Bio 319: Antibiotics                                      Lecture Six                                         Topic:      ...
DNA Technology and Antibiotics                       Production                                        • Worldwide over 10...
Advantages     • DNA tec. Can be used to produce new       structurally unique antibiotics with;            – Increased ac...
Organism of Choice• Streptomyces• Unlike E-coli not exist as  individual cells but as extended  aggregates of mycelial fil...
Isolation of antibiotic biosynthesis genes:            by complementation  • Mutant cells (not able to produce    antibiot...
cntd    •Plasmid DNA from the clone that supplied a functional    gene and gene product (i.e. complements the mutant) is  ...
Engineering Polyketide Antibiotics (PA) • Antibiotics synthesized through succesive   enzymatic condensation of small carb...
FA biosynthesis, quick reminder:Fatty acid biosynthesis is a stepwise assemblyFatty acid biosynthesis is a stepwise assemb...
Initiation       Overall Reaction                               Malonyl-CoA + ACP      CH3C~SCo        -                  ...
β-Carbon                               Elongation           CH3C- CH2C~S- ACP              O     O                        ...
Biosynthesis PA     • Analogous to synthesis of long-chain FA     • Each condensation cycle results in the       formation...
Classes of polyketide biosynthetic                         enzymes             i.      Those involve in the synthesis of a...
Example: Engineering of Erythromycin                               production• Is synthesized by saccharopolyspora erythra...
Terrorism• Terrorism is defined as the unlawful use of force or  violence against persons or property to intimidate or  co...
Delivery Mechanisms• Aerosol route  – Easiest to disperse  – Highest number of people exposed  – Most infectious  – Undete...
• Biologic agents are likely to be used  by terrorists as weapons because:  – They are capable of damaging populations,   ...
BIOLOGICAL AGENTS• There are several types of agents. They are  classified as:  – Bacteria  – Rickettsia  – Viruses  – Bio...
BACTERIA• Single celled organisms             EXAMPLES  capable of causing disease.                                •   ANT...
RICKETTSIA• Vector borne (ticks, lice,    EXAMPLES  mosquitos) parasitic         • TYPHUS  form of bacteria             • ...
VIRUSES• Smaller than bacteria                                  EXAMPLES• RNA or DNA in a protein                         ...
BIOTOXINS• Are poisonous by-                              EXAMPLES  products of bacteria,  fungi, marine animals  or plant...
Biological Agents most likely to be     used in a terrorist attack• Bacteria - anthrax, plague, tularemia• Virus - small p...
Brief history        • Caused by Bacillus anthracis        • Human zoonotic disease              – Spores found in soil wo...
Bioweapon Potential           • Many countries have weaponized             anthrax                  – Former bioweapon pro...
Features of anthrax suitable as BTagent      – Fairly easy to obtain, produce and store      – Spores easily dispersed as ...
PLAGUE• In nature, fleas living on rodents spread  infection to humans. As a bioterrorist weapon –  inhalation of aerosol ...
RashesA tentative diagnosis of plague is made
PLAGUE• Transmission: may occur person to person by  respiratory droplet inhalation• Incubation period: 2 to 3 days• Morta...
TULAREMIA• A zoonotic, bacterial infection caused by  Francisella tularensis, a gram negative  coccobacillus• In nature, b...
TULAREMIA• Symptoms:sudden and influenza-like with  fever,chills, headache and nausea• Transmission: not usually person to...
BOTULISM• Infectious agent: Clostridium botulinum – a  spore forming, anaerobic bacillus• In nature, may be food borne, wo...
BOTULISM• Symptom: fatigue, weakness, blurred vision, difficulty  in swallowing and speaking, descending muscle  paralysis...
Emergency Preparedness /      Stockpiling
Strategic National Stockpile• Repository of   – Antibiotics   – Vaccines   – Immunoglobulins   – Chemical antidotes   – An...
Containers designed to facilitatetransport by roads and railways.      And also by airways.
• The Problem with Stockpiling   – Antidotes and treatments are expensive   – Have limited shelf-lives   – Unlikely to be ...
Antibiotics to         Counteract Biologic Weapons• Often older agents are still the  most effective.• Dosage regimens var...
Post-exposure ProphylaxisBacteria      1st choice      Alternatives                              rifampin, penicillin,    ...
TreatmentBacteria    1st choice      Alternatives            Ciprofloxacin   rifampin, vancomycin, penicillin,Anthrax     ...
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ANTIBIOTICS Lecture 06

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  • Release of biological weapons could be accomplished in a number of ways. However, it is expected that an aerosol release is the most likely to be encountered in a large-scale bioterrorism event if the goal is to sicken or kill large numbers of people. This is because aerosols are odorless, colorless and relatively easy to disperse over vast areas, and for most of the diseases, it is the route that is most contagious and causes the most severe disease. Many of these agents could theoretically be used to target food or water supplies, however, the psychological impact would likely be more of a concern than the clinical effect because of water purification processes, dilution of infectious material into large volumes, stability of the organisms and the logistical difficulties of dispersion [2].
  • Bacteria are single-celled organisms capable of causing a variety of diseases in animals, plants and humans. They may also produce potent toxins inside the human body. These single-celled organisms may also be cultured in nutrient media and be used as a bioterrorist agent.
  • A parasitic form of bacteria. They are smaller than typical bacteria, but larger than viruses. They are gram stain negative and can only multiply inside living cells.
  • They are smaller than bacteria, and unlike bacteria and rickettsia, they are totally dependent on the host’s living cells for reproduction
  • A National Repository of Life-Saving Pharmaceuticals and Medical Material The SNS is a national repository of antibiotics, chemical antidotes, vaccines, immunogloblins, antitoxins, life-support medications, IV administration, airway maintenance supplies, and medical/surgical items. The SNS is designed to supplement and re-supply state and local public health agencies in the event of a national emergency anywhere and at anytime within the U.S. or its territories.
  • Temporary stockpiles have been assembled infrequently in the past. They have typically been assembled for important events that represented a potential terrorist target, such as the 1996 Olympic Games. Factors mitigating against stockpiles have been cost, outdating, and low probability of actual use.
  • For example: Cipro 500 mg cost $2.25 each Doxycycline 100mg costs $0.08 each
  • ANTIBIOTICS Lecture 06

    1. 1. Bio 319: Antibiotics Lecture Six Topic: •Biotechnology production •Engineering polyketide antibiotics •Bioterrorism/Stockpiling Dr. G. Kattam Maiyoh GKM/bio 319/antibiotics/2013Wednesday, April 3, 2013 1
    2. 2. DNA Technology and Antibiotics Production • Worldwide over 100,000 tons per year • Sales ~ Ksh. 400b • Annually 100-200 antibiotics are discovered through labor intensive laboratory research. • Involves screening of different organisms for unique antibiotics. • This is a very costly process yet only 1-2% of antibiotics so discovered adds to the disease fighting arsenal. • Recombinant DNA technology can improve this situation.Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 2
    3. 3. Advantages • DNA tec. Can be used to produce new structurally unique antibiotics with; – Increased activities against selected targets – Decreased side effects – Increased yields – Decreased cost of productionWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 3
    4. 4. Organism of Choice• Streptomyces• Unlike E-coli not exist as individual cells but as extended aggregates of mycelial filaments• Must remove cell wall to release individual cells – to allow distinction between transformed cells and non transfomed cells.• Cells are transformed with different genes based on the desired antibiotic to be produced. Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 4
    5. 5. Isolation of antibiotic biosynthesis genes: by complementation • Mutant cells (not able to produce antibiotics) are transformed with DNA from a clone bank constructed from wild-type chromosomal DNA • Transformants are then screened for ability to produce antibiotics • e.g. for the antibiotic undecylprodigiosin, this involves color change to red due to antibiotic presenceWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 5
    6. 6. cntd •Plasmid DNA from the clone that supplied a functional gene and gene product (i.e. complements the mutant) is used as a hybridization probe to screen another clone bank of wild-type chromosomal DNA •Isolate clones with regions that overlap the probe sequence. •If antibiotic biosynthetic genes are clustered, then genes adjacent to complementing gene are likely to be involved in the biosynthesis of target antibiotic. •If scattered, more than one mutant are required to identify the rest of the genes.Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 6
    7. 7. Engineering Polyketide Antibiotics (PA) • Antibiotics synthesized through succesive enzymatic condensation of small carboxylic acids e.g. acetate, propionate and butyrate • Some PA are produce by plants and fungi • Most are produced by actenomycetes as sec. metabolites • To create new PA; I. study the functions of the enzymes involved in the biosynthetic pathway II. Manipulate the genes that encode this enzymes Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 7
    8. 8. FA biosynthesis, quick reminder:Fatty acid biosynthesis is a stepwise assemblyFatty acid biosynthesis is a stepwise assemblyof acetyl-CoA units (mostly as malonyl-CoA)of acetyl-CoA units (mostly as malonyl-CoA)ending with palmitate (C16 saturated)ending with palmitate (C16 saturated)3 Phases Activation Elongation Termination
    9. 9. Initiation Overall Reaction Malonyl-CoA + ACP CH3C~SCo - OOC-CH2C~S- ACP + HS-CoA A O O Acyl Carrier CO2 HS-CoA Protein CH3C- CH2C~S- ACP O O NOTE: Malonyl-CoA carbons become new COOH end Nascent chain remains tethered to ACP CO2, HS-CoA are released at each condensation
    10. 10. β-Carbon Elongation CH3C- CH2C~S- ACP O O ReductionNADPH β-Ketoacyl-ACP reductaseD isomer H CH3C- CH2C~S- ACP HO O Dehydration -H2O β -Hydroxyacyl-ACP dehydrase H CH3C- = C- C~S- ACPNADPH H Reduction O Enoyl-ACP reductase CH3CH2CH2C~S- ACP O
    11. 11. Biosynthesis PA • Analogous to synthesis of long-chain FA • Each condensation cycle results in the formation of , on the growing chain, of a β-keto group. • The repeated steps include; – Ketoreduction – Dehydration – Enoylreduction of the β-keto group of the growing polyketide chainWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 11
    12. 12. Classes of polyketide biosynthetic enzymes i. Those involve in the synthesis of aromatic polyketides (aromatic polyketide syntheses) - have active sites on same polypeptide ii. Those with the active sites on separate domains • In either case, the alteration of a catalytic domain whose function is known allows for predictable changes on the structure of the antibioticWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 12
    13. 13. Example: Engineering of Erythromycin production• Is synthesized by saccharopolyspora erythraea• The entire 56kb DNA that contains the ery gene cluster has been sequenced.• The erythromycin polyketide synthase gene altered as follows; 1. DNA encoding beta reductase activity deleted 2. Mutation of DNA region encoding enoylreductase activity Results; 1. Carbonyl group instead of hydroxyl at C-5 2. Carbon-carbon double bond at C6 and C7Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 13
    14. 14. Terrorism• Terrorism is defined as the unlawful use of force or violence against persons or property to intimidate or coerce a government or civilian population in the furtherance of political or social objectives. Bioterrorism• The use, or threatened use, of a micro-organism or the product of a micro-organism in order to generate fear, morbidity or mortality in a population.
    15. 15. Delivery Mechanisms• Aerosol route – Easiest to disperse – Highest number of people exposed – Most infectious – Undetectable to humans• Food / Waterborne less likely – Larger volumes required – More technically difficult
    16. 16. • Biologic agents are likely to be used by terrorists as weapons because: – They are capable of damaging populations, economies, and food supplies – Certain agents are inexpensive to make – They can be directed at a small group of people or an entire population – They can be used to attack people, economies and food supplies – They cause fear, panic and social disruption
    17. 17. BIOLOGICAL AGENTS• There are several types of agents. They are classified as: – Bacteria – Rickettsia – Viruses – Biotoxins
    18. 18. BACTERIA• Single celled organisms EXAMPLES capable of causing disease. • ANTHRAX These agents, grown on culture to produce large • SMALL POX quantities, can be modified • PLAGUE or “weaponized” for greater destruction • TYPHOID• Produces inflamation in • CHOLERA tissues and/or toxins • TULAREMIA
    19. 19. RICKETTSIA• Vector borne (ticks, lice, EXAMPLES mosquitos) parasitic • TYPHUS form of bacteria • ROCKY MT. SPOTTED• Diseases are difficult to FEVER treat • Q FEVER• Variants exist • INDIA TICK FEVER worldwide • MEDITERANEAN TICK FEVER
    20. 20. VIRUSES• Smaller than bacteria EXAMPLES• RNA or DNA in a protein • EBOLA coat • LASSA FEVER• Use living cells to reproduce • INFLUENZA• Not affected by • VIRAL HEPATITIS antibiotics • VIRAL HEMORRHAGIC FEVERS
    21. 21. BIOTOXINS• Are poisonous by- EXAMPLES products of bacteria, fungi, marine animals or plants • BOTULINUM• Do not replicate in the • STAPHLOCCOCAL host ENTEROTOXIN B• Are not communicable • RICIN• Highly toxic when delivered as an aerosol
    22. 22. Biological Agents most likely to be used in a terrorist attack• Bacteria - anthrax, plague, tularemia• Virus - small pox, viral hemorrhagic fever• Biotoxin - botulism
    23. 23. Brief history • Caused by Bacillus anthracis • Human zoonotic disease – Spores found in soil worldwide – Primarily disease of herbivorous animals • Sheep, goats, cattle – Occasional human disease • Epidemics have occurred but uncommonWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 23
    24. 24. Bioweapon Potential • Many countries have weaponized anthrax – Former bioweapon programs • U.S.S.R.,U.S.,U.K., and Japan – Recent bioweapon programs • Iraq – Attempted uses as bioterrorism agent • WW I: Germans inoculated livestock • WW II: Alleged Japanese use on prisonersWednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 24
    25. 25. Features of anthrax suitable as BTagent – Fairly easy to obtain, produce and store – Spores easily dispersed as aerosol – Moderately infectious – High mortality for inhalational (86-100%)Wednesday, April 3, 2013 GKM/bio 319/antibiotics/2013 25
    26. 26. PLAGUE• In nature, fleas living on rodents spread infection to humans. As a bioterrorist weapon – inhalation of aerosol leads to pneumonia, sepsis and infections of bodily organs• Infectious agent: Yersinia pestis – a gram neg., non-motile bacillus• May be bubonic ( infection of lymph nodes) or pneumonic (infection of lungs)or septicemic• Symptoms: cough with bloody sputum, fever, chill, shortness of breath
    27. 27. RashesA tentative diagnosis of plague is made
    28. 28. PLAGUE• Transmission: may occur person to person by respiratory droplet inhalation• Incubation period: 2 to 3 days• Mortality: 50 to 60%• Treatment: antibiotics• Prevention: vaccine ineffective against aerosol exposure
    29. 29. TULAREMIA• A zoonotic, bacterial infection caused by Francisella tularensis, a gram negative coccobacillus• In nature, bacteria is commonly found in ticks living on rabbits and transmitted by handling the animal or by tick bite. Inhalation of aerosol leads to pneumonia and sepsis
    30. 30. TULAREMIA• Symptoms:sudden and influenza-like with fever,chills, headache and nausea• Transmission: not usually person to person• Incubation period:3 to 5 days(range 1 to 14)• Mortality:low unless untreated• Treatment:antibiotics if early, vaccine available• Prevention: in nature, avoid tick bites and using gloves when handling infected animals
    31. 31. BOTULISM• Infectious agent: Clostridium botulinum – a spore forming, anaerobic bacillus• In nature, may be food borne, wound, or intestinal. As a bioterrorist weapon, ingestion or inhalation leads to production of the neurotoxin and resulting flaccid paralysis
    32. 32. BOTULISM• Symptom: fatigue, weakness, blurred vision, difficulty in swallowing and speaking, descending muscle paralysis and respiratory failure• Transmission: none person to person• Incubation period: 12 to 72 hours• Mortality: most lethal compound per weight• Treatment: antitoxins, respiratory support• Prevention: vaccine available for types A and B
    33. 33. Emergency Preparedness / Stockpiling
    34. 34. Strategic National Stockpile• Repository of – Antibiotics – Vaccines – Immunoglobulins – Chemical antidotes – Antitoxins – Life-support medications – IV administration – Airway maintenance supplies – Medical/surgical items
    35. 35. Containers designed to facilitatetransport by roads and railways. And also by airways.
    36. 36. • The Problem with Stockpiling – Antidotes and treatments are expensive – Have limited shelf-lives – Unlikely to be used in large quantities • 350,000 for prophylaxis • 2 doses daily for > 7 days of Cipro or Doxycycline • 4.9 million doses • Clearly exceeds local supply• What plans currently exist for such a disaster?
    37. 37. Antibiotics to Counteract Biologic Weapons• Often older agents are still the most effective.• Dosage regimens vary depending on – Bacterial agent being treated – Treatment vs. prophylaxis• Most expensive drug is not necessarily the better drug!
    38. 38. Post-exposure ProphylaxisBacteria 1st choice Alternatives rifampin, penicillin, Ciprofloxacin ampicillin, chloramphenicol,Anthrax Doxycycline clindamycin, and clarithromycin. DoxycyclinePlague Chloramphenical Ciprofloxacin DoxycyclineTularemia Ciprofloxacin
    39. 39. TreatmentBacteria 1st choice Alternatives Ciprofloxacin rifampin, vancomycin, penicillin,Anthrax ampicillin, chloramphenicol, imipenem, Doxycycline clindamycin, and clarithromycin. Gentamicin Doxycycline, CiprofloxacinPlague Streptomycin Chloramphenical Doxycycline, Ciprofloxacin, GentamicinTularemia Chloramphenicol Streptomycin
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