CHPT 9

How do antimicrobial agents act against cells walls, cytoplasmic membranes, proteins and nucleic acids?

1. Alteration of cell walls and membranes Since the cell wall maintains the integrity of the cell, if it bursts due to osmotic effect, or is dissolved (thin membrane, Gram +), then the cell dies. Furthermore, damage to the cell membrane could lead to the leakage of cellular content. Damage to the envelope of a virus could fatally interrupt viral replication as the virus requires the envelope (composed of proteins and phospholipids) to attach to its target cell. 2. Damage of proteins and nucleic acids Heat, chemicals, or radiation can denature the 3D shape of proteins within a cell and cause fatal consequences. Remember that proteins within a cell include: DNA, enzymes such as ribozyme. Hence destruction of these proteins halt protein synthesis through action on RNA.

Suppose a scientist treats a broth containing 1 billion micobes (10^9) with an agent that kills 90% of them in 1 minute. 1. Which type of microbes would die within the first minute first? A. Hardier B. More Susceptible 2. Moreover, upon 2 minutes, how many cells will remain in the broth?

1. B. The most susceptible cells die first, then the hardier. 2. After 1 minute, 100 million will exist (10^8). After a 2nd minute, (10^6) will remain. Microbial death rate is usually constant for a particular agent acting against a single microbe, death rates do vary - sometimes dramatically - among microorganisms and viruses.

List the 5 Physical Methods of Microbial Control?

1. Exposure of microbes to extreme heat or cold 2. Desiccation 3. Filtration 4. Osmotic Pressure 5. Radiation

List practical conditions of antimicrobial agents.

1. Inexpensive 2. Fast- acting 3. Stable during storage 4. Capable of controlling microbial growth while being harmless to humans, animals, and objects.

Complications of development of resistant microbes

1. Little evidence that products containing antiseptic and disinfecting chemicals add to human or animal health 2. Use of such products promotes development of resistant microbes

What are the 4 methods used to measure the effectiveness of disinfectants and antiseptics?

1. Phenol Coefficient 2. Use-dilution Test 3. Kelsey-Sykes Capacity Test 4. In-Use test

Often, scientists and medical personnel select a method to kill the hardiest microorganisms present, assuming that such a treatment will kill more fragile microbes as well. What are some of the most resistant to most susceptible microbes?

1. Prions - The most resistant. They cause degenerative diseases of the brain. 2. Bacterial endospores - Bacillus and Clostridium. 3. Mycobacteria - Myobacterium tuuberculosis. They contain a large waxy lipid that prevents the cell from drying and protect against water based chemicals. Cysts of protozoa - A protozoans cyst's wall prevents entry of most disinfectants, protects from drying, and shields against radiation. Active-stage protozoa (trophozoites) Most Gram-negative bacteria Fungi Non-enveloped viruses Most gram-positive bacteria Enveloped viruses (keys)

In general, list the factors affecting the Efficacy if Antimicrobial control.

1. Site to be treated 2. Relative susceptibility of Microorgansims. 3. Environmental Conditions.

The effectiveness of a particular antimicrobial agent is regulated by time &

1. The number of microbes-more microbes require more time 2. The nature of the microbes present 3. The temperature and pH of the environment 4. Concentration (dosage ,intensity) of the agent 5. The mode of action of the agent (how does it kill the microbe?) 6. The presence of solvents, interfering organic matter and inhibitors. Saliva, blood and feces can inhibit the actions of disinfectants and even heat

What are the levels of germicide classification?

A high level germicide will kill all pathogens, including endospores. Used to clean catheters, implants, and parts of heart-lung machines. Intermediate-level germicide: Kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria, but not bacterial endospores. Used to disinfect instruments that come in contact with mucous membranes but are noninvasive, such as respiratory equipment and endoscopes. Low- level germicides: kill vegetative bacteria, fungi, protozoa, and some viruses. Used only to disinfect items that contact only the skin of patients, such as furniture and electrodes.

Antisepsis

A process by which a chemical is used on skin or other tissue. The chemical is called an antiseptic

3. Kelsey-Sykes Capacity Test

Alternative assessment approved by the European Union Bacterial suspensions added to the chemical being tested Samples removed at predetermined times and incubated Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective

What are some chemical controls?

Antimicrobial agents 1. chemotherapeutic agents: used to treat disease 2. germicides and germistats: disinfectants and antiseptics 3. 1200 germicides - sold by 330 manufacturers in U.S. Selection: -damage the tissue or object -control the target microorganism(s) -purpose of treatment

What is a technique in evaluating the efficacy of an antimicrobial agent?

Calculate the Microbial Death Rate. This is usually found to be constant over time for any particular microorganism under a particular set of conditions. Graph: negative slope: y = -x + 10^9 Constant percentage of the extant population is killed each minute.

What is decimal reduction time?

Cell death occurs logarithmically. When measuring the effectiveness of heat sterilization, researchers calculate the decimal reduction time (D), which is the time required to destroy 90% of microbes in a sample. In reducing the number of endospores produced by C botulinum, researchers calculate the time to reduce the number of endospores from 10^12 to 10^0. The D-value for C. botulinum endospores at 121 C is 0.204/minute. Therefore it takes 2.5 minutes (0.204 x 12) to reduce 10^12 to 1 endospore.

Germicide

Chemical agents that destroy pathogenic microorganisms in general. Can be used on inanimate (nonliving) materials or living tissues and usually does NOT kill resistant microbes. Any physical or chemical agent that kills germs has gemicide properties.

Degermation

Cleansing technique that removes micro organisms and debris from living tissue. Surgical handscrub, alcohol wipes.

Disinfectants

Destroys most microorganisms and viruses on nonliving tissue. Unlike sterilization, disinfection does not guarantee that all pathogens are eliminated. For example disinfectants cannot inhibit endospores and some viruses. Terms is used ONLY to treat inanimate objects. Reduces the number of pathogens -pose no danger of disease Phenolics; alcohols; aldehydes; soaps

Sterilization

Destruction of all microorganisms and viruses in or on an object; the term is not usually applied to the destruction of prions. Iodine/Alcohol

An aseptic

Environment or procedure free of pathogenic contaminants Preparation of a surgical field; hand washing; flame sterilization of laboratory equipment.

Why are hyperthermophilic microorganisms viable but harmless in canned foods?

Hyperthermophilic microorganisms cannot grow at room temperature, which is the temperature of canned food. SO im assuming they cannot grow to the extent of being harmful or they cannot conduct binary fission.

Disinfection

Refers to the use of physical or chemical agents known as disinfectants. Disinfectants destroy vegetative pathogens but not bacterial endospores. Used only on inanimate objects because it is toxic to living tissues. ex. Bleaching or boiling.

Degerming

Removal of microbes by mechanical means, scrubbing. The actual action of scrubbing may be more than the chemical in removing microbes. Hand washing; alcohol swabbing at site of injection.

Sanitization

Removal or disinfection of pathogens from objects (places or utensils)to reduce the number of pathogenic microbes to meet public health standards Washing tableware in scaling water in restaurants.

Decontamination

Renders (makes) contaminated surfaces safe to handle

Which two types of microbial control are used to reduce the number of microbes?

Sanitization Degermation

What are the three major types of microbial control?

Sterilization (elimination of all microbes) Antisepsis Disinfection All of which denote the destruction of vegetative (nonspore) cells and many viruses. Modification of disinfection are: degerming, sanitization, and pasteurization.

-cide -cidal

Suffixes indicating destruction of a type of microbe. Refers to agents that destroy or permanently inactivate a particular type of microbe. Bactericide; fungicide; germicide; virucide Example: a virucide in activated viruses. A bactericide kills bacteria, fungicides kill fungal hyphae, spores and yeasts.

-stasis -static

Suffixes indication inhibition but not complete destruction of a type of microbe. Indicates that a chemical or physical agent that inhibits microbial metabolism and growth but does not necessarily kill microbes. Bacteriostatic; fungistatic; virustatic Example: refrigeration is bacteriostatic for most bacterial species. It inhibits their growth, but they can resume metabolism when the optimal temperature is restored.

4. In-Use test

Swabs taken from objects before and after application of disinfectant or antiseptic Swabs inoculated into growth medium and incubated Medium monitored for growth Accurate determination of proper strength and application procedure for each specific situation

What are the four Biosafety levels in labs dealing with pathogens?

The CDC has established guidelines for four levels of safety in microbiological laboratories dealing with pathogens. Each level raises personnel and environmental safety by specifying increasingly strict laboratory techniques, use of safety equipment, and design of facilities. 1. Biosafety Level 1 (BSL-1) Handling pathogens that do not cause disease in healthy humans. This includes handwashing with antimicrobial soap and washing surfaces with disinfectants. 2. Biosafety Level 2 (BSL-2) Handling moderately hazardous agents 3. Biosafety Level 3 (BSL-3) Handling microbes in safety cabinets. Hence laboratories are especially designed with double sets of doors and ventilation. Air leaving the room is HEPA-filtered before being discharged outside the room. 4. Biosafety Level 4 (BSL-4) Handling dangerous or exotic microbes that cause severe or fatal disease. This includes ebola, smallpox, and lassa fever virus. Researchers wear "space suits" supplied with air hoes. Suits and the laboratory itself are pressurized such that microbes are swept away from workers.

Microbial Death

The permanent loss of reproductive ability, even under optimum growth/ environmental conditions.

Heat is one of the older and more common means of microbial control. Heat can be used for sterilization, in which case all cells and viruses are deactivated, or for commercial preparation of canned goods. What is the difference between thermal death point and thermal death time?

Thermal death point: Lowest temperature that kills all cells in broth in 10 min Thermal death time: Time to completely sterilize a particular volume of "liquid" at a set temperature. Hence it is the minimal time. Effects of high temperatures: 1. Denature proteins 2. Interfere with integrity of cytoplasmic membrane and cell wall 3. Disrupt structure and function of nucleic acids

Pasteurization

Use of heat to destroy pathogens and reduce the number of spoilage microorganisms in foods and beverages. Pasteurized milks, fruit juices, wine and/or beer, etc.

1. Phenol Coefficient

Uses a phenol coefficient. Evaluates efficacy of disinfectants and antiseptics Compares an agent's ability to control microbes to phenol Greater than 1.0 indicates agent is more effective than phenol Has been replaced by newer methods

What factors affect the efficacy of antimicrobial methods?

In each situation, microbioligsts, laboratory personnel, and medical staff must consider at least three factors: the nature of the sites to be treated, the degree of susceptibility of the microbes involved, and the environmental conditions. 1. Caution to the site treated. Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects. The method of microbial control is based on the site of medical procedure. Hence, depending on the conditions, certain procedures may or may not be used to ensure the safety of the site treated. 2. Relative susceptibility of Microorganisms a. Nonenveloped? Endospore?Waxy lipid? Type of cell wall? b. Germicide classification - see the flashcard with the list of the most susceptible microbes and separate care for germicide classification 3. Environmental Conditions Temperature and pH affect microbial death rates and the efficacy of antimicrobial methods. Warm disinfectants work better than cool ones because chemicals react faster at higher temperatures. Acidic conditions enhance the antimicrobial effect of heat. For example, household chlorine bleach is more effective at a low pH. **Organic materials such as fat, feces, vomit, blood, and the intercellular matrix of biofilms, interfere with the penetration of heat, chemicals, and some forms of radiation, and in some cases these materials inactivate chemical disinfectants. For this reason it is important to clean objects before sterilization or disinfection so that antimicrobial agents can throughly contact all the object's surfaces.

Microbiostatic

Inhibiting the growth of microorganisms

Microbiocidal

Kills of microorganisms

Antiseptic

Kills/inhibits microorganisms (particularly pathogens) on skin or tissue by the use of a chemical antiseptic, whereas disinfection refers to the use of agents to inhibit microbes on inanimate objects.

Use Dilution test

Metal cylinders dipped into broth cultures of bacteria Contaminated cylinder immersed into dilution of disinfectant Cylinders removed, washed, and placed into tube of medium Most effective agents entirely prevent growth at highest dilution Current standard test in the U.S. New standard procedure being developed