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Industrial microbiology presentation ppt
- 1. SCHOOL OF BIOSCIENCEAND TECHNOLOGY DEPARTMENT OF BIOLOGY Production of Antibiotics and Antitumor Agents Seminar on the Course of Industrial Microbiology (BIOLM) By: Gedefaw Wubie June, 2019 Dessie; Ethiopia 1
- 2. Outline of thepresentation Key terms Definition of antibiotics Production of antibiotics Uses of antibiotics Classification Drug susceptibility testing • Classification of antibiotics 2
- 3. Key terms andphrases – Chemotherapy • The use of drugs to treat a disease – Antimicrobial drug • A chemical that destroys pathogens without damaging body tissues – Antibiotic • A substance produced by microorganisms, that in small amount, inhibits another microorganisms – Chemotherapeutic agent • A synthetic chemical (drug) used in the treatment of disease 3
- 4. Meaning of antibiotics Literal translation – anti – against – biotic – living things It is a chemical substance produced by a microorganism that inhibits the growth of or kills other microorganisms. The term antibiotic was coined from the word antibiosis which literally means against life. It defined as a substance, produced by one microorganism (Denyer et al., 2004), or of biological origin (Schlegel, 2003) which at low concentrations can inhibit the growth of, or are lethal to other microorganisms (Russell, 2004). 4
- 5. The history ofantibiotics 1928 – Alexander Fleming discovered penicillin, produced by Penicillium The early work done by Fleming, who discovered the effect of penicillin Development of this and other antibiotics such as actinomycin and aureomycin and other tetracyclines solved many problems during the second world war. 1940 – Howard Florey and Ernst Chain performed 1st clinical trials of penicillin 5
- 6. History of chemotherapy –Paul Ehrlich • In the early 20th century, while attempting to stain bacteria without staining the surrounding tissue, he speculated about “Magic bullet” – That would selectively find and destroy pathogens but not harm the host • The father of chemotherapy • With Sahachiro Hata developed Salvarsan (Arsphenamine) against syphilis in 1910 6
- 7. Sources of Antibiotics Natural microbial production using fermentation technology. Example: Penicillin Semi synthetic production (post production modification of natural antibiotics). Example: Ampicillin Synthetic production of antibiotics made synthetically in the lab. Example: Quinoline 7
- 8. Cont… – More thanhalf of our antibiotics are produced by species of Streptomyces, filamentous bacteria that commonly inhibit soil – It is interesting to note that practically all antibiotic producing microbes have some sort of sporulation process 8
- 9. Table 20.1 Representativesources of antibiotics 9 Microorganism Antibiotic Gram + rods Bacillus subtilis Bacitracin Bacillus polymyxa Polymyxin Actinomycetes Streptomyces nodosus Amphotericin B S. venezuelae Chloramphenicol S. aureofaciens Chlortetracycline & tetracycline S. erythraeus Erythromycin S. fradiae Neomycin S. griseus Streptomycin Micromonospora purpureae Gentamicin Fungi Cephalosporium spp. Cephalotin Penicillium griseofulvum Griseofulvin P. notatum Penicillin
- 10. Antibiotics production Microbialproduction of antibiotics by secondary metabolisms Because the production of antibiotics is non-growth associated, using cell immobilization to uncouple cell growth and metabolite production is an effective method of improving the process. 10
- 11. Steps in theProduction of Antibiotics It is long and a basic research proposal with d/t manufacturing requirements 1st species are screened for any sign of antibacterial action. The species is tested against a variety of known infectious bacteria. the organism is grown on a large scale so the compound responsible for the antibiotic effect can be isolated. Go to clinical testing to prove that the antibiotic works in animals and humans and is not harmful. If these tests are passed, the (FDA) must then approve the antibiotic as a new drug. This whole process can take many years (Stinson and Stephen, 1996). 11
- 12. Raw Materials fermentationbroth 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. These materials are needed as a food source for the organisms. Nitrogen is another necessary compound in the metabolic cycles of the organisms. phosphorus, sulfur, magnesium, zinc, iron, and copper introduced through water soluble salts. Anti-foaming agents such as lard oil, octadecanol, and silicones are used. 12
- 13. The Manufacture Process It involves Isolating a desired microorganism fueling growth of the culture and refining and isolating the final antibiotic product. Sterile conditions must throughout the manufacturing process If no, there is contamination by foreign microbes will ruin the fermentation. 13
- 14. Starting the Culture Beforefermentation, the desired antibiotic-producing organism must be isolated and its numbers must be increased by many times. 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. 14
- 15. Fermentation MOs areallowed to grow and multiply. 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. 15
- 16. Isolation and purification After 3to 5days, 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. 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. 16
- 17. Refining Antibiotic productscan take on many different forms. They can be sold in solutions for – intravenous bags or syringes, – in pill or gel capsule form, – 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. the antibiotic product is then Packed and transported to various distributors, hospitals, and pharmacies. The entire process of fermentation, recovery, and processing can take anywhere from five to eight days. 17
- 18. Quality Control • Nocontamination at any point during production. • Medium + all of the processing equipment = steam sterilized. • Physical + chemical properties of the finished product are checked such as • pH • MP and • MC (Crueger, 1998). • FDA requires that for certain antibiotics: – Must be checked the effectiveness and purity. – Only after they have certified the batch can it be sold for general consumption. • In fact the development of a new drug is a costly proposition, However, an alarming development has encouraged a re-energized interest in the development of new antibiotics. 18
- 19. Therapeutic index mustbe calculated – The ratio between toxic dose and therapeutic dose – High therapeutic index less toxic or the safer the drug High therapeutic dose less toxic dose is safer 19
- 20. Antitumor Antibiotics • Antitumorantibiotics (cytotoxic/anticancer antibiotics) are drugs that inhibit and combat the development of tumors. • Anthracyclines are important group of antitumor antibiotics and seven members of this group have been shown to be clinically important in cancer treatment which include: » daunorubicin » doxorubicin » epirubicin » idarubicin » pirarubicin » zorubicin and aclarubicin (Fischer et al., 2003). 20
- 21. Cont… • Anthracycline firstisolated as red substances from microorganisms in 1939 and their antibiotic properties were studied in the 1950s. • These antibiotics killed bacteria quite readily but were too toxic to be used against the infections in humans. • It was after 1960s that anthracycline antibiotics were tested for antitumor properties and found to be active against cancer cells (Taatjes et al., 1997). • Among all groups of microorganisms, the antitumor antibiotics produced by Streptomyces are invaluable in the medical field (Mueller and Nicole 2002). • All the seven members of anthracyclines are produced by Streptomyces species (Martins and Souto- Maior, 2003). 21
- 22. Cont… • Mostly producedby staged fermentations where the source microorganism is grown in large containers containing a liquid growth medium (Madigan and Martinko, 2005). • Batch fermentation used for the production of anthracyclines (Ciclamycin) from Streptomyces capoamus (Martins and Souto- Maior, 2003). • Industrial production of daunorubicin and doxorubicin has also been reported (White and Stroshane, 1984) but very little has been published on process and media development for maximal titre production of anthracyclines in commercial fermentations (Martins and Souto-Maior, 2003). • Increasing the shaking speed and decreasing the medium volume improves antitumor production. • HIGH glucose uses in catabolite repression of the biosynthesis of the antitumor antibiotic. • Also increasing antibiotic activity was observed both intra and extracellularly during growth under the carbon and nitrogen limiting conditions (Lilley et al., 1981). 22
- 23. Role of Antibiotics Bacteriostatic = To inhibit multiplication Inhibit bacterial growth rely on host immunity Bacteriocidal = Kill bacteria – Most useful in situations when host defenses cannot control pathogen » (walsh, 2003) 23
- 24. Cont… Bacteriostatic antibiotics Tetracyclines Spectinomycin Sulphonamides Macrolides Chloramphenicol Trimethoprim Bactericidal antibiotics Penicillins Cephalosporins Fluoroquinolones (Ciprofloxacin) Glycopeptides (Vancomycin) Monobactams Carbapenems 24
- 25. Spectrum of activity •For antibiotic to be an effective drug: – It must cause a significantly greater harm to the pathogen than to the host being treated (selective toxicity) Narrow spectrum – Work on narrow range of organisms » Gram+VE only OR Gram-VE only – Advantage: effects pathogen only – Disadvantage: requires identification of pathogen Broad spectrum – Advantage: Work on broad range of organisms – Disadvantage : disruption of normal flora – This require the targeting of features of the pathogen that differ from the host’s cells • Bacteria have several such targets such as cell wall and ribosomes • Viruses and eukaryotic pathogens are much more challenging to treat since they have less features that can be safely targeted 25
- 26. Antibiotic Classes Penicillins penicillin amoxicillin Cephalosporins cephalexin(Keflex) Sulfonamides co-trimoxazole (Bactrim) Fluoroquinolones ciprofolxacin(Cipro) levofloxacin (Levaquin) ofloxacin (Floxin) Tetracyclines Tetracycline(Sumycin) doxycycline (Vibramycin) Aminoglycosides gentamicin (Garamycin) kanamycin tobramycin(Tobrex) Macrolides erythromycin (E-Mycin) azithromycin (Zithromax) Prevents bacteria from making cell walls Inhibits DNA replication Inhibits protein synthesis Inhibits Folate synthesis
- 27. The major modesof action of antimicrobial drugs 27
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- 29. Inhibition of CellWall Synthesis – Bacterial cell wall is made of peptidoglycan – PG is found only in bacteria – Human cell don’t have peptidoglycan cell wall – Since they affects cell wall synthesis process • Only actively growing cells are affected 29
- 30. 30 Figure:The structure ofpenicillins
- 31. 31 Figure 20.8 Theeffect of penicillinase on penicillins
- 32. Inhibition of ProteinSynthesis – Protein synthesis is a common feature for all cells – But there is difference in ribosomes structure • Prokaryotes have 70S ribosomes (30S + 50S) • Eukaryotes have 80S ribosomes (40S + 60S) – “S” for Svedberg unit, the relative rate of sedimentation in a high speed centrifuge – Mitochondria – important eukaryotic organelles also contain 70S ribosomes similar to prokaryotes » Therefore, antibiotics targeting the 70S can have adverse effect on the host cells 32
- 33. 33 Figure 20.4 Theinhibition of protein synthesis by antibiotics
- 34.
- 35. Cont… – Most drugsthat inhibit protein synthesis have a broad spectrum of activity • Chloramphenicol • Aminoglycosides – Streptomycin, neomycin, gentamycin • Tetracyclines • Erythromycin is an exception – Since it doesn’t penetrate gram negative cell wall 35
- 36. Injury to thePlasma Membrane – Especially polypeptide antibiotics • Cause change in the permeability of the plasma membrane – Polymyxin B • Cause disruption of the plasma membrane by attaching to the phospholipids of the membrane – Against gram negative bacteria – Amphotericin B, miconazole, ketoconazole • Antifungal drugs • By combining with sterols … disrupts fungal plasma membrane • Don’t act on bacteria since bacteria lack sterol • Can be toxic to host cell since animal cell have sterols 36
- 37. • Animal cellhave mostly cholesterol Vs • Fungal cell have mostly ergosterol against which the drug is most effective – So that the balance of toxicity is tilted against the fungus 37 Figure. Injury to the plasma membrane of a yeast cell caused by an antifungal drug, miconazole
- 38. Inhibition of NucleicAcid Synthesis – Rifampin and quinolones • Interfere with the process of DNA replication and transcription in microorganisms • More selectively toxic – Rifampin • Inhibit synthesis of mRNA • Treatment for tuberculosis – Quinolones • Inhibit DNA synthesis • Broad spectrum • Treatment for urinary tract infections 38
- 39. Inhibition of theSynthesis of Essential Metabolites – Para-aminobenzoic acid (PABA) • Substrate for an enzymatic reaction leading to the synthesis of folic acid • Folic acid – A vitamin that functions as a coenzyme for the synthesis of nucleic acids and many amino-acids – Important for the growth of the microorganism – Sulfanilamide • Competitor to PABA • In its presence, the enzyme combine with sulfanilamide in steady of PABA … which prevents folic acid formation 39
- 40. • Human don’tproduce folic acid as a result sulfanilamide selectively affects microorganisms that synthesize their own folic acid 40 Figure 5.7 Enzyme Inhibitors
- 41. Antimicrobial Sensitivity Test –Necessary only: • When susceptibility of a pathogen is not predictable or • When antibiotic drug resistance problems develop • The Diffusion Methods – The most widely used method – The method of testing is disc-diffusion method • Also known as the Kirby-Bauer test 41
- 42. Results reportedas: – Sensitive – Intermediate – Resistant 42 Figure: The disc-diffusion method for determining the activity of antimicrobials • Results are often inadequate for many clinical purposes • The test is simple and inexpensive • Mostly used when more sophisticated lab facilities are not available
- 43. – E-test • Amore advanced diffusion method • Enable to estimate the minimal inhibitory concentration (MIC) – The lowest antibiotic concentration that prevents visible bacterial growth 43 Figure 20.18 The E test (for epsilometer), a gradient diffusion method that determines antibiotic sensitivity and estimates MIC
- 44. E-test • A moreadvanced diffusion method • Enable to estimate the minimal inhibitory concentration (MIC) – The lowest antibiotic concentration that prevents visible bacterial growth 44 Figure : The E test (for epsilometer), a gradient diffusion method that determines antibiotic sensitivity and estimates MIC
- 45. The Broth DilutionTests – A weakness of the diffusion method is that it doesn’t determine whether a drug is: • Bactericidal or bacteriostatic – But this method determine • MIC – By making a sequence of decreasing concentration • Minimal bactericidal concentration (MBC) – By culturing wells that don’t show growth or higher concentration than the MIC in drug free broth 45
- 46. 46 Figure 20.19 Amicrodilution or microtiter, plate used for testing for MIC of antibiotics
- 47. Antibiotic Resistance Meaning: The ability of a microorganism to survive at a given concentration of an antimicrobial agent at which the normal population of the microorganism would be killed this is called the “Epidemiological breakpoint”. 47
- 48. cont… A variety ofmutations can lead to resistance – Mechanisms of antibiotic resistance: 48
- 49.
- 50. Emerging problems – Fluoroquinolones-resistantSalmonella – 3rd gen. – Cephalosporin-resistant Salmonella– Fluoroquinolone- and – macrolide-resistant Campylobacter – Vancomycin resistant enterococci (VRE) – (Multiresistant E. coli) – 50
- 51. Cont… – Evolution ofdrug resistance could be • Vertical … due to spontaneous mutation • Horizontal … due to gene transfer e.g. gene in plasmids – Misuse of antibiotics selects resistance mutants – Misuse includes: • Using outdated or weakened antibiotics • Using antibiotics for the common cold and other inappropriate conditions • Using antibiotics in animal feed • Failing complete the prescribed regimen • Using someone else's leftover prescription 51
- 52. Effect of combinationof drugs Combination sometimes used to treat infections Synergism (Synergistic: whole is > sum) • When the effect of two drugs together is greater than the effect of either alone – E.g. penicillin and streptomycin for bacterial endocarditis Antagonism (Antagonistic: whole is < sum) • When the effect of two drugs together is less than the effect of either alone – E.g. penicillin and tetracycline Additive: whole is the sum 52
- 53. Antibiotic resistance crisis Anincrease in resistant organisms + A limited number of new antimicrobial drugs = a problematic scenario “The pharmaceutical industry has largely turned away from antibiotic research due to the low likelihood of getting a return on investment. Any new class of antibiotics would need to be used sparingly to conserve their effectiveness, meaning sales would be slow.” - WSJ
- 54. Post-antibiotic era Currently: - 80%of gonorrhoeal infections are now resistant to antibiotics. - 440,000 new cases of drug-resistant tuberculosis annually. Sally Davies (Britain’s Chief Medical Officer) “Antibiotic resistance should be added to the list of national emergencies”. In the future: - - The cost to treat drug-resistant cases is estimated to be at least double.
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Editor's Notes
- #27 Mutations in target site of enzymes