1. MICROBIAL PRODUCTION OF CHEMOTHERAPEUTIC AGENTS

2.  Antibiotic Antibiotic as originally defined was a chemical substance produced by various species of microorganisms that was capable of inhibiting the growth or kill the microorganism.  Antimicrobial Agents Antimicrobial agents is a chemical substance which inhibit or kill the microorganism.

3. Introduction  Antibiotics are a special category of chemotherapeutic agents that are administered to fight infections, usually microbial, of humans and animals.  the classical definition of antibiotics associates it with secondary metabolite, the term antibiotic today includes many similar but chemically synthesized chemotherapeutic compounds also.  Over 8000 antibiotics are known and several hundreds discovered yearly.  Nearly 3000 antibiotically active substances have been detected in lichens, algae, higher animals and plants.

4.  Each year about 300 new antibiotically active substances are detected of which 30-35% are secondary components from fermentation with known antibiotics.  only 123 are currently being produced by fermentation. In addition, some 50 antibiotics are produced as semisynthetic antibiotics.  Three antibiotics, viz., phosphonomycin, pyrrolnitrin and chloramphenicol are produced completely synthetically.

5. Examples of organisms capable of producing antibiotics

6. Ideal Characteristics of antibiotic  Wide spectrum: it must be active against a wide range of pathogens  Prevent the development of resistant forms: pathogens should not easily gain resistance to the antibiotic in question  Selective nature: it must act only against the target and not the host organism  Not disturb the normal gut flora when orally administered

7. Classification of Antibiotic

10. STREPTOMYCIN  Streptomycin is an aminoglycoside antibiotic produced by selected strains of Streptomyces griseus.  The antibiotic works by inhibiting the synthesis of DNA and proteins.

11. CHEMISTRY  It is basic in nature, with solubility in water at the rate of 20 g/L.  It is stable to pH changes.  It can withstand boiling temperature.  Being a base, streptomycin is usually produced as salt, normally of HCl and sulfate.  One unit of streptomycin is equal to 1 μg of free base.  Streptomycin is composed of 3 subunits: (i) aminocyclitol (= streptidine), (ii) L-streptose, and (iii) Nmethyl- L-glucosamine.

13. Some streptomycins and their R groups

14. USES  In the treatment of tuberculosis, urinary tract infection, systemic infection by Gram positive bacteria  for bacteria that have gained resistance to penicillin.  Non-medical uses include preparation of selective media  in cloning experiments  as laboratory standard for quantitative analysis of streptomycin.

15. LIMITATIONS  The antibiotic exerts a neurotoxic reaction upon prolonged use.  It can lead to hearing loss and loss of balance (that is, it is ototoxic).  Streptomycin may sometimes damage kidney also.  The drug may lead to development of streptomycin-resistant forms. It is therefore advisable to use the drug along with p- aminosalicylic acid or isoniazid.  Dihydrostreptomycin has lesser side effects than streptomycin.

16. MODE OF ACTION  It affect protein synthesis, the target of this antibiotic is the 30S subunit of the 70S ribosome of the prokaryotes.  It strongly inhibits initiation and elongation of peptide chains.  It also causes misreading of mRNA thereby leading to insertion of wrong amino acids  Finally, under the influence of streptomycin, some molecules of nucleic acids (e.g., rRNA, tRNA, and denatured DNA) also act as mRNA although they ordinarily do not have this property.

18. BIOSYNTHESIS OF STREPTOMYCIN  It follows three pathway.  Glucose act as sole source of carbon.  It involves 28 enzymes.  The final intermediate of the pathway, streptomycin phosphate, is biologically inactive but becomes active following removal of the phosphate group.  Many organisms synthesize mannosidostreptomycin before the actual formation of streptomycin.

19.  mannosidostreptomycin is degraded by the organism’s own enzyme mannosidostreptomycinase to yield streptomycin.  In fermentation, the concentration of mannosidostreptomycin can reach up to 40%.  Biosynthesis of streptomycin is regulated by an inducer called Factor A.  The antibiotic is synthesized in the idiophase and this occurs only after factor A has reached a critical concentration.

21. GENERAL PRODUCTION METHOD a. MICROBIAL STRAIN  Streptomyces griseus is used for production of streptomycin  Walksman’s discovered the species and till now it has been using  The productivity of Streptomyces griseus has increased by over 100 fold.  Classical mutation programs are used for the improvement of the strain

22. b. CULTURE MEDIUM  Glucose is the carbon source of choice, because it is precursor for streptomycin.  The preferred nitrogen source is soybean flour meal.  Minerals are automatically inclusive because of the complex nature of the medium.

23. PRODUCTION  The inoculum is built up in a stepwise manner at 27°C.  The process starts with the plate culturing of lyophilized spore cultures in soy flour agar medium.  Incubation is done at 27°C for 2-3 weeks.  The spores are then transferred to shaker flask.  After growth for some time the whole is again transferred to propagator for biomass build up.  The medium is sterilized as usual.  The fermenter is inoculated at the rate of 5-10% vol/vol. The process is aerobic.

24.  Inadequate supply of air (O2) leads to accumulation of lactate and pyruvate, which is undesirable.  The pH is maintained at around 7 and fermentation carried out at 27°C.  The fermentation is triphasic. Trophophase lasts for 24 hrs.  The pH increases preferential utilization of soybean meal.  Growth and concomitant accumulation of Factor Aalso rapid.  Idiophase lasts for 2-7 days during which streptomycin is rapidly synthesized. Glucose utilization is very rapid.  The third phase marks the cessation of antibiotic synthesis.  Cells begin to lyse, and pH rises due to NH3 liberation.  Harvesting is done before the third phase commences.  The yield is about 1200 μg/ml.

25. RECOVERY  The broth (beer) is filtered in rotary vacuum filter to remove mycelia.  Water is added to the liquor in the ratio 1:1 and passed through adsorption column.  Through the same column, EDTA solution is passed to remove metal ions.  The adsorbed, pure streptomycin is eluted from the column with 2.5 N H2SO4.  Further processing entails decolorizing with carbon, antigen removal by filtration, concentration, and drying.  The final product is either sulfate- or hydrochloride salt of streptomycin.  The purity will be of the order of 98%.

27. TETRACYCLINE

28. Introduction  Tetracyclines are a group of broad-spectrum antibiotics  They can be prepared microbiologically as well as chemically  Tetracycline has low toxicity and good oral absorption.  It is bacteriostatic and requires high dosage.  Examples of Tetracycline and organism

29. Structure of Tetracycline

30. Uses  This antibiotic is used in the treatment of shigellosis, salmonellosis, typhoid fever, brucellosis, etc.  It is also used in feed to eliminate parasites (and thus help weight gain in animals).  The antibiotic also finds use in the preservation of fish (the ice in which the fish is kept is treated with tetracycline).

31. PRODUCTION OF TETRACYCLINE  Tetracycline can be produced chemically as well as microbiologically.  The microbial production of all tetracyclines is similar.  Chlortetracycline production, however, is comparatively simpler than the production of other tetracyclines.  In particular, production of tetracycline is very sensitive to chloride content in the medium: it leads to the production of chlortetracycline rather than the tetracycline

32. FERMENTATION  For trade fermentations, UV mutants of Streptomyces aureofaciens are used.  The organism comes in the form of lyophilized spores.  The inoculum preparation requires several stages.  Starting from the spores dried on sand or lyophil vials, one or more shake flask stages may be used and then one or two inoculum tank stages.  The sporulation medium, inoculum build-up medium and the production medium have different compositions

34.  During inoculum build-up, the organism remains in the shake-flask for 24 hours at 28°C.  The final propagator uses medium of the same composition.  About 5% inoculum is added and propagation carried out for 19-24 hours at pH 5.2-6.2.  The main fermenter receives 2-10% of inoculum from the final propagator.  The fermenter has a nominal capacity of 5000 to 15000 gallons.  Fermentation is carried out in sterilized medium (121°C for 12 hour).  The main fermentation runs for 60-65 hours at 28°C.

35.  The pH is around 5.8-6.  It is a submerged fermentation requiring 0.5-2 vol/vol/min of aeration.  Agitation is carried out with mechanical agitators.  Lard is used as antifoam.  Glucose is generally not used in the main fermentation as this exerts catabolite repression.  The yield is around 15000 units per ml.

36. HARVESTING AND PURIFICATION