ANTIBIOTICSClick to edit Master subtitle style

AntibioticsNow most of the antibiotics were discovered from soil microorganisms especially in microorganism Streptomyces spp.

Engineered genes into production strains.

Modification of existing antibiotics.

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.

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.

Classification of AntibioticsThe 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.Grouping of antibiotics based on their chemical structures

The nomenclature of antibioticsThe 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.

Some Antibiotics Produced By Microorganisms

BETA-LACTAM ANTIBIOTICSThe 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 -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

The Beta-lactam antibiotics include the well-established and clinically important penicillin and cephalosporin 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.

The Commercial Production Of Penicillin

First discovered by Fleming in 1932, 19% of worldwide antibiotic market.

Superior inhibitory action on bacterial cell wall synthesis.

Low toxicity.

Broad spectrum of antibacterial activity.

Outstanding efficacy against various bacterial strains.

Excessive use has led to development of resistant pathogens

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

COMMERCIAL PRODUCTION OF PENICILLINOriginally used Penicillium notatum , now use Penicillium chrysogenum.

Initially produced via surface mat culture.

Problems!

Inefficient, slow penicillin synthesis and contamination.

Inoculum prepared until it represents ~ 5-10% of the volume of the fermenter.

About 3-5 tones of wet mycelial mass will be used to inoculate a 50,000 liter fermenter. Mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. The fermenters vary from 38,000-380,000 liters

Composition of early mediaCorn steep liquor (cotton seeds, peanut,Linseed or soybean meals)2-4%Lactose, glucose or beet molasses2-4%Caco3 or phosphates (buffer) 0.5-1%Precursor0.1-0.5%

DEEP LIQUID CULTURE

Three distinct phases:1.Trophophase: Rapid mycelial growth (30-40 hrs)Idiophase: Penicillin production via fed batch fermentation (5-7 days).Carbon and nitrogen sources are depleted, antibiotic production ceases.The mycelia lyse releasing ammonia and the pH rises. The pH is maintained between 6.8-7.4 by the automatic addition of H2SO4 or NaOH as necessary. Fermentor cooled by internal coils or external jackets (25-27oC). Oxygen added and mixed with mycelium.

Catabolite repression of the enzymes responsible for penicillin biosynthesis occurs in high concentrations of glucose.Use of precursors to increase penicillin yield.

Use of precursors: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.

Extraction Of Penicillin After FermentationThe 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.

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.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.

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.

NATURAL PENICILLINSThey are destroyed by acid in the stomach.

Sensitive to the enzyme penicillinase

Effective against Gram +ve bacteria only.

Penicillin G6 - APASide Chain ModificationAmoxycillinAUGMENTINClavulanic acidPenicillinase (E.coli)b-lactamase resistant

The Need for New AntibioticsThe 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

Antibiotic ProductionIsolation or collection of culturesScreening of cultures to detect those with antimicrobial activityDevelopment of methods for submerged-culture productionDevelopment of methods for isolation and purification of antibioticDetermination of antibiotic properties (physical: adsorption and absorption, chemical: reactions, solubility in solvents, stability to acids, alkalis, heat etc.)Evaluation of antibioticPharmacological testsAntimicrobial activityComparison with existing antibioticDevelopment of pilot plant production methodsSubmission of licence for clinical trialsTesting of purified antibioticDevelopment of plant scale production methodsObtaining a product licence for clinical useOther considerations:Development of methods to control production of antibioticDevelopment of new applicationsDevelopment of marketing and distribution systemFinancing of business

Towards a new definition of antibioticThe 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.

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