1. Definition of Terms Sterilization - process of destroying all forms of microbial life Disinfection - elimination of microorganisms from inanimate objects and surfaces Antimicrobial agents - agents that inhibit the growth or completely destroy the life of microorganisms

2. Anti-microbial agents could be: 1. Physical agents 2. Chemical agents 3. Chemotherapeutic agents Disinfectant - chemical agents that kills the growing forms but not necessarily the resistant spore forming bacteria 3. Antiseptic/Germicide - chemical agents that kills or inhibits the growth and metabolism of the microorganisms - used in the body without damaging the tissues Sanitizer - agent that reduces the microbial population to safe level 4. Bactericidal/Bactericide - total killing of the microorganisms Bacteriostatic/ Bacteriostasis - condition in which bacterial growth are prevented Decontamination - treatment of an object or surfaces to make it safe to handle 5. Uses of Sterilization - Culture media, reagents, containers, loops and needles, glasswares, etc. - Medical and surgical equipment or materials used in penetrating normally sterile portions of the human body Uses of Disinfection - in cases in which sterility is unnecessary - if steriliztion is impractical 6. A. Physical Antimicrobial Control 1. Heat sterilization - most reliable and universally applicable - divided into Moist heat and Dry heat a. Moist heat 1. Autoclaving - steam under pressure - 121 C, 15 psi, 10 15 minutes 7. 2. Boiling - commonly employed but incompletely effective 3. Free flowing steam (Arnold sterilizer) - also known as Intermittent sterilization or Tyndallisation or fractionation - consist of heating 80 to 100 C for 30 mins. For 3 consecutive days - first heating: vegetative cells and some spores will be killed subsequent heating: more resistant spore 8. 4. Pasteurization - process used in food industries for milk and and other dairy products, fruit juices, wine beer and other heat sensitive substances - exposing the product to 62 C for 3o mins. - 71 C for 15 seconds (flash pasteurization) 9. b. Dry heat (protein denaturation) 1. Flaming - used in bacteriological loop and needles 2. Hot air oven - for glasswares, petridishes, pipettes, flasks - 180 C for 1 2 hours 3. Incineration (Burning) 10. 2. Radiation sterilization - electromagnetic radiation (U.V., X-rays) - causing DNA breaks a. Ionizing radiation - generates electrons, hydroxyl radicals, etc. - alters DNA, cell death 11. 3 . Filter sterilization - for heat-sensitive substances - consist of pores too small for passage of microorganisms but large enough to allow the passage of gas o liquid e.g. Membrane Filter - composed of polymers with high tensile strength Nucleation tuck filter (Nucleopore) 12. - other filters - Seitz filter (disks of asbestos-cellulose mixture) - Chamberland filter (unglazed porcelain) - Barkefeld filter (diatomaceous earth) c. Low temperature (Freezing) d. Desiccation - Lyophilization (extreme dehydration) e. Ultrasonic and Sonic Vibration (20 1000 kc) 13. B. Chemical Antimicrobial Agents Mode of action 1. Damage to cell wall or inhibition of cell wall synthesis 2. Alteration of the cytoplasmic membrane permeability 3. Alteration of the physical and chemical state of proteins and nucleic acid 4. Inhibition of enzyme function 5. Inhibition of protein and nucleic acid synthesis 14. Factors affecting the antimicrobial activity 1. Concentration of the agent 2. Temperature (increase Temp., increase reaction) 3. Contact time 4. pH 5. Nature of microorganism (species) 6. Growth phase of microorganisms. 7. Presence of special structure (spore, capsule) 8. Number of microorganisms 9. Presence of extraneous materials(blood,pus,etc) 15. Qualities of good disinfectant 1. High co-effecient of disinfection 2. stable 3. water soluble 4. non-toxic, non-corrosive 5. easy to use 6. cheap 16. Chemical Agents Mode of action Use Alcohol (Ethanol, Isopropanol) Protein denaturant - topical antiseptic - Disinfectant for medical instrument Phenol-containing compounds Disrupt cell membrane Protein denaturant -topical disinfectant -Laboratory surfaces Cat-ionic detergents (Benzalkonium chlroide) Interact with phospholipid of cell membrane -topical disinfectant -Disinfectant for medical instrument, food and dairy Hydrogen peroxide Oxidizing agent Topical antiseptics Iodine-containing compounds Oxidizing agent Topical antiseptics Chlorine gas/compound Oxidizing agent Disinfectant for food, dairy and water purification Copper sulfate Oxidizing agent Algicide disinfectant in swimming pools/water 17. Ethylene oxide Alkylating agent Steilant for heat-sensitive materials Formaldehde (3 8 %) Alkylating agent Surface disinfectant Glutaraldehyde (2%) Alkylating agent Mercuric chloride Combines with -SH group Disinfectant (surfaces) Ozone Strong oxidizing agent Disinfectant for drinking water Orthoparaldehyde (OPA) High-level disinfectant for medical instrument Alkylating agent 18. C. Chemotherapeutic Agents - chemical substances used in treating diseases Antibiotics - special type of chemotherapeutic agents used in treating infectious diseases - usually obtained from living microorganisms 19. Historical Highlight 1630 - European use natural quinine (from bark of Cinchona tree) for malaria 1910 - Use of Salvarsan for syphilis by Paul Erlich 1935 - Use of Sulfonamides for large variety of pathogens 20. 1945 - use of Sulfadiazine and Sulfamerazine (derivative of sulfonamides) for its anti- bacterial effect 1929 - significant discovery of the miracle drug, Penicillin by Alexander Fleming - the drug was first out in the market in 1950s 21. Mechanisms of Action of Antibiotics 1. Inhibitionn of cell wall synthesis 2. Protein synthesis inhibition -the beta-lactams: penicillin, cephalosphorins, carbapenems and monobactams) - binding to 50S ribosomal subunit (exhibited by macrolides, chloramphenicol and clindamycin) or to the 30S subunit (the aminoglycosides, and tetracycline 22. 3. Interference with nucleic acid synthesis 4. Inhibition of metabolic pathway - sulphonamides 5. Disruption of bacterial membrane structure -RNA, by rifampin, DNA, by fluoroquinolones -by polymyxins, daptomycin 23. Properties of a good Antibiotic 1. Selective toxicity - harmful to microorganism but without being harmful to the host - relative rather than absolute 2. Broad spectrum - inhibits many different species of pathogenic microorganisms 3. Prevents development of genetically and phenotypically resistant strains 24. 4. Bactericidal rather than Bacteriostatic 5. Non-allergenic and no adverse reaction 6. Should remain active in the presence of plasma and other body fluids. 7. Stable and water soluble 25. Different Groups of Antibiotics 1. Pinicillins 2. Cephalosphorins 3. Quinolons 4. Sulfonamides 5. Aminoglycosides 6. Macrolides (e.g.. Erythromycin) 7. Tetracyclin 8. Chloramphenicol 9. Anti-fungal 10. Anti-viral 26. Why is drug resistance a concern? 1. Proliferation of resistant strains in Health care institution 2. Treatment failure which causes serious consequences especially critically ill person 3. Broader infection control problems for both in Healthcare Institutions and communities as well Antibiotic resistance - the ability of bacteria or other microbes to resist the effects of antibiotic. 27. Reasons why antibiotic resistance occurs 1. Over-prescription of antibiotics 2. Non-completion of prescribed antibiotics 3. Use of antibiotics in animals as growth enhancers 4. Poor hospital hygiene 5. Indiscriminate use of antibiotics 28. Mechanisms of Resistance to Antibiotics 1. Up-regulating the production of enzymes that inactivate the anti- microbial agent. 2. Up-regulating pumps that expel the drugs from the cell. . 29. AcrAB-TolC Complex 30. 3. Acquisition of genes for antibiotic resistance through horizontal gene transfer 4. Down regulating or altering an outer membrane protein channel that the drug requires 31. Antimicrobial activity in vivo A. Drug-Pathogen relationship 1. Environment - influence the effect to microorganism (microorganisms are located in different tissues and parts of the body a. State of metabolic activity - diverse biosynthetic activity of microorganisms may results to dormancy which often survive upon exposure to drugs (causes clinical relapse 32. b. Distribution of drugs - unequal distribution of drugs to the tissues and body fluids c. Location of organisms - drugs enter the cells at different rates d. Interfering substances present in the host - drugs may react with blood, tissue proteins, exudates, necrotic debris which is unfavourable to drug action 33. 2. Concentration - organisms inside the body may not be exposed to constant concentration of drugs - factors: a. Absorption b. Distribution c. Variability of concentration d. Post-antibiotic effect - delayed re-growth of bacteria after exposure to antimicrobial agent 34. B. Host-Pathogen Relationship 1. Alteration in Tissue response (Inflammatory) 2. Alteration of Immune response 3. Alteration of Microbial flora C. Host Drug Relationship 1. Absorption 2. Metabolism 4. Toxicity 3. Distribution 5. Excretion 35. Dangers of indiscriminate use of Antibiotics 1. Widespread sensitization (Hypersensitivity rxn) 2. Changes in normal flora of the body (super-infection due to drug-resistant strain) 3. Masking serious infection 4. Direct drug toxicity (renal damage, auditory nerve damage) 5. Development of drug resistance in microbial population 36. Antimicrobial Chemoprophylaxis - administration of antimicrobial drugs to prevent infection - use of specific drugs to specific microorganisms - the risk of acquiring infection is weighed against the toxicity, cost, inconvenience and enhanced risk of super-infection 37. Antimicrobial Activity in vitro Antimicrobial Susceptibility Test -used to select effective antimicrobial drugs for treatment and control of infectious diseases especially when caused by resistant pathogens - predicts the in-vivo success or failure of antibiotic therapy - MIC and MBC determination 38. Methods A. Dilution Method - cumbersome, limited to special circumstances - provides quantitative information on MIC and MBC 1. Two-fold dilution 2. Agar dilution 39. Tube dilution technique 40. B. Diffusion Method - most widely used is the disk diffusion test - filter paper disk containing a measured quantity of a drug is placed on the surface of a solid medium that has been inoculated on the surface with the test organism - after incubation, the diameter of the clear zone of inhibition surrounding the disk is taken as a measure of the inhibitory power of the drug against the particular test organism - subject to many physical and chemical factors in addition to the simple interaction of drug and organisms 41. Kirby-Bauer Technique - use of commercially available antibiotic disk - culture medium: MHA - inoculum: turbidity = 0.5/1.0 McFarland = 3.8 x 10 raised 8 cfu - indicator: zone of inhibition (mm units) - Interpretation: Susceptible/Sensitive Intermediate Resistant 42. Factors affecting the antimicrobial activity in vitro 1. pH of the environment - some drugs are active at acidic condition some at alkaline like aminoglycosides 2. component of the media - some additives will inhibit the growth of other microorganisms (e.g., PABA from tissue extract: antigonize sulfonamides, NaCl: enhance growth of MRSA) 3. stability of the antibiotic 43. 4. Size of the inoculums - larger bacterial inoculums the lower the apparent susceptibility 5. Length of incubation - the longer the incubation the greater the chance for resistant mutant strain to immerge 6. metabolic activity of the microorganism - in general, actively growing microorganisms are more susceptible to those in resting phase 44. END