc.)
• Evaluation of antibiotic
• Pharmacological tests
• Antimicrobial activity
• Comparison with existing antibiotic

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The classical method for searching for antibiotics

• By random search in the soil.
• Although the first important commercially
  produced antibiotic was discovered by chance,
  most present day antibiotics were discovered by
  systematic search.
• The soil is a vast repository of microorganisms
  and it is to the soil that search is turned when
  antibiotics are being sought.
• The stages to be discussed below are not
  necessarily rigidly followed they are merely
  meant to indicate in a general manner some of the
  activities involved in the development of
  antibiotics.

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(i) The primary screening

• Several methods have been employed in primary
  screening.
• The crowded plate
• The direct-soil-inoculation method
• The cross-streak method
• The agar plug method
• The replica plating method

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(a) The crowded plate

• A heavy aqueous suspension (110 1100) of soil
  is plated on agar.
• Organisms showing clear zones around themselves
  are isolated for further study.
• This method has the disadvantage that
  slow-growing antibiotic-producing organisms such
  as actinomycetes are usually over grown and are
  therefore hardly isolated.
• The susceptibility of soil organisms to the
  antibiotics produced in the test, may be
  unrelated to the susceptibility of clinically
  important organisms.

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(b) The direct-soil-inoculation method

• This method is used when the aim is to isolate
  antibiotics against a known organism or
  organisms.
• Pour plates containing the test organisms are
  prepared.
• Soil crumbs or soil dilutions are then placed on
  the plates.
• Antibiotic producing organisms develop which then
  inhibit the growth of the organisms in the plate.
  
• They are recognized by the cleared zone which
  they produce around themselves and they may then
  be picked out.

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(c) The cross-streak method

• This method is used for testing individual
  isolates, especially actinomycetes which may be
  obtained from soil without any previous knowledge
  of their antibiotic-producing potential.
• The organism may come from one of the two methods
  already indicated above.
• The purified isolate is streaked across the upper
  third of plate containing a medium which supports
  its growth as well as that of the test organisms.
  
• A variety of media may be used for streaking the
  antibiotic producer.
• It is allowed to grow for up to seven days, in
  which time any antibiotic produced would have
  diffused a considerable distance from the streak.
  
• Test organisms are streaked at right angles to
  the original isolates and the extent of the
  inhibition of the various test organisms observed.

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The Cross Streak Method for the Primary Search of
Antibiotic Producing Organisms
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Testing of Antibiotic Producing Strains
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(d) The agar plug method

• This method is particularly useful when the test
  organism grows poorly in the medium of the growth
  of the isolate such as fungi.
• Plugs about 0.5 cm in diameter are made with a
  sterile cork borer at progressive distances from
  the fungus.
• These plugs are then placed on plates with pure
  cultures of different organisms.
• The diameters of zones of clearing are used as a
  measure of antibiotic production of the isolate.
• The method may be used with actinomycetes.

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(e) The replica plating method

• If a large number of organisms are to undergo
  primary screening, one rapid method is the use of
  replica plating.
• The method consists of placing a sterile velvet
  pad on the colonies formed in the crowded plate
  or soil inoculation plate, or on series of
  discrete colonies to be tested for antibiotic
  properties.
• The pad is thereafter carefully touched on four
  or five plates seeded with the test organisms.
• As a landmark is placed on the pad as well as on
  the plates it is possible to tell which colonies
  are causing the cleared zones on the tested
  plates.

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Velvet pad
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Replica Plating Method of Testing Antibiotic
Producing Colonies
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Secondary screening

• Organisms showing suitably wide zones of clearing
  against selected target organisms are cultivated
  in broth culture in shake flasks using components
  of the solid medium in which the isolate grew
  best.
• Crude methods of isolating the active antibiotic
  are developed by extracting the broth using a
  wide range of extractive methods.
• With each extraction the resultant material is
  assessed for activity against the target
  organisms at various dilutions.
• The extract is either spotted on filter paper
  discs placed on agar seeded with the test
  organism or introduced into wells dug out from
  the seeded agar with sterile cork borers.

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• The most efficient extractive methods and the
  spectrum of activity of the organisms are
  determined.
• Secondary screening is aimed at eliminating at an
  early stage any antibiotic which does not appear
  promising either by virtue of low activity, other
  undesirable properties or because it has been
  discovered previously.

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Antibiotic spectrum

• The minimal inhibitory concentration (MIC) is a
  means of determining the activity of the isolated
  antibiotic and comparing this activity with those
  of existing antibiotics.
• Tests involving agar diffusion such as filter
  paper discs or agar wells described above are
  rapid and very useful for initial screening.
• Its ability to diffuse through agar.
• The MIC has the advantage that it is performed in
  broth thereby eliminating the disadvantage of
  large-molecule slower-diffusing antibiotics.

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Other properties

• Toxicity to mammals, determined by intra
  peritoneal injection into animals
• Haemolysis is tested by observing the effect on
  blood agar
• Serum binding is tested by adding serum to the
  broth before testing against susceptible
  organisms
• The inactivation of the antibiotic by several
  enzymes from various organs is tested by exposing
  the antibiotic to them
• Acid stability is tested if the antibiotic is
  meant for oral fermentation
• For plant antibiotics, phytotoxicity as shown by
  damage to leaves in the laboratory and in the
  green house, is determined
• For feed antibiotics, low absorbability and low
  toxicity are desirable and are tested.

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• (iv) Further laboratory evaluation
• If and after all the above, the antibiotics is
  promising, then further experimentation is done
  in shake flasks as a preparation for pilot
  production.
• The optimal conditions of growth are determined
  the most suitable medium, optimal pH,
  temperature, length of fermentation etc. are all
  determined.
• (v) Pilot plant production
• The results obtained in previous experimentation
  are fed into the pilot plant.
• The material produced is subjected to further
  safety tests and chemical analysis.

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• (vi) Plant production
• The production plant utilizes all the information
  obtained in the pilot experimentation.
• (vii) Certification
• A government agency must approve the antibiotic
  before it becomes available for general use.
• (viii) Marketing and financing

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Towards a new definition of antibiotic

• The current definition of the term antibiotic
  which restricts them to chemicals produced by
  microorganisms
• However, the higher organisms have been shown to
  produce anti-microbial substances.
• Such substances are low molecular weight
  secondary metabolites in the same way as regular
  antibiotics are.
• Due to this, there is now a tendency to extend
  the term antibiotic to all secondary metabolites,
  irrespective of their origin, which are able to
  inhibit various growth processes at low
  concentration.
• Even wholly synthetic antimicrobials such as
  ciprofloxacin are now legitimately termed
  antibiotics.
• The word antibiotic derives from two origins,
  anti (against) and bios (life). Nothing in the
  word itself restricts antibiotics both in origin
  or in use to microbial life.