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Sterilization, disinfection and antisepsis

Sterilization, disinfection and antisepsis. Original methods for disinfection. For lecture only. BC Yang. In 1866 J lister suggested antiseptic surgery. His rational was ( 典型的三段式論證 ) : Putrefaction is caused by microbes Wound sepsis is a form of putrefaction

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Sterilization, disinfection and antisepsis

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  1. Sterilization, disinfection and antisepsis Original methods for disinfection For lecture only BC Yang

  2. In 1866 J lister suggested antiseptic surgery. His rational was (典型的三段式論證): • Putrefaction is caused by microbes • Wound sepsis is a form of putrefaction • Wound sepsis is caused by microbes Carbolic acid, phenol For lecture only BC Yang

  3. In the previous year Lister had heard that 'carbolic acid' was being used to treat sewage in Carlise, and that fields treated with the affluent were freed of a parasite causing disease in cattle. • Lister then began to clean wounds and dress them using a solution of carbolic acid. He was able to announce at a British Medical Association meeting, in 1867, that his wards at the Glasgow Royal Infirmary had remained clear of sepsis for nine months. • Opposition was great In England and the United States mainly against Lister's germ theory rather than against his "carbolic treatment."

  4. Phenols and phenolics • Carbolic acid • Low level disinfetanct • O-phenylphenol (Lysol) • Hexachlorophene (Phisohex) • High level for Staphylococcus sp. and Streptococcus sp. • Surgical scrubs • Damage membrane, inactive enzymes, denature proteins For lecture only BC Yang

  5. Mechanism of antimicrobial action • Damage to DNA: radiation, UV, DNA-reactive agents • Protein denaturation: Heat, pH, metals • Disruption of cell membrane or cell wall: detergents, enzymes • Chemical antagonism: analogs • Removal of free sulfhydryl groups: oxidizing agents For lecture only BC Yang

  6. To die or not to die, that is a question • Sepsis: microbes contamination • Asepsis: absence of microbes contamination • Sterilization: 100% killing • Disinfection: loss the ability to cause disease • -cide or cidal: killing of microorganism • -static or stasis: inhibit growth or multiplication of microbes For lecture only BC Yang

  7. Killing is a probability • Die at a constant rate (logarithmically) • Depend on exposure time and microbial load • Rare will reach 100% (on purpose and money consuming) For lecture only BC Yang

  8. Parameters of sterilization/disinfection • Action process • The rate/kinetics: the death rate of a microbial population follows a logarithmic path. • Resistances: pure empirical, varied from culture condition, nature of the environment • Control/indicators: • Living: • heat : spores, themophilic bacteria • ethylene oxide and radiation: bacillus subtilits var. nigar. • Chemical:succinic anhydride (120oC), sulphur (115oC); color change; etc.. For lecture only BC Yang

  9. Action index • A spoonful work already well? (一匙靈) • Why not LD50? • Phenol coefficient (PC) • American official analytical chemist’s use-dilution method • Staphylococcus aureus, pseudomonas aeruginosa, salmonella choleraesuis • Filter paper method For lecture only BC Yang

  10. As a matter of temperature • Heat • Boiling water • Moist heat (steam under pressure, autoclave) • Hot-air sterilization (Oven) • Pasteurization • Tyndallization • Incineration • Low temperature • Refrigerator temp (4-7oC) • Deep-freeze (-20oC to -40oC); liquid nitrogen (-196oC) • Lyophilization (desiccation in low temperature) For lecture only BC Yang

  11. Pasteurization 別呆了, 不要喝尚青尚新鮮的牛奶! • 1909 John Ross Robertson finances the installation of Toronto's first milk pasteurization plant in the College Street hospital, 30 years before it becomes mandatory. For lecture only BC Yang

  12. Tyndallization (Fractional sterilization) • John Tyndall was a man of science—draftsman, surveyor, physics professor, mathematician, geologist, atmospheric scientist, public lecturer, and mountaineer. • For killing both vegetative and spore forming bacteria at temperature of 100oC or below for 30 min on the consecutive days For lecture only BC Yang

  13. Filtration For lecture only BC Yang

  14. Filtration • Liquid • When subjects are heat-labile • 0.22/0.45 mm membrane filter • Air • Large space Alternative: electrostatic precipitation • Particles passed through a high voltage electric field become charged, and attracted to and held by the plate of opposite potential (remove ~97% of bacteria) For lecture only BC Yang

  15. Wash away: do you really clean your hands? Surfactants: breaks oily film on skin For lecture only BC Yang

  16. UV radiation • Ultraviolet is light with very high energy levels and a wavelength of 200-400 nm. • One of the most effective wavelengths for disinfection is that of 254 nm. For lecture only BC Yang

  17. Alcohols • High level disinfectant • Disrupt protein, disrupt membranes, dissolve lipids • 70% ethanol For lecture only BC Yang

  18. 碘酒 Halogens (Iodine) • Iodine combines with tyrosine & oxidized SH groups on other amino acids • Tincture: 1-2% I2 + 2% Na2I in 70% ethanol (for surgical site disinfection). • Povidone-iodines: Betadine • Skin disinfection • Surgical scrub For lecture only BC Yang

  19. The choices?It always depends! • The materials • The purpose • The nature of microbes For lecture only BC Yang

  20. Antibiotics Antibiotics and vaccines are among the biggest medical advances since 1000. (Culver Pictures) For lecture only BC Yang

  21. Diagrammatic representation of the results of treatment related to specific chemotherapy Patients with normal immunity and uncomplicated mild to moderate infections Patients with serious life-threatening infections For lecture only BC Yang

  22. A brief history of antibiotics • 1495, mercury to treat syphilis. • 1630, quinine (chinchona tree) for malarial fever by South American Indians. • 1889, Buillemin defined antibiosis. • 1910, Paul Ehrlich developed arsenical compound (Salvarsan) for syphilis, term: the chemical knife. • 1929, Alexander Fleming found penicillin. • 1935, Gerhard Domagk showed the value of sulfonamides. • 1940, Ernst Chain and Howard Flory demonstrated the effect of penicillin. • 1940-1970, then searching for new antibiotics (based on killing) • ~ recent year: modifying old drugs, finding new discipline in antibacterial combats • Early time in war: thanks penicillin, we can go home now…. • Now a day? Antibiotics resistance For lecture only BC Yang

  23. A. Fleming E. Chain H. Florey Thanks to work by Alexander Fleming (1881-1955), Howard Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin was first produced on a large scale for human use in 1943. At this time, the development of a pill that could reliably kill bacteria was a remarkable development and many lives were saved during World War II because this medication was available. For lecture only BC Yang

  24. A tale by A. Fleming • He took a sample of the mold from the contaminated plate. He found that it was from the penicillium family, later specified as Penicillium notatum. Fleming presented his findings in 1929, but they raised little interest. He published a report on penicillin and its potential uses in the British Journal of Experimental Pathology. For lecture only BC Yang

  25. Scenario of penicillin action on E. coli 3 1 2 4 5 6 1: ordinary appearance 2-4: globular extrusions emerge 5: rabbit-ear forms 6: Ghost form For lecture only BC Yang

  26. An ideal antibiotics • Broad-spectrum • Did not induce resistance • Selective toxicity, low side effects • Preserve normal microbial flora For lecture only BC Yang

  27. Susceptibility test • Tube dilution method • Minimal inhibitory concentration (MIC): the smallest amount of chemotherapeutic agent required to inhibit the growth of organism in vitro • Disk diffusion method • Zone of inhibition (ZOI): the correlation of ZOI and MIC has been established by FAD ETest. This commercially-prepared strip creates a gradient of antibiotic concentration when placed on an agar plate For lecture only BC Yang

  28. Guidance of antimicrobial therapy • Minimum inhibitory concentration: lowest concentration of antibiotic that inhibits visible growth • Minimum bactericidal concentration: lowest concentration of antibiotic that kills 99.9% of the inoculum • Serum bactericidal title: dilution of serum that kills 99.9% of the inoculum • Synergy test: synergistic activity of multiple antibiotics For lecture only BC Yang

  29. In vitro: Factors for optimal antibiotic action • pH of environment: • Nitrofurantoin is more active in acid pH; sulfonamides and aminoglycoside are more active in alkaline pH. • Components of medium: • Anionic detergents inhibit aminoglycosides, serum proteins bind to penicillin in varying degrees. • Stability of drug: • Aminoglycosides and chloramphenical are stable for long period in vivo. • Size of inoculums: • The larger the bacterial inoculum, the greater the chance for resistnat mutant to emerge. • Metablic activity of microorganisms: • Actively and rapidly growing organisms are more susceptible to drug action For lecture only BC Yang

  30. Affecting factors in vivo • Abscess: circulation is blocked off. • Foreign bodies: obstruction of the urinary, biliary or respiratory tracts etc. • Immunity. For lecture only BC Yang

  31. Sites of action For lecture only BC Yang

  32. Modes of action (1) • Inhibitors of cell wall synthesis.Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin. • Inhibitors of Cell Membrane.Polyenes - Amphotericin B, nystatin, and condicidin.Imidazole - Miconazole, ketoconazole and clotrimazole.Polymixin E and B. • Inhibitors of Protein Synthesis.Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin.Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline.Erythromycin, lincomycin, chloramphenicol and clindamycin. vancomycin Amphotericin Aminoglycosides Tetracyclines For lecture only BC Yang

  33. rifamycin Modes of action (2) • Inhibitors of metabolites (Antimetabolites).Sulfonamides - Sulfanilamide, sulfadiazine silver and sulfamethoxazole.Trimethoprim, ethambutol, isoniazid. • Inhibitors of nucleic acids (DNA/RNA polymerase).Quinolones - Nalidixic acid, norfloxacin and ciprofloxacin.Rifamycin and flucytosine.  For lecture only BC Yang

  34. Penicillin: an extensively studied example For lecture only BC Yang

  35. Action mechanism of penicillin • Action target: cell wall • on penicillin binding proteins (PBPs) • Transpeptidases (form cross-links in peptidoglycan) • Beta-lactam ring attached to 5-membered thiazolidine ring • Accessibility of PBPs differ in gram+ and gram- bacteria • Amino acyl side chain groups determine spectrum, adsorption, susceptibility to lactamase • Bactericidal inhibitors For lecture only BC Yang

  36. Metallo-b-Lactamase Serine-b-Lactamase Resistance • Failure to bind to PBPs • Cannot penetrate porins (gram-) • Production of lactamase (penicillinase) • Lack autolytic enzyme • B-lactamase Types: • Different substrate specificity • Penicillinases • cephalosporinases Location: • Gram+: extracellularly • Gram-: periplasmic space • By Dr. Osnat Herzberg • University of Maryland Biotechnology Institute (UMBI) For lecture only BC Yang

  37. In Rwanda 聯合報 八十五年 四月十三日 頭版新聞 For lecture only BC Yang

  38. Resistances • Natural (inherent) resistance • Structural barrel • Lack of target • Transport system • Acquired resistance • Mutation • Gene exchange (conjugation in most) For lecture only BC Yang

  39. Transferable antibiotic resistance in bacteria • Reduced uptake into cell (chloramphenicol) • Active efflux from cell (tetracycline) • Modification of antibiotic targets (b-lactam, erythromycin) • inactivation of antibiotic by anzymic modification: hydrolysis (b-lactam, erythromycin); derivatization (aminoglycosides) • Sequestration of antibiotic by protein binding (b-lactam) • Metabolic bypass (sulfonamides) • Overproduction of antibiotic target (titration: sulfonamides) For lecture only BC Yang

  40. Some probable overuse/misuse of antibiotics • Prophylatic use before surgery • Empiric use (blinded use) • Increased use of broad spectrum agents • Pediatric use for viral infections • Patients who do not complete course (chronic disease, eg. TB, AIDS) • Antibiotics in animal feeds For lecture only BC Yang

  41. Policy to deal drug resistance (1) • Ideally, bacteriological management of clinical infection should involve: • Identification of causative organism • Sensitivity test • Follow-up the drug effect • Monitor antibiotic level to avoid toxicity. • In reality, most patients requiring antimicrobial therapy are treated empirically. In serious infections immediate chemotherapy may be life-saving. For lecture only BC Yang

  42. Policy to deal drug resistance (2) • Periodic changes of antibiotics used might change selective pressure and thus avoid the emergence of resistance and retain the therapeutic value of antibiotics over a longer period. • The unnecessary prophylactic or animal feeds use should be discouraged. • Distribution of information on current/updated infectious microbes (consult microbiologists): use more targeted antibiotics • Patient education (不隨便吃藥, 停藥) For lecture only BC Yang

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