Control of Microbial growth Dr. Hala Al Daghistani

1. Control of Microbial growth Dr. Hala Al Daghistani

2. Terminology • Sepsis: Characterized by the presence of pathogenic microbes in living tissues or associated fluids. • Asepsis: absence of significant contamination. • Asepticsurgery techniques prevent microbial contamination of wounds. • Antimicrobial : chemicals, expected to destroy pathogens but not to achieve sterilization • Disinfectant: used on objects (reduce the number of viable microorganisms) • Antiseptic: used on living tissue, destroys or inhibits the growth of microorganisms • Nosocomial Infection(Hospital Acquired Infection) an infection that is contracted from the environment or staff of a healthcare facility.

3. Sterilization: A defined process used to render a surface or product free from viable organisms, including bacterial spores. • Biocide: A chemical or physical agent, usually broad spectrum, that inactivates (kill) microorganisms. Chemical biocides include hydrogen peroxide, alcohols, bleach, cycloheximide, and phenols physical biocides include heat and radiation. Fungicide, Virucide, Germicide, bactericide • Sanitization: Lowering of microbial counts to prevent transmission in public setting (e.g., restaurants & public rest rooms)

4. Antibiotics: Naturally occurring and synthetically derived organic compounds that inhibit or destroy selective bacteria, generally at low concentrations. • Bacteriostatic: Inhibits bacterial reproduction • Bactericidal: Kills bacteria

6. Factors affect the antimicrobial treatment • Time it takes to kill a microbial population is proportional to number of microbes. • Microbial species and life cycle phases (e.g.: endospores) have different susceptibilities to physical and chemical controls. • Organic matter may interfere with heat treatments and chemical control agents.(heat treatment is efficient at low pH) • Exposure time: Longer exposure to lower heat produces same effect as shorter time at higher heat.

7. Actions of Microbial Control Agents • Disruption of the Cell Membrane permeability(damage protein or lipids of PM) • Disruption of the cell Wall synthesis(either by inhibit the enzyme involved in cell wall synthesis or interfere with CW building block synthesis). E.g. penicillin interfere with transpeptidase and prevent the assembly of PG layer. • Damage to proteins (break down H- bonds, destruction of three dimensional structure of a protein , inhibition of translation and transcription of genetic material).

8. Damage to nucleic acids(include ionizing radiations, ultraviolet light, and DNA-reactive chemicals. Ultraviolet light, induces cross-linking between adjacent pyrimidines on one or the other of the two DNA strands, forming thiamine dimers • Antimetabolite groups(sulfa drugs interfere with folic acid synthesis). Antimetabolites are substances that interfere with the normal metabolism of an organism, thereby causing its death. Evolution of drug resistance: * spontaneous mutation * gene transfer

9. Antibiotic Resistance • Mechanisms of antibiotic resistance • Enzymatic destruction of drug • Prevention of penetration of drug • Alteration of drug's target site • Rapid ejection of the drug • Resistance genes are often on plasmids or transposons that can be transferred between bacteria.

10. Resistance to Antibiotics Fig 20.20

11. The disk-diffusion methodAntibiotic Susceptibility test Kirby-Bauer test determines susceptibility of an organism to a series of antibiotics OR CHEMICALS Disk-diffusion method is used in teaching laboratories to evaluate the efficacy of a chemical agent. A disk of filter paper is soaked with a chemical and placed on an agar plate that has been previously inoculated and incubated with the test organism After incubation, if the chemical is effective, a clear zonerepresenting inhibition of growth can be seen around the disk (Inhibition Zone)

13. Physical Methods of Microbial Control • A)Heat (used for media, food, glass wares) • Two types (moist and dry heat) • Heat is very effective (fast and cheap). • Thermal death point (TDP):Lowest temperature at which all cells in a culture are killed in 10 min. • Thermal death time (TDT):Min Time to kill all cells in a culture at a given temp. • Decimal Reduction Time (DRT):Time in minutes in which 90% of a bacterial population are killed at a given Temp.

14. Moist heat • A temperature of 100°C (Boiling) will kill bacteria, viruses, and fungi spores, but not spore forms of bacteria within 10 minutes in laboratory-scale cultures. • a temperature of 121°C, pressure of 15 lb/sq inches for 15 minutes is used to kill spores. Steam is generally used, both because bacteria are more quickly killed when moist heat are used, and because steam provides a means for distributing heat to all parts of the sterilizing vessel. Autoclave: Steam under pressure, Most dependable sterilization method • Prions (infectious proteins) need 134°C and NaOH solution for 4-5 h. • Moist heatkill M.O. by denaturing of proteins(breakdown H bonds that hold 3-dimentional structures).

15. Pasteurization • Moist heat method • Significant number reduction (esp. spoilage and pathogenic organisms)  does not sterilize • Denaturation of proteins by breaking H-bonds • Historical goal(LOUIS PASTEUR): destruction of M. tuberculosis • Classic pasterization method: 63C for 30 min • Flash pasteurization (HTST): 72C for 15 sec. Most common method (milk pasteurization). Thermoduric(heat resistant) organisms survive • Ultra High Temperature (UHT):140C for 4 sec.(liquid is sprayed with high T –steam under pressure then rapidly cooled).

16. Dry heat sterilization • For sterilizing materials that must remain dry, circulating hot air electric ovens are available. because heat is less effective on dry material, we use to apply a temperature of 160–170°C for 2 h. • Flaming of loop • Incineration of carcasses (Anthrax, Foot and mouth disease, Bird flu) • Hot-air sterilization • Dry heat kill M.O. by oxidation

17. Filtration • Used for heat sensitive materials(vaccines, antibiotics) • Air filtration using high efficiency particulate air (HEPA) filters. Effective to 0.3 m • Membrane filters for fluids. • Pore size for bacteria: 0.22 – 0.45 m • Pore size for viruses: 0.01 m

19. Low Temperature • Refrigeration(0-7C) (reduce metabolic rate and prevent toxin production).Freezing forms ice crystals that damage microbial cells • Deep freezing(-50,-95C) bacteriostatic effect • Lyophilization( removing water at low T) • Three methods used for food and drugs) Various Other Methods • High pressure in liquids: denatures bacterial proteins and CH2O and inactivation of the cells, preserves flavor • Desiccation prevents metabolism • Osmotic pressure causes plasmolysis (hypertonic solution). Molds and yeast can grow in high osmotic pressure environment.

20. Radiation Ionizing radiation (X-rays, -rays, electron beams) • have short wavelength, penetrate deeply, ionize water forming OH¯ radicals and other highly reactive molecules • Salmonella and Pseudomonas are particularly sensitive • Used for plastic syringes, surgical gloves, suturing materials, and catheters. Non ionizing radiation ( UV light) Most effective wave legnth ~ 260 nm, Effect: thymine dimers (inhibit correct replication) Used to limit air and surface contamination. Use at close range to directly exposed microorganisms. e.g.: germicidal lamps in the lab used for sterilization of heat sensitive materials: drugs, vitamins, herbs, suture material

22. Chemical Methods of Microbial Control • Few chemical agents achieve sterility. • We should considered the followings: • The presence of organic matter • The degree of contact with microorganisms • The temperature

23. Types of Disinfectants • Phenol = carbolic acid (historic importance) • Phenolics: Cresols (Lysol) - disinfectant • Bisphenols • Hexachlorophene • Used in hospitals • Triclosan (toothpaste, antibacerial soaps, etc.) Fig 7.7 • Phenol and derivatives disrupt plasma membranes (lipids) and lipid rich cell walls • Remain active in presence of organic compounds

24. Halogens Chlorine • Oxidizing agent • Widely used as disinfectant • Forms bleach (hypochlorous acid) when added to water. • Broad spectrum, not sporicidal(pools, drinking water) Iodine More reactive, more germicidal. Alters protein synthesis and membranes. Tincture of iodine (solution with alcohol)  wound antiseptic Iodophorscombined with an organic molecule  iodine detergent complex. Occasional skin sensitivity, partially inactivated by organic debris, poor sporicidal activity.

25. Alcohols • Ethyl (60 – 80% solutions) and isopropyl alcohol • Denature proteins, dissolve lipids • No activity against spores and poorly effective against viruses and fungi • Easily inactivated by organic debris • Also used in hand sanitizers and cosmetics

26. Heavy Metals Oligodynamic action: Oligodynamic action: the ability of very small amounts of heavy metals to exert antimicrobial activity toxic effect due to metal ions combining with sulfhydryl (—SH) and other groups  proteins are denatured. • Mercury (HgCl2, used for skin lesions) • Copperagainst chlorophyll containing organisms  Algicides • Silver(AgNO3): Antiseptic for eyes of newborns • Zinc(ZnCl2) in mouthwashes

27. Surface Acting Ingredients / Surfactants • Soaps and Detergents • Major purpose of soap: Mechanical removal and use as wetting agent • Definition of detergents • Acidic-Anionic detergents Anion reacts with plasma membrane. Nontoxic, non-corrosive, and fast acting. Laundry soap, dairy industry. • Cationic detergents Quaternary ammonium compounds. Strongly bactericidal against a wide range, but esp. Gram+ bacteria

28. Aldehydes (alkylating agents) • Inactivate proteins by cross-linking with functional groups (–NH2, –OH, –COOH, –SH) • Glutaraldehyde: Sterilant for delicate surgical instruments Formaldehyde: Virus inactivation, use for vaccines

29. Plasma • The four state of matter in which a gas is excited by electromagnetic field to make a mixture of nuclei with assorted electrical charges and free electrons. • used for metal or plastic surgical instruments (Tubular instruments). • Free radicals in plasma gases are used to sterilize plastic instruments and destroy microbes Supercritical Fluids Supercritical fluids, which have properties of liquid and gas, can sterilize at low temperatures. Peroxygens and Other Forms of Oxygen • Hydrogen peroxide, peracetic acid, benzoyl peroxide, and ozone exert their antimicrobial effect by oxidizing molecules inside cells.

30. Hydrogen Peroxide: Oxidizing agent Inactivated by catalase Not good for open wounds Good for inanimate objects; packaging for food industry (containers etc.) 3% solution is used Effective against anaerobic bacteria

31. Microbial Characteristics and Microbial Control