Bacterial Vaccines- Sterilization and Disinfection

1. Bacterial Vaccines- Sterilization and Disinfection Samuel Aguazim( MD)

5. Active immunity: • Bacterial vaccines are composed of capsular polysaccharides, inactivated protein, exotoxins(**toxoids), killed bacteria, or live attenuated bacteria. • The available bacterial vaccines and their indications are as follows: **Toxoid is a modified form of a toxin that is no longer toxic but is able to stimulate antibody production.

6. A. Capsular Polysaccharide Vaccines: (1) Streptococcus pneumoniae (2) Neisseriameningitidis (3) Haemophilusinfluenzae

7. (1) Streptococcus pneumoniae vaccine • Contains the capsular polysaccharides of the 23 prevalent types. • Recommended for patients over 60 years of age and patients of any age with such chronic diseases as diabetes and cirrhosis. • A second vaccine containing the capsular polysaccharide coupled to a carrier protein (diphtheria toxoid) is available for the protection of young children who do not respond well to the unconjugated vaccine.

8. (2) Neisseriameningitidis: • vaccine contains capsular polysaccharide of four important types. • It is given when there is a high risk of meningitis.

9. (3) Haemophilusinfluenzae: • vaccine contains the type b polysaccharide conjugated to diphtheria toxoid or other carrier protein. • It is given to children between the ages of 2 and 15months to prevent meningitis. • Capsular polysaccharide alone is a poor immunogen in young children but coupling it to a carrier protein greatly enhances its immunogenicity.

10. B.Toxoid Vaccines: (1) Corynebacteriumdiphtheriae: • vaccine contains the toxoid • indicated for every child and is given in three doses at 2, 4, and 6 months of age, with boosters given 1 year later and at intervals thereafter. (2) Clostridium tetani: • vaccine contains tetanus toxoid • everyone both early in life and later as boosters every 10 years for protection against tetanus. • Bordetellapertussis: • Vaccine contains pertussistoxoid • Includes other proteins as well

11. C. Purified Protein Vaccines (1) Bordetellapertussis (2) Bacillus anthracis (3) Borreliaburgdoiferi

12. (1)Two types ofBordetellapertussisvaccines: • containing killed bacteria • containing purified proteins from the organism (acellular vaccine). • The acellular vaccine is now recommended in the United States. • first vaccine to contain a genetically inactivated toxoid. • Indicated for every child as a protection against whooping cough. • It is usually given in combination with diphtheria and tetanus toxoids (DPT vaccine or DtaP vaccine). • principal antigen in the vaccine is inactivated pertussis toxin (pertussistoxoid), but other proteins, such as filamentous hemagglutinin and pertactin are also required for full protection

13. (2) Bacillus anthracis • vaccine contains “protective antigen” purified from the organism. • It is given to persons whose occupations place them at risk of anthrax. (3)The vaccine against Lyme disease: • contains a purified outer surface protein (OspA) of Borreliaburgdoiferi as the immunogen. • OspA is made by recombinant DNA techniques.

14. D. Live, attenuated bacterial vaccines: (1) The vaccine against tuberculosis: • Contains a live, attenuated strain of Mycobacterium boviscalled BCG • recommended for children at high risk of exposure to active tuberculosis in some countries. (2) The vaccine against tularemia: • Contains live, attenuatedFrancisellatularensisorganisms • Used primarily in laboratory personnel, veterinarians, and hunters.

15. E. Killed bacterial vaccines: 1) Vibriocholeraevaccine: • given to persons traveling to areas where cholera is endemic. (2) Yersiniapestisvaccine: • indicated for persons at high risk of contracting plague. (3) The vaccine against typhus: • contains killed Rickettsiarickettsiae organisms • used primarily to immunize members of the armed forces. (4)The vaccine against Q fever • contains killed Coxiellaburnetti organisms • used to immunize those who are at high risk of being exposed to animals infected with the organism.

16. F. Killed, live, attenuated and polysaccharide capsule vaccines: (1) Salmonella typhivaccine: • Given to persons living or traveling in areas in which there is a high risk of typhoid fever • persons in close contact with infected patients and carriers. • Three types of the vaccine are available: one contains the polysaccharide capsule of S typhi, one contains live, attenuated S typhi, and the third contains killed S typhi.

17. Passive Immunity: Antitoxins (immune globulins) can be used for either the treatment or prevention of certain bacterial diseases. The following preparations are available: (1) Tetanus (2) Botulinum (3) Diphtheria

18. (1) Tetanus: • antitoxin (passive immunity)is used in the treatment of tetanus and in its prevention (prophylaxis). • Intreatment, because the goal is to neutralize any unbound toxin to prevent the disease from getting worse, the antitoxin should be given promptly. • In prevention, the antitoxin is given to inadequately immunized persons with contaminated (“dirty”) wounds. • The antitoxin is made in humans to avoid hypersensitivity reactions. • In addition to the antitoxin, these people should receive tetanus toxoid (active immunity). • This is an example of passive-activeimmunity. The toxoid and the antitoxin should be given at different sites in the body to prevent the antitoxin from neutralizing the toxoid.

19. (2) Botulinum: • antitoxin is used in the treatment of botulism. • Because the antitoxin can neutralize unbound toxin to prevent the disease from progressing. (3) Diphtheria:antitoxin is used in the treatment of diphtheria. • The antitoxin can neutralize unbound toxin to prevent the disease from progressing

20. Sterilization and Disinfection Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object. Heating is the most commonly used method of sterilization. Commercial Sterilization: Heat treatment that kills endospores of Clostridium botulinum the causative agent of botulism, in canned food. Does not kill endospores of thermophiles, which are not pathogens and may grow at temperatures above 45oC.

22. Control of Microbial Growth:Definitions Disinfection:Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases. Usually involves the removal of vegetative or non-endosporeformingpathogens. May use physical or chemical methods. • Disinfectant: Applied to inanimate objects. • Antiseptic: Applied to living tissue (antisepsis). • Degerming: Mechanical removal of most microbes in a limited area. Example: Alcohol swab on skin. • Sanitization: Use of chemical agent on food-handling equipment to meet public health standards and minimize chances of disease transmission. E.g: Hot soap & water

23. Control of Microbial Growth:Definitions Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination. Aseptic techniquesare used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery. Aseptic techniques are also used to prevent bacterial contamination in food industry.

24. Control of Microbial Growth:Definitions Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them. Suffix stasis: To stop or steady. Germicide: An agent that kills certain microorganisms. • Bactericide: An agent that kills bacteria. Most do not kill endospores. • Viricide: An agent that inactivates viruses. • Fungicide: An agent that kills fungi. • Sporicide: An agent that kills bacterial endospores of fungal spores.

25. Control of Microbial Growth:Rate of Microbial Death Several factors influence the effectiveness of antimicrobial treatment. 1. Number of Microbes: The more microbes present, the more time it takes to eliminate population. 2. Type of Microbes: Endospores are very difficult to destroy. Vegetative pathogens vary widely in susceptibility to different methods of microbial control. 3. Environmental influences: Presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials, pH etc. 4. Time of Exposure: Chemical antimicrobials and radiation treatments are more effective at longer times. In heat treatments, longer exposure compensates for lower temperatures.

26. CHEMICAL AGENTS • Chemicals vary greatly in their ability to kill microorganisms. • Chemical agents act primarily by one of three mechanisms: 1. disruption of the lipid-containing cell membrane 2. modification of proteins 3. modification of DNA.

27. DISRUPTION OF CELL MEMBRANES Alcohols • Ethanol is widely used to clean the skin before immunization or venipuncture. • disorganizing the lipid structure in membranes, but it denatures proteins as well. • Ethanol requires the presence of water for maximal activity: ie, it is far more effective at 70%.

28. Detergents • “surface-active” agents • composed of a long-chain, lipid-soluble, hydrophobic portion and a polar hydrophilic group. • These surfactants interact with the lipid in the cell membrane through their hydrophobic chain and with the surrounding water through their polar group and thus disrupt the membrane

29. MODIFICATION OF PROTEINS Chlorine • Used as a disinfectant to purify the water supply and to treat swimming pools. • Active component of hypochlorite (bleach, Clorox), which is used as a disinfectant in the home and in hospitals. • Powerful oxidizing agent that kills by cross-linking essential sulfhydryl groups in enzymes to form the inactive disulfide

30. Iodine • most effective skin antiseptic used in medical practice. It is supplied in two forms: (1) Tincture of iodine (2% solution of iodine and potassium iodide in ethanol) is used to prepare the skin prior to blood culture. (2) lodophorsare complexes of iodine with detergents that are frequently used to prepare the skin prior to surgery because they are less irritating than tincture of iodine.

31. Heavy Metals • Mercury and silver have the greatest antibacterial activity of the heavy metals and are the most widely used in medicine. • They act by binding to sulfhydryl groups, thereby blocking enzymatic activity. • Silver nitrate drops are useful in preventing gonococcalophthalmianeonatorum. • Silver sulfadiazine is used to prevent infection of burn wounds.

32. Hydrogen Peroxide • Antiseptic to clean wounds • Disinfect contact lenses. • Effectiveness is limited by the organism’s ability to produce catalase, an enzyme that degrades H2O2 (The bubbles produced when peroxide is used on wounds are formed by oxygen arising from the breakdown of H2O2 by tissue catalase.) • Hydrogen peroxide is an oxidizing agent that attacks sulfhydryl groups, thereby inhibiting enzymatic activity

33. Ethylene Oxide • Ethylene oxide gas is used extensively in hospitals for the sterilization of heat-sensitive materials such as surgical instruments and plastics.

34. Physical Agents The physical agents act either by imparting energy in the form of: • heat • radiation • removing organisms through filtration

35. HEAT Heat energy can be applied in three ways: • moist heat (either boiling or autoclaving) • dry heat or by pasteurization. • In general, heat kills by denaturing proteins, but membrane damage and enzymatic cleavage of DNA may also be involved

36. Moist heat • sterilizes at a lower temperature than dry heat, because water aids in the disruption of noncovalent bonds, eg, hydrogen bonds, which hold protein chains together in their secondary and tertiary structures. • Boiling: Heat to 100oC or more at sea level. Kills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores within 10 minutes or less. Endospores and some viruses are not destroyed this quickly. However brief boiling will kill most pathogens. • Hepatitis virus: Can survive up to 30 minutes of boiling. • Endospores: Can survive up to 20 hours or more of boiling.

37. Autoclaving • Moist-heat sterilization, usually autoclaving,is the most frequently used method of sterilization. • Because bacterial spores are resistant to boiling(100 0C at sea level), they must be exposed to a higher temperature; this cannot be achieved unless the pressure is increased. • For this purpose, an autoclave chamber is used in which steam, at a pressure of 15 lb/in2, reaches a temperature of 121 0C and is held for 15—20minutes. This kills even the highly heat-resistant spores of Clostridium botulinum.

38. Sterilization by dry heat, • temperatures in the range of 180 0C for 2 hours. • This process is used primarily for glassware and is used less frequently than autoclaving. Dry Heat: Kills by oxidationeffects. • Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow. • Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste. • Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat.

39. Pasteurization • used primarily for milk, consists of heating the milk to 62 0C for 30 minutes followed by rapid cooling. • “Flash” pasteurization at 72 0C for 15 seconds is often used. • Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3 seconds and then cooled very quickly in a vacuum chamber. • Advantage: Milk can be stored at room temperature for several months. • This is sufficient to kill the vegetative cells of the milk-borne pathogens, eg, M bovis, Salmonella, Streptococcus, Listeria, and Brucella,but not to sterilize the milk

40. Radiation • The two types of radiation used to kill microorganisms are ultraviolet (UV) light and x-rays. • The most significant lesion caused by UV irradiation is the formation ofthymine dimers, but addition of hydroxyl groups to the bases also occurs. • As a result, DNA replication is inhibited and the organism cannot grow.

41. Cells have repair mechanisms against UV-induced damage • involve either cleavage of dimers in the presence of visible light or excision of damaged bases, which is not dependent upon visible light (dark repair). • Because UV radiation can damage the cornea and skin, the use of UV irradiation in medicine is limited.

42. X-rays • have higher energy and penetrating power than UV radiation and kill mainly by the production of free radicals,eg, production of hydroxyl radicals by the hydrolysis of water. • These highly reactive radicals can break covalent bonds in DNA, thereby killing the organism. • kill vegetative cells readily, but spores are remarkably resistant, probably because of their lower water content. • X-rays are used in medicine for sterilization of heat-sensitive items, such as sutures and surgical gloves, and plastic items, such as syringes.

43. FILTRATION • is the preferred method of sterilizing certain solutions. eg, those with heat-sensitive components. • In the past, solutions for intravenous use were autoclaved but heat-resistant endotoxin in the cell walls of the dead gram-negative bacteria caused fever in recipients of the solutions. • Therefore, solutions are now filtered to make them “pyrogen-free” prior to autoclaving • Used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media.

44. FILTRATION • High Efficiency Particulate Air Filters (HEPA): Used in operating rooms and burn units to remove bacteria from air. • Membrane Filters: Uniform pore size. Used in industry and research. Different sizes: • 0.22 and 0.45um Pores: Used to filter most bacteria. Don’t retain spirochetes, mycoplasmas and viruses. • 0.01 um Pores: Retain all viruses and some large proteins.