Bio 319: Antibiotics             Lecture Five             Topics:               •Mechanisms of Antibiotic Resistance      ...
Antimicrobial Resistance• Relative or complete lack of effect of  antimicrobial against a previously  susceptible microbeA...
Antibiotic ResistanceTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013          3                                             ...
Principal resistance strategies                           for bacterial survival.  Drugs such as tetracyclines or erythro...
A. Efflux pumpsTuesday, March 26, 2013     GKM/ANTIBIOTIC/2013   5
B. Enzymatic destruction of drugTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   6
Enzymatic modification     Aminoglycosides such as KanamycinTuesday, March 26, 2013         GKM/ANTIBIOTIC/2013   7
Target modification   target structure in the bacterium can be reprogrammed to have a low affinity for   antibiotic recogn...
Tuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   9
Antibiotic Selection for Resistant BacteriaTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   11
What Factors Promote AntimicrobialResistance?  • Exposure to sub-optimal levels of    antimicrobial – innapropriate antibi...
Inappropriate Antimicrobial Use   • Prescribing practices of providers. The use of     antibiotics for viral infections, u...
Tuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   14
Inappropriate Antimicrobial Use           • Lack of quality control in manufacture or             outdated antimicrobial  ...
Uses of antibiotics in agriculture?     – Growth promotion     – Disease prevention     – Sick animal treatment/plants – v...
Should antibiotics for growth promotion anddisease prevention be banned?     • Adverse effect on animal industry        • ...
Consequences of Antimicrobial Resistance                          • Infections resistant to                            ava...
Current problems of Resistance/MDR bacteriaHospital                                      CommunityGram Negative           ...
Multi-Drug Resistant TBTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   20
MRSA “mer-sah”                                • Methicillin-Resistant                                  Staphylococcus aure...
Proposals to Combat AntimicrobialResistance • Speed development of new   antibiotics • Track resistance data nationwide • ...
Proposals to combat antimicrobialresistance       •   Use more narrow spectrum antibiotics       •   Use antimicrobial coc...
The Future of Chemotherapeutic Agents   • Antimicrobial peptides     – Antibiotics from plants and animals               •...
Production of                                                  AntibioticsTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   ...
Production of Antibiotics                          • The mass production of antibiotics                            began d...
Production of Antibiotics     • The mold is strained out of the fermentation broth, and       then the antibiotic is remov...
Raw Materials                          • The compounds that make the fermentation                            broth are the...
Other elements Tuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   29
For E.g. Scheme for Penicillin Production Tuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   30
Steps in Production•     The production of a new antibiotic - lengthy and costly.        – First, the organism that makes ...
Commercial Production over view                             • After the antibiotic has been shown to                      ...
Steel Vats                                                •The seed tanks are                                             ...
Fermentation   • Microorganisms are allowed to grow and multiply.   • During this process, they excrete large quantities o...
Isolation and Purification• 3-5days, the maximum amount of antibiotic will  have been produced• The isolation process can ...
Water soluble Antibiotic• For example, for antibiotic compounds that  are water soluble, an ion-exchange method  may be us...
Organic Antibiotics                          • To isolate an oil-soluble antibiotic such                            as pen...
Refining/Packaging    • Antibiotic products can take on many different forms. They      can be sold in solutions for intra...
Antibiotics packagingTuesday, March 26, 2013     GKM/ANTIBIOTIC/2013   39
Pharmacology and Toxicity                          • After purification, the effect of the                            anti...
Production                           •   Once these steps have been completed,                               the manufactu...
Quality Control• Quality control is of utmost importance in the production of   antibiotics.• Since it involves a fermenta...
Penicillin – Industrial production                                           The antibiotic substance,                   ...
 When penicillin was first                                         made at the end of thePenicilium notatum              ...
Antibiotic Production Methods                                   Penicillin is produced by the fungus                     ...
Colony growth and penicillin ProductionTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   46
Purification  Downstream processing is relatively easy since   penicillin is secreted into the medium  So there is no ne...
Purification                            GKM/ANTIBIOTIC/2013Tuesday, March 26, 2013                           48
Batch-fed fermenterTuesday, March 26, 2013   GKM/ANTIBIOTIC/2013   49
Products          The resulting penicillin (called penicillin G) can be chemically           and enzymatically modified t...
1. What is the Carbon source? lactose2. What is the nitrogen source? yeast3. What is the energy source?        glucose4. I...
Biosynthesis of Penicillin                                                                            •Three main and impo...
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ANTIBIOTICS Lecture 05

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  • Antibiotic Resistance in the Farm
  • Antibiotic Resistance in the Farm Increased disease incidence, may see more foodborne pathogens.
  • ANTIBIOTICS Lecture 05

    1. 1. Bio 319: Antibiotics Lecture Five Topics: •Mechanisms of Antibiotic Resistance •Production of antibiotics •Commercial production of penicillinsis Dr. G. Kattam MaiyohTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 1
    2. 2. Antimicrobial Resistance• Relative or complete lack of effect of antimicrobial against a previously susceptible microbeAntibiotic resistance• Relative or complete lack of effect of antibiotic against a previously susceptible bacreriaTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 2
    3. 3. Antibiotic ResistanceTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 3 Figure 20.20
    4. 4. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 4
    5. 5. A. Efflux pumpsTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 5
    6. 6. B. Enzymatic destruction of drugTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 6
    7. 7. Enzymatic modification Aminoglycosides such as KanamycinTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 7
    8. 8. Target modification target structure in the bacterium can be reprogrammed to have a low affinity for antibiotic recognitionTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 8
    9. 9. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 9
    10. 10. Antibiotic Selection for Resistant BacteriaTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 11
    11. 11. What Factors Promote AntimicrobialResistance? • Exposure to sub-optimal levels of antimicrobial – innapropriate antibiotic use (see next page) • Exposure to microbes carrying resistance genesTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 12
    12. 12. 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).Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 13
    13. 13. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 14
    14. 14. 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 foodsTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 15
    15. 15. 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 plantsTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 16
    16. 16. Should antibiotics for growth promotion anddisease 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. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 17
    17. 17. Consequences of Antimicrobial Resistance • Infections resistant to available antibiotics • Increased cost of treatmentTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 18
    18. 18. Current problems of Resistance/MDR bacteriaHospital CommunityGram Negative Gram NegativeAcinetobactor sp. E. ColiCitrobacter sp. Neisseria gonorrhoeaeEnterobacter sp. S. typhiKlebsiella sp. S. tythimuriumP. aeruginosaSerratia marcescensGram Positive Gram PositiveEnterococcus sp.: vancomycin resistant Enterococcus sp.: vancomycin resistantenterococci (VRE) enterococci (VRE)Coagulase negative staphylococcus Mycobacterium turberculosisMRSA MRSAMRSA heterogenously resistant to Streptococcus pneumoniaevancomycin Streptococcus pyogenesTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 19
    19. 19. Multi-Drug Resistant TBTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 20
    20. 20. MRSA “mer-sah” • Methicillin-Resistant Staphylococcus aureus • Most frequent nosocomial (hospital-acquired) pathogen • Usually resistant to several other antibioticsTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 21
    21. 21. Proposals to Combat AntimicrobialResistance • Speed development of new antibiotics • Track resistance data nationwide • Restrict antimicrobial use • Direct observed dosing (TB)Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 22
    22. 22. Proposals to combat antimicrobialresistance • Use more narrow spectrum antibiotics • Use antimicrobial cocktails • Tx only the sick or at risk • Producer education • Further research before imposing bansTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 23
    23. 23. 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 pathogens virulence gene(s) and prevents transcription – Phage therapy - use of bacteriophages to treat pathogenic bacterial infections Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 24
    24. 24. Production of AntibioticsTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 25
    25. 25. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 26
    26. 26. 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. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 27
    27. 27. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 28
    28. 28. Other elements Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 29
    29. 29. For E.g. Scheme for Penicillin Production Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 30
    30. 30. 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. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 31
    31. 31. 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 largeLarge scale antibiotics steel vats, in submerged cultures withproduction forced aeration. • The naturally fermented product may be modified chemically to produce a semisynthetic antibiotic. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 32
    32. 32. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 33
    33. 33. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 34
    34. 34. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 35
    35. 35. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 36
    36. 36. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 37
    37. 37. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 38
    38. 38. Antibiotics packagingTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 39
    39. 39. 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..Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 40
    40. 40. 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. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 41
    41. 41. 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 Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 42
    42. 42. 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 fermentationTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 43
    43. 43.  When penicillin was first made at the end of thePenicilium 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 44
    44. 44. 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 usedof a growth limiting nutrient to prolong the stationary period andsubstrate to a culture. so increase production. Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 45
    45. 45. Colony growth and penicillin ProductionTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 46
    46. 46. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 47
    47. 47. Purification GKM/ANTIBIOTIC/2013Tuesday, March 26, 2013 48
    48. 48. Batch-fed fermenterTuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 49
    49. 49. 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.Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 50
    50. 50. 1. What is the Carbon source? lactose2. What is the nitrogen source? yeast3. What is the energy source? glucose4. Is the fermentation aerobic or anaerobic? aerobic5. What is the optimum temperature? 25 - 27ºC6. Is penicillin a primary or secondary metabolite?secondary7. What volume fermenter is used? 40 – 200 dm38. Why isnt a larger fermenter used? Too difficult to aerate9. When is penicillin produced? 40 hours – after main increase in fungal mass10.How long can it be produced for? 140 hours (180 – 40 hours)11.What was the first fungus known to produce penicillin? Penicillin notatum12.What species produces about 60mg/dm3 of penicillin? Penicillin chrysogenum13.How did scientists improve the yield still further? Genetic modification14.What is the substrate?Corn steep liquor15.Why is batch culture used? Secondary metabolite16.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 salts17.Why cant penicillin be taken orally? Destroyed by stomach acid18.Name the form of penicillin which can be taken orally. Penicillin V, ampicillin19.How does Penicillin kill bacteria? Stops production of cell wall20.Why are Gram negative bacteria not killed by penicillin?Different cell wall Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 51
    51. 51. 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 thenδ-(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 Tuesday, March 26, 2013 GKM/ANTIBIOTIC/2013 52
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