Physical and Chemical Control of Microbes Chapter 9

Transcription

1 Physical and Chemical Control of Microbes Chapter 9 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2 Controlling Microorganisms Controlling our degree of exposure to potentially harmful microbes is a monumental concern in our lives The methods of microbial control used outside of the body are designed to result in four possible outcomes - sterilization - disinfection - decontamination (also called sanitization) - antisepsis

3 Concepts in Antimicrobial Control Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.1 Concepts in Antimicrobial Control Techniques and chemicals that are capable of sterilizing are highlighted with a pink background. Term Defnition Key Points Examples of Agents Sterilization Process that destroys or removes all viable microorganisms (including viruses) The term sterile should be used only in the strictest sense to refer to materials that have been subjected to the process of sterilization (there is no such thing as slightly sterile) Generally reserved for inanimate objects as it would be impractical or dangerous to sterilize parts of the human body Common uses: surgical instruments, syringes, commercially packaged food Heat (autoclave) Sterilants (chemical agents capable of destroying spores) Disinfection Physical process or a chemical agent to destroy vegetative pathogens but not bacterial endospores Removes harmful products of microorganisms (toxins) from material Normally used on inanimate objects because the concentration of disinfectants required to be effective is harmful to human tissue Common uses: boiling food utensils, applying 5% bleach solution to an examining table, immersing thermometers in an iodine solution between uses Bleach Iodine Heat (boiling) Decontamination/ Sanitization Cleansing technique that mechanically removes microorganisms as well as other debris to reduce contamination to safe levels Important to restaurants, dairies, breweries, and other commercial entities handle large numbers of soiled utensils/containers Common uses: cooking utensils, dishes, bottles, and cans must be sanitized for reuse Soaps Detergents Commercial dish washers Antisepsis/ Degermation Reduces the number of microbes on the human skin A form of decontamination but on living tissues Involves scrubbing the skin (mechanical friction) or immersing it in chemicals (or both) Alcohol Surgical hand scrubs

4 Relative Resistance of Different Microbial Types to Microbial Control Agents More resistant Prions Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Bacterial endospores Mycobacterium Staphylococcus and Pseudomonas Protozoan cysts Protozoan trophozoites Most gram-negative bacteria Fungi and fungal spores Nonenveloped viruses Most gram-positive bacteria Less resistant Enveloped viruses

5 Comparative Resistance of Bacterial Endospores to Control Agents

6 Differentiation: Agents vs. Processes (cont d) Antiseptics: chemical agents applied directly to exposed body surfaces (skin and mucous membranes), wounds, and surgical incisions to prevent vegetative pathogens - preparing the skin before surgical incisions with iodine compounds - swabbing an open root canal with hydrogen peroxide - ordinary hand washing with a germicidal soap

7 Differentiation: Agents vs. Processes (cont d) Stasis and static mean to stand still Bacteristatic: chemical agents that prevent the growth of bacteria on tissues or on objects in the environment Fungistatic: chemicals that inhibit fungal growth Antiseptics and drugs often have microbiostatic effects because microbicidal compounds can be toxic to human cells

8 What Is Microbial Death? Death: permanent termination of an organism s vital processes - microbes have no conspicuous vital processes, therefore death is difficult to determine - permanent loss of reproductive capability, even under optimum growth conditions has become the accepted microbiological definition of death

9 Factors Affecting Death Rate (cont d) The number of microbes - higher load of contaminants takes longer to destroy The nature of the microorganisms in the population - target population is usually a mixture of bacteria, fungi, spores, and viruses Temperature and ph of the environment The concentration (dose, intensity) of the agent - UV radiation is most effective at 260 nm - most disinfectants are more active at higher concentrations

10 Modes of Action of Antimicrobial Agents Antimicrobials have a range of cellular targets - least selective agents tend to be effective against the widest range of microbes (heat and radiation) - selective agents target only a single cellular component (drugs) Cellular targets of physical and chemical agents - cell wall - cell membrane - cellular synthetic processes - proteins

11 Actions of Various Physical and Chemical Agents Upon the Cell Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.3 Actions of Various Physical and Chemical Agents upon the Cell Cellular Target Cell Wall Effects of Agents Chemical agents can damage the cell wall by: Blocking its synthesis Digesting the cell wall Examples of Agents Used Chemicals Detergents Alcohol Cell Membrane Cellular Synthesis Proteins Agents physically bind to lipid layer of the cell membrane, opening up the cell membrane and allowing injurious chemicals to enter the cell and important ions to exit the cell. Agents can interrupt the synthesis Formaldehyde of proteins via the ribosomes, Radiation inhibiting proteins needed for growth Ethylene oxide and metabolism and preventing multiplication. Agents can change genetic codes (mutation). Some agents are capable of denaturing proteins (breaking of protein bonds, which results in breakdown of the protein structure). Agents may attach to the active site of a protein, preventing it from interacting with its chemical substrate. Detergents Moist heat Alcohol Phenolics

12 Mode of Action of Surfactants on the Cell Membrane Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Surfactant molecules Membrane phospholipids Cytoplasm

13 Methods of Physical Control: Heat Elevated temperatures are microbicidal Lower temperatures are microbiostatic Moist heat: hot water, boiling water, or steam - between 60 C and 135 C Dry heat: hot air or an open flame - ranges from 160 C to thousands of degrees Celsius

14 Comparison of Times and Temperatures to Achieve Sterilization with Moist and Dry Heat

15 Heat Resistance and Thermal Death: Spores and Vegetative Cells Bacterial endospores - exhibit greatest resistance - destruction of spores usually requires temperatures above boiling - resistance varies Vegetative cells - vary in sensitivity to heat - death times vary from 50 C for 3 minutes to 60 C for 60 minutes

16 Moist Heat Methods Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.5 Moist Heat Methods Techniques and chemicals that are capable of sterilizing are highlighted with a pink background. Method Applications Boiling Water: Disinfection A simple boiling water bath or chamber can quickly decontaminate items in the clinic and home. Because a single processing at 100 C will not kill all resistant cells, this method can be relied on only for disinfection and not for sterilization. Exposing materials to boiling water for 30 minutes will kill most nonspore-forming pathogens, including resistant species such as the tubercle bacillus and staphylococci. Probably the greatest disadvantage with this method is that the items can be easily recontaminated when removed from the water. Useful in the home for disinfection of water, materials for babies, food and utensils, bedding, and clothing from the sickroom Pasteurization: Disinfection of Beverages Fresh beverages such as milk, fruit juices, beer, and wine are easily contaminated during collection and processing. Because microbes have the potential for spoiling these foods or causing illness, heat is frequently used to reduce the microbial load and destroy pathogens. Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while at the same time retaining the liquid s flavor and food value. Milk, wine, beer, other beverages Ordinary pasteurization techniques require special heat exchangers that expose the liquid to 71.6 C for 15 seconds (flash method) or to 63 C to 66 C for 30 minutes (batch method). The first method is preferable because it is less likely to change flavor and nutrient content, and it is more effective against certain resistant pathogens such as Coxiella and Mycobacterium. Although these treatments inactivate most viruses and destroy the vegetative stages of 97% to 99% of bacteria and fungi, they do not kill endospores or particularly heat-resistant microbes (mostly nonpathogenic lactobacilli, micrococci, and yeasts). Milk is not sterile after regular pasteurization. In fact, it can contain 20,000 microbes per milliliter or more, which explains why even an unopened carton of milk will eventually spoil. (Newer techniques can also produce sterile milk that has a storage life of 3 months. This milk is processed with ultrahigh temperature [UHT] 134 C for 1 to 2 seconds.) This is not generally considered pasteurization, so we don t consider pasteurization a sterilization method. Monday Tuesday Wednesday Nonpressurized Steam Selected substances that cannot withstand the high temperature of the autoclave can be subjected to intermittent sterilization, also called tyndallization. This technique requires a chamber to hold the materials and a reservoir for boiling water. Items in the chamber are exposed to free-flowing steam for 30 to 60 minutes. This temperature is not sufficient to reliably kill spores, so a single exposure will not suffice. On the assumption that surviving spores will germinate into less resistant vegetative cells, the items are incubated at appropriate temperatures for 23 to 24 hours, and then again subjected to steam treatment. This cycle is repeated for 3 days in a row. Because the temperature never gets above 100 C, highly resistant spores that do not germinate may survive even after 3 days of this treatment. Heat-sensitive culture media, such as those containing sera, egg, or carbohydrates (which can break down at higher temperatures) and some canned foods. Probably not effective in sterilizing items such as instruments and dressings that provide no environment for spore germination, but it certainly can disinfect them. Thursday Friday Even though this is sometimes called intermittent sterilization, sterilization is not guaranteed so we don t consider it a reliable sterilization method. (pot): The McGraw-Hill companies, Inc./Charles D. Winters, photographer; (pasteurization): James King-Holmes/Photo Researchers; (beer): John A. Rizzo/Getty Images (RF);

17 Moist Heat Methods (cont d) Table 9.5 (continued) Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Method Steam Under Pressure: Sterilization At sea level, normal atmospheric pressure is 15 pounds per square inch (psi), or 1 atmosphere. At this pressure, water will boil (change from a liquid to a gas) at 100 C, and the resultant steam will remain at exactly that temperature, which is unfortunately too low to reliably kill all microbes. In order to raise the temperature of steam, the pressure at which it is generated must be increased. As the pressure is increased, the temperature at which water boils and the temperature of the steam produced both rise. For example, at a pressure of 20 psi (5 psi above normal), the temperature of steam is 109 C. As the pressure is increased to 10 psi above normal, the steam s temperature rises to 115 C, and at 15 psi above normal (a total of 2 atmospheres), it will be 121 C. It is not the pressure by itself that is killing microbes but the increased temperature it produces. Applications Heat-resistant materials such as glassware, cloth (surgical dressings), metallic instruments, liquids, paper, some media, and some heatresistant plastics. If items are heat-sensitive (plastic Petri dishes) but will be discarded, the autoclave is still a good choice. However, it is ineffective for sterilizing substances that repel moisture (oils, waxes), or for those that are harmed by it (powders). Such pressure-temperature combinations can be achieved only with a special device that can subject pure steam to pressures greater than 1 atmosphere. Health and commercial industries use an autoclave for this purpose, and a comparable home appliance is the pressure cooker. The most efficient pressure-temperature combination for achieving sterilization is 15 psi, which yields 121 C. It is important to avoid overpacking or haphazardly loading the chamber, which prevents steam from circulating freely around the contents and impedes the full contact that is necessary. The duration of the process is adjusted according to the bulkiness of the items in the load (thick bundles of material or large flasks of liquid) and how full the chamber is. The range of holding times varies from 10 minutes for light loads to 40 minutes for heavy or bulky ones; the average time is 20 minutes. Pressure regulator Recorder Control handle Safety valve Exhaust to atmosphere Steam from jacket to chamber or exhaust from chamber Steam to jacket Steam from jacket to chamber Door gasket Strainer Discharge Steam supply valve Temperaturesensing bulb Steam trap Jacket condensate return Condensate to waste Steam jacket Steam supply Trap (autoclave): Science VU/Visuals Unlimited

18 Dry Heat Methods Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.6 Dry Heat Methods Techniques and chemicals that are capable of sterilizing are highlighted with a pink background. Method Applications Incineration in a flame is perhaps the most rigorous of all heat treatments. The flame of a Bunsen burner reaches 1,870 C at its hottest point, and furnaces/incinerators operate at temperatures of 800 C to 6,500 C. Direct exposure to such intense heat ignites and reduces microbes and other substances to ashes and gas. Bunsen burners/small incinerators: laboratory instruments such as inoculating loops. Large incinerators: syringes, needles, culture materials, dressings, bandages, bedding, animal carcasses, and pathology samples. Incineration of microbial samples on inoculating loops and needles using a Bunsen burner is a very common practice in the microbiology laboratory. This method is fast and effective, but it is also limited to metals and heatresistant glass materials. This method also presents hazards to the operator (an open flame) and to the environment (contaminants on needle or loop often spatter when placed in flame). Tabletop infrared incinerators have replaced Bunsen burners in many labs for these reasons. Large incinerators are regularly employed in hospitals and research labs for complete destruction of infectious materials. The hot-air oven provides another means of dry-heat sterilization. The so-called dry oven is usually electric (occasionally gas) and has coils that radiate heat within an enclosed compartment. Heated, circulated air transfers its heat to the materials in the oven. Sterilization requires exposure to 150 C to 180 C for 2 to 4 hours, which ensures thorough heating of the objects and destruction of endospores. Glassware, metallic instruments, powders, and oils that steam does not penetrate well. Not Suitable for plastics, cotton, and paper, which may burn at the high temperatures, or for liquids, which Will evaporate. (top): Raymond B. Otero/Visuals Unlimited; (bottom): Steve Allen/Brand X Pictures (RF)

19 The Effects of Cold and Desiccation Principal benefit of cold treatment is to slow growth of cultures and microbes in food during processing and storage Cold merely retards the activities of most microbes Most microbes are not adversely affected by gradual cooling, long-term refrigeration, or deep-freezing Temperatures from -70 C to -135 C can preserve cultures of bacteria, viruses, and fungi for long periods

20 The Effects of Cold and Desiccation (cont d) Psychrophiles grow slowly at freezing temperatures and can secrete toxic products Pathogens able to survive several months in the refrigerator - Staphylococcus aureus - Clostridium species - Streptococcus species - Salmonella - yeasts, molds, and viruses

21 The Effects of Cold and Desiccation (cont d) Desiccation: vegetative cells directly exposed to normal room temperature gradually become dehydrated - Streptococcus pneumoniae, the spirochete of syphilis, and Neisseria gonorrhoeae die after a few hours of air drying - endospores of Bacillus and Clostridium are viable for thousands of years under extremely arid conditions - staphylococci, streptococci, and the tubercle bacillus surrounded by sputum remain viable in air and dust - many viruses and fungi can also withstand long periods of desiccation

22 The Effects of Cold and Desiccation (cont d) Lyophilization - combination of freezing and drying - method of preserving microorganisms in a viable state for many years - pure cultures are frozen instantaneously and exposed to a vacuum that removes water, avoiding the formation of ice crystals

23 Radiation Energy emitted from atomic activities and dispersed at high velocity through matter or space - gamma rays - X rays - ultraviolet radiation

24 Radiation Methods Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.7 Radiation Methods Techniques and chemicals that are capable of sterilizing are highlighted with a pink background. Method Applications Ionizing Radiation: Gamma Rays and X Rays Ionizing radiation is a highly effective alternative for sterilizing materials that are sensitive to heat or chemicals. Devices that emit ionizing rays include gamma-ray machines containing radioactive cobalt, X-ray machines similar to those used in medical diagnosis, and cathode-ray machines. Items are placed in these machines and irradiated for a short time with a carefully chosen dosage. The dosage of radiation is measured in Grays (which has replaced the older term, rads). Depending on the application, exposure ranges from 5 to 50 kilograys (kgray; a kilogray is equal to 1,000 Grays). Although all ionizing radiation can penetrate liquids and most solid materials, gamma rays are most penetrating, X rays are intermediate, and cathode rays are least penetrating. Drugs, vaccines, medical instruments (especially plastics), syringes, surgical gloves, tissues such as bone and skin, and heart valves for grafting. After the anthrax attacks of 2001, mail delivered to certain Washington, D.C., zip codes was irradiated with ionizing radiation. Its main advantages include speed, high penetrating power (it can sterilize materials through outer packages and wrappings), and the absence of heat. Its main disadvantages are potential dangers to radiation machine operators from exposure to radiation and possible damage to some materials. Foods have been subject to irradiation in limited circumstances for more than 50 years. From flour to pork and ground beef, to fruits and vegetables, radiation is used to kill not only bacterial pathogens but also insects and worms and even to inhibit the sprouting of white potatoes. Irradiated food has been extensively studied, and found to be safe and nonradioactive. Irradiation may lead to a small decrease in the amount of thiamine (vitamin B1) in food, but this change is small enough to be inconsequential. The irradiation process does produce short-lived free radical oxidants, which disappear almost immediately (this same type of chemical intermediate is produced through cooking as well). Certain foods do not irradiate well and are not good candidates for this type of antimicrobial control. The white of eggs becomes milky and liquid, grapefruit gets mushy, and alfalfa seeds do not germinate properly. Lastly, it is important to remember that food is not made radioactive by the irradiation process, and many studies, in both animals and humans, have concluded that there are no ill effects from eating irradiated food. In fact, NASA relies on irradiated meat for its astronauts. Nonionizing Radiation: Ultraviolet Rays Ultraviolet (UV) radiation ranges in wavelength from approximately 100 to 400 nm. It is most lethal from 240 to 280 nm (with a peak at 260 nm). Owing to its lower energy state, UV radiation is not as penetrating as ionizing radiation. Because UV radiation passes readily through air, slightly through liquids, and only poorly through solids, the object to be disinfected must be directly exposed to it for full effect. Ultraviolet rays are a powerful tool for destroying fungal cells and spores, bacterial vegetative cells, protozoa, and viruses. Bacterial spores are about 10 times more resistant to radiation than are vegetative cells, but they can be killed by increasing the time of exposure. Even though it is possible to sterilize with UV, it is so technically challenging that we don t regularly call it a sterilizing technology. Usually directed at disinfection rather than sterilization. Germicidal lamps can cut down on the concentration of airborne microbes as much as 99%. They are used in hospital rooms, operating rooms, schools, food preparation areas, and dental offices. Ultraviolet disinfection of air has proved effective in reducing postoperative infections, preventing the transmission of infections by respiratory droplets, and curtailing the growth of microbes in foodprocessing plants and slaughterhouses. Ultraviolet irradiation of liquids requires special equipment to spread the liquid into a thin, flowing film that is exposed directly to a lamp. This method can be used to treat drinking water and to purify other liquids (milk and fruit juices) as an alternative to heat. The photo shows a UV treatment system for the disinfection of water. (top): Adam Hart-Davis/Photo Researchers; (bottom): Tom Pantages

25 Formation of Pyrimidine Dimers by the Action of Ultraviolet (UV) Radiation Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Normal Segment of DNA A C A A C T G T T G Thymine Dimer UV A C A A C T G T T G O CH 3 CH 3 O O T T Details of bonding O

26 Other Physical Methods: Filtration Effective method to remove microbes from air and liquids - fluid is strained through a filter with openings large enough for the fluid to pass, too small for microbes - thin membranes of cellulose acetate, polycarbonate, and plastics whose pore size is carefully controlled - charcoal, diatomaceous earth, or unglazed porcelain are also used - pore sizes can be controlled to permit true sterilization by trapping viruses or large proteins

27 Membrane Filtration Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Liquid Filter Pore Filter Sterilized fluid (b) (a) Vacuum pump suction Fred Hossler/Visuals Unlimited

28 Osmotic Pressure Adding large amounts of salt or sugar to foods creates a hypertonic environment for bacteria, causing plasmolysis Pickling, smoking, and drying foods have been used for centuries to preserve foods Osmotic pressure is never a sterilizing technique

29 Selecting a Microbicidal Chemical Rapid action even in low concentrations Solubility in water or alcohol and long-term stability Broad-spectrum action without being toxic to human and animal tissues Penetration of inanimate surfaces to sustain a cumulative or persistent action Resistance to becoming inactivated by organic matter

30 Required Concentrations and Times for Chemical Destruction of Selected Microbes

31 Germicidal Categories According to Chemical Group Sterilizing Agents Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 9.9 Germicidal Categories According to Chemical Group Techniques and chemicals that are capable of sterilizing are highlighted with a pink background. Agent Halogens: chlorine Target Microbes Can kill spores (slowly); all other microbes Form(s) Liquid/gaseous chlorine (Cl 2 ), hypochlorites (OCl), chloramines (NH 2 Cl) Mode of Action In solution, these compounds combine with water and release hypochlorous acid (HOCl); denature enzymes permanently and suspend metabolic reactions Indications for Use Limitations Chlorine kills bacteria, Less effective if exposed to endospores, fungi, and viruses; light, alkaline ph and gaseous/ liquid chlorine: used excess organic matter todisinfect drinking water, sewage and waste water; hypochlorites: used in health care to treat wounds, disinfect bedding and instruments, sanitize food equipment and in restaurants, pools and spas; chloramines: alternative to pure chlorine in treating drinking water; also used to treat wounds and skin surfaces Halogens: iodine Can kill spores (slowly); all other microbes Free iodine in solution (I 2 ) Iodophors (complexes of iodine and alcohol) Penetrates cells of microorganisms where it interferes with a variety of metabolic functions; interferes with the hydrogen and disulfide bonding of proteins 2% iodine, 2.4% sodium iodide (aqueous iodine) is used as a topical antiseptic 5% iodine, 10% potassium iodide used as a disinfectant for plastic and rubber instruments, cutting blades, etc. Iodophor products contain 2% to 10% of available iodine, which is released slowly; used to prepare skin for surgery, in surgical scrubs, to treat burns, and as a disinfectant Can be extremely irritating to the skin and is toxic when absorbed Hydrogen peroxide (H 2 O 2 ) Kills spores and all other microbes Colorless, caustic liquid Decomposes in the presence of light metals or catalase into water, and oxygen gas Oxygen forms free radicals ( OH), which are highly toxic and reactive to cells As an antiseptic, 3% hydrogen Sporicidal only in high peroxide is used for skin and concentrations wound cleansing, mouth washing, bedsore care Used to treat infections caused by anaerobic bacteria 35% hydrogen peroxide is used in low temperature sterilizing cabinets for delicate instruments Aldehydes Kill spores and all other microbes Organic substances bearing a CHO functional group on the terminal carbon Glutaraldehyde can irreversibly disrupt the activity of enzymes and other proteins within the cell Formaldehyde is a sharp irritating gas that readily dissolves in water to form an aqueous solution called formalin; attaches to nucleic acids and functional groups of amino acids Glutaraldehyde kills rapidly and is broad-spectrum; used to sterilize respiratory equipment, scopes, kidney dialysis machines, dental instruments Formaldehyde kills more slowly than glutaraldehyde; used to disinfect surgical instruments Glutaraldehyde is somewhat unstable, especially with increased ph and temperature Formaldehyde is extremely toxic and is irritating to skin and mucous membranes Gaseous sterilants/ disinfectants Ethylene oxide kills spores; other gases less effective Ethylene oxide is a colorless substance that exists as a gas at room temperature Ethylene oxide reacts vigorously with functional groups of DNA and proteins, blocking both DNA replication and enzymatic actions Chlorine dioxide is a strong alkylating agent Ethylene oxide is used to disinfect plastic materials and delicate instruments; can also be used to sterilize syringes, surgical supplies, and medical devices that are prepackaged Ethylene oxide is explosive it must be combined with a high percentage of carbon dioxide or fluorocarbon It can damage lungs, eyes, and mucous membranes if contacted directly Ethylene oxide is rated as a carcinogen by the government (top): Richard Hutchings (RF); (Bottom): The McGraw-Hill Companies, Inc./Jill Braaten, photographer

32 Germicidal Categories According to Chemical Group Disinfection Only Table 9.9 (continued) Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Agent Target Microbes Form(s) Mode of Action Indications for Use Limitations Phenol (carbolic acid) Some bacteria, viruses, fungi Derived from the distillation of coal tar Phenols consist of one or more aromatic carbon rings with added functional groups In high concentrations they are cellular poisons, disrupting cell walls and membranes, proteins In lower concentrations they inactivate certain critical enzyme systems Phenol remains one standard against which other (less toxic) phenolic disinfectants are rated; the phenol coefficient quantitatively compares a chemical s antimicrobial properties to those of phenol Phenol is now used only in certain limited cases, such as in drains, cesspools, and animal quarters Toxicity of many phenolics makes them dangerous to use as antiseptics Chlorhexidine Most bacteria, viruses, fungi Complex organic base containing chlorine and two phenolic rings Targets both bacterial membranes, where selective permeability is lost, and proteins, resulting in denaturation Mildness, low toxicity and rapid action make chlorhexidine a popular choice of agents Used in hand scrubs, prepping skin for surgery, as an obstetric antiseptic, as a mucous membrane irrigant, etc. Effects on viruses and fungi are variable Alcohol Most bacteria, viruses, fungi Colorless hydrocarbons with one or more OH functional groups Ethyl and isopropyl alcohol are suitable for antimicrobial control Concentrations of 50% and higher dissolve membrane lipids, disrupt cell surface tension and compromise membrane integrity Germicidal, nonirritating, and inexpensive Routinely used as skin degerming agents (70% to 95% solutions) Rate of evaporation decreases effectiveness Inhalation of vapors can affect the nervous system Detergents Some bacteria, viruses, fungi Polar molecules that act as surfactants Anionic detergents have limited microbial power Cationic detergents, such as quaternary ammonium compounds ( quats ), are much more effective antimicrobials Positively charged end of the molecule binds well with the predominantly negatively charged bacterial surface proteins Long, uncharged hydrocarbon chain allows the detergent to disrupt the cell membrane Cell membrane loses selective permeability, causing cell death Effective against viruses, algae, fungi, and gram-positive bacteria Rated only for low-level disinfection in the clinical setting Used to clean restaurant utensils, dairy equipment, equipment surfaces, restrooms Ineffective against tuberculosis bacterium, hepatitis virus, Pseudomonas, and spores Activity is greatly reduced in presence of organic matter Detergents function best in alkaline solutions Heavy metal compounds Some bacteria, viruses, fungi Heavy metal germicides contain either an inorganic or an organic metallic salt; may come in tinctures, soaps, ointment, or aqueous solution Mercury, silver, and other metals exert microbial effects by binding onto functional groups of proteins and inactivating them Organic mercury tinctures are fairly effective antiseptics Organic mercurials serve as preservatives in cosmetics, ophthalmic solutions, and other substances Silver nitrate solutions are used for topical germicides and ointments Microbes can develop resistance to metals Not effective against endospores Can be toxic if inhaled, ingested, or absorbed May cause allergic reactions in susceptible individuals (alcohol): Richard Hutchings (RF); (heavy metal): The McGraw-Hill Companies, Inc./Stephen Frisch, photographer

33 The Structure of Detergents Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Charged Head R 1 + Uncharged hydrocarbon chain (C number from 8 to 18) R 2 N R 4 (a) R 3 + CH 3 C N H 2N + N + CH Cl 2 CH 3 (b) Benzalkonium chloride

34 Active Ingredients of Various Commercial Antimicrobial Products

35 Antimicrobial Treatment Chapter 10 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

36 Principles of Antimicrobial Therapy The introduction of modern drugs to control infections was a medical revolution in the 1930s Antimicrobial drugs have reduced the incidence of certain infections, but they have not eradicated infectious disease and probably never will Today, doctors are worried that we are dangerously close to a postantibiotic era where the drugs we have are no longer effective

37 Characteristics of the Ideal Antimicrobial Drug

38 The Origins of Antimicrobial Drugs Antibiotics are common metabolic products of aerobic bacteria and fungi - produced to inhibit the growth of competing microbes in the same habitat Derived from - bacteria in the genera Streptomyces and Bacillus - molds in the genera Penicillium and Cephalosporium

39 The Origins of Antimicrobial Drugs (cont d) Before actual antimicrobial therapy can begin, three factors must be known - the nature of the microorganism causing the infection - the degree of the microorganism s susceptibility (or sensitivity) to various drugs - the overall medical condition of the patient

40 Identifying the Agent Specimens from the patient must be taken before any antimicrobial drug is given - body fluids, sputum, stool Doctors often begin therapy on the basis of initial detection methods, or on the basis of an informed guess - if a sore throat appears to be caused by Streptococcus pyogenes, penicillin will be prescribed - Streptococcus pneumoniae accounts for the majority of cases of meningitis in children, followed by Neisseria meningitidis

41 Testing for Drug Susceptibility Necessary for bacteria commonly showing resistance Staphylococcus species, Neisseria gonorrhoeae, Streptococcus pneumoniae, Enterococcus faecalis, and aerobic gram-negative enteric bacilli Difficult and unnecessary for fungal or protozoan infections Not necessary if the patient is allergic to certain antibiotics

42 Testing for Drug Susceptibility (cont d) Kirby-Bauer technique -surface of an agar plate is spread with bacteria -small discs containing a prepared amount of antibiotic are placed on the plate -zone of inhibition surrounding the discs is measured and compared with a standard for each drug -antibiogram provides data for drug selection -this method is less effective for anaerobic, fastidious, or slow-growing bacteria

43 Technique for Preparation and Interpretation of Disc Diffusion Tests Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. = Zone of Inhibition (agar is uncolonized) Enrofloxacin 5 g (R < 17 mm; S 22 mm) = Region of bacterial growth ENR 5 = Antibiotic carrier (disc) imprinted with abbreviation and concentration R = resistant, I = intermediate, S = sensitive 0 mm ENR S Oxytetracycline 30 g (R < 17 mm; S 22 mm) OT 30 R I GN 10 Gentamicin 10 g (R < 17 mm; S 21 mm) (b) Cefotaxime 30 g (R < 14 mm; S 23 mm) CTX 30 S R C 30 I AMP 10 Ampicillin 10 g (R < 14 mm; S 22 mm) (a) *R and S values differ from table 12.7 due to differing concentrations of the antimicrobials. Chloramphenicol 30 g (R < 21 mm; S 21 mm) b: Kathy Park Talaro

44 Tube Dilution Test for Determining MIC Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Same inoculum size of test bacteria added Control 0 Negative control µg/ml 6.4 Increasing concentration of drug 12.8 Growth No growth (a) (b) (b): Courtesy of David Ellis

45 Mechanisms of Drug Action Goals of chemotherapy: identifying structural and metabolic needs of a living cell and removing, disrupting, or interfering with these requirements Antimicrobial drug categories - inhibition of cell wall synthesis - inhibition of nucleic acid structure and function - inhibition of protein synthesis - interference with cell membrane structure and function - inhibition of folic acid synthesis

46 Primary Sites of Action of Antimicrobial Drugs on Bacterial Cells Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Protein Synthesis Inhibitors Acting on Ribosomes Site of action: 50S subunit Erythromycin Clindamycin Synercid Pleuromutilins Site of action: 30S subunit Aminoglycosides Gentamicin Streptomycin Tetracyclines Glycylcyclines Both 30S and 50S Blocks initiation of protein synthesis Linezolid Substrate Enzyme Product Cell Membrane Cell Wall Inhibitors Block synthesis and repair Penicillins Cephalosporins Carbapenems Vancomycin Bacitracin Fosfomycin Isoniazid Cause loss of selective permeability Polymyxins Daptomycin DNA DNA/RNA Folic Acid Synthesis in the Cytoplasm Block pathways and inhibit metabolism Sulfonamides (sulfa drugs) Trimethoprim mrna Inhibit replication and transcription Inhibit gyrase (unwinding enzyme) Quinolones Inhibit RNA polymerase Rifampin

47 Specific Drugs and Their Metabolic Targets

48 Specific Drugs and Their Metabolic Targets

49 Spectrum of Activity for Antibiotics

50 Characteristics of Selected Penicillin Drugs Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 10.6 Characteristics of Selected Penicillin Drugs Name Spectrum of Action Uses, Advantages Disadvantages Penicillin G H 2 Beta-lactam ring S Narrow Best drug of choice when bacteria are sensitive; low cost; low toxicity Can be hydrolyzed by penicillinase; allergies occur; requires injection CH 2 CO N CH 3 CH 3 O N COOH Penicillin V Narrow Good absorption from intestine; otherwise, similar to penicillin G Hydrolysis by penicillinase; allergies Oxacillin, cloxacillin Cl CO N S CH 3 CH 3 Not susceptible to penicillinase; good absorption Allergies; expensive N O CH 3 O N COOH Methicillin, nafcillin Narrow Not usually susceptible to penicillinase Poor absorption; allergies; growing resistance S CO N CH 3 CH 3 O N COOH Ampicillin Broad Works on gram-negative bacilli Can be hydrolyzed by penicillinase; allergies; only fair absorption Amoxicillin Broad Gram-negative infections; good absorption Hydrolysis by penicillinase; allergies Carbenicillin S Broad Same as ampicillin Poor absorption; used only parenterally CH CO COONa N CH 3 CH 3 O N COOH Azlocillin, mezlocillin, ticarcillin CH CO N COONa S CH 3 CH 3 Very broad Effective against Pseudomonas species; low toxicity compared with aminoglycosides Allergies; susceptible to many betalactamases S O N COOH

51 How Does Drug Resistance Develop? Resistance to penicillin developed in some bacteria as early as 1940 In the 1980s and 1990s scientists began to observe treatment failures on a large scale Microbes become newly resistant to a drug after one of the following occurs - spontaneous mutations in critical chromosomal genes - acquisition of entire new genes or sets of genes via horizontal transfer from another species

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53 Mechanisms of Drug Resistance Table 10.9 Mechanisms of Drug Resistance Mechanism Example Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. New enzymes are synthesized, inactivating the drug (occurs when new genes are acquired). Bacterial exoenzymes called beta-lactamases R hydrolyze the betalactam ring structure of some penicillins and cephalosporins, rendering the drugs inactive. O N S COOH Penicillinase R O C OH N H S CH 3 CH 3 COOH Active penicillin Inactive penicillin Permeability or uptake of the drug into the bacterium is decreased (occurs via mutation). Drug Cell surface of microbe Normal receptor Cell surface of microbe Differently-shaped receptor Drug is immediately eliminated (occurs through the acquisition of new genes). Many bacteria possess multidrug-resistant (MDR) Drug pumps that actively transport drugs out of cells, conferring drug resistance on many gram-positive and gram-negative pathogens. Cell surface of microbe Cell surface of microbe New active drug pump Binding sites for drugs are decreased in number and/or affinity (occurs via mutation or through the acquisition of new genes). Erythromycin and clindamycin resistance is associated with an alteration on the 50S ribosomal binding site. An affected metabolic pathway is shut down, or an alternative pathway is used (occurs via mutation of original enzymes). Sulfonamide and trimethoprim resistance develop when microbes deviate from the usual patterns of folic acid synthesis. Drug acts A B C D Product C 1 D 1

54 New Approaches to Antimicrobial Therapy (cont d) Mimicking defense peptide molecules - peptides of amino acids secreted as part of the mammalian innate immune system called defensin, magainins, and protegrins - bacteria also produce defense peptides called bacteriocins and lantibiotics - insert into membranes and target other structures in cells - may be more effective than narrowly targeted drugs and less likely to foster resistance

55 New Approaches to Antimicrobial Therapy (cont d) Using bacteriophages - Eastern European countries use mixtures of bacteriophages as medicine, but these drugs have never been approved for use in the West - Biophage-PA used to treat ear infections caused by Pseudomonas aeruginosa biofilms - other researchers are incorporating bacteriophages into wound dressings - advantage to bacteriophage is their narrow specificity; only infect one species of bacterium

56 Major Adverse Toxic Reactions to Common Drug Groups