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  Physical and Chemical Control of Microorganisms

I. In most medical settings, the control of microorganisms is of paramount concern. Decontamination refers to the destruction or removal of microorganisms from instruments, materials, body surfaces, etc. Many agents and procedures have been developed to accomplish this end. It is imperative that you, as a medical professional, understand the modes of action, level of activity and other factors which influence the effectiveness of these procedures and agents.

A. Generally, decontamination involves physical and/or chemical agents. Physical agents include high temperature, radiation, filtration or cavitating sound waves. A myriad of chemical decontamination agents exists. For the most part, they are substances that react with and thus alter some important molecular component of the cell.

B. Microorganisms are not uniformly affected by physical and chemical decontamination. Susceptibility to the effects of physical and chemical agents depends upon the type of microorganism and at what stage in the microorganism’s lifecycle they are exposed to the agent. When choosing and applying a method of decontaminating materials, it is important that you understand what type of organism is being targeted and the relative resistance of that organism.

1. The target with the highest resistance is the bacterial endospores. Endospores are ubiquitous in the environment. Many bacteria found in the soil are capable of forming these structures. Introduced into deep wounds or during surgical procedures, these spores can cause severe problems. Thus surgical equipment and other materials used in invasive procedures need to be decontaminated in such a way as to destroy these agents.

2. Targets with the moderate resistance include protist cysts, sexual fungal spores, nonenveloped viruses (many enteric viruses including those responsible for polio, Hepatitis A and Hepatitis E), Mycobacterium tuberculosis, Staphylococcus aureus and members of the genus Pseudomonas.

3. Targets with the least resistance include vegetative cells of most microbes, enveloped viruses (including those viruses responsible for AIDS and Hepatitis B), and asexual fungal spores.

C. There are several terms that have precise meanings. When these terms are used in a product description or as part of procedural instructions it is important that you are aware of these precise meanings.

1. Sterilization refers to any process that destroys or removes all infectious organisms including endospores and viruses.

2.  Disinfection refers to any physical process or application of any chemical that will kill the growing (vegetative) microbial cells. These processes need not kill or inactivate endospores. A disinfectant is a chemical capable of killing microbial cells. It should be understood that if a chemical is referred to as a disinfectant, it is to be used on inanimate objects and not to be used on body surfaces.

3. Sanitize refers to any mechanical process (scrubbing, rinsing, etc.) that reduces the microbial load on a surface. Sanitizers are chemical agents that assist in this task. These are usually soaps or detergents.

4. Microbicidal agents are chemicals that will kill or destroy microorganisms. Among the microbicidal agents are those that target specific microorganisms including:

a. fungicidal agents which are designed to kill fungi;

b. bactericidal agents which are designed to kill bacteria;

c. sporicidal agents which are designed to destroy endospores;

d. viricidal agents which are designed to destroy viruses.

5. Microbiostasis refers to the inhibition of growth of microorganisms. This does not mean that the organisms are killed simply that they are unable to grow. Refrigeration and many antimicrobial drugs exert a microbistatic effect.

a. Bacteriostatic agents are chemicals that inhibit the growth of bacteria.

b. Fungistatic agents are chemicals that inhibit growth of fungi.

5. Disinfection refers to any physical process or application of any chemical that will kill the growing (vegetative) microbial cells. These processes need not kill or inactivate endospores. A disinfectant is a chemical capable of killing microbial cells. It should be understood that if a chemical is referred to as a disinfectant, it is to be used on inanimate objects and not to be used on body surfaces.

6. Antisepsis refers to those practices that keep microorganism from entering the sterile tissues. The application of these practices is referred to as aseptic technique. Antiseptics are those chemicals that can be applied to tissue surfaces to kill or inhibit the growth of microorganisms.

II. There are several factors that will influence the effectiveness of antimicrobial agents. When attempting to sterilize, disinfect or sanitize a surface and in the application of aseptic technique, these factors must be taken into consideration.

A. Time of exposure The amount of time that the microorganisms are exposed to any agent (physical or chemical) will greatly affect how many microorganism are destroyed. Short exposures often kill the most susceptible organism and thus select for the more robust organisms. This can be counter productive in that the robust organism will then come to dominate the population of microorganisms and will often rapidly replace the organisms killed by the brief exposure.

B. Microbial load The number of microorganisms must be also considered. Highly contaminated substances will require more protracted exposure to eliminate all living contaminates.

C. Type organism or organisms As mentioned earlier, different organisms display differing susceptibilities to antimicrobial agents. If elimination of vegetative cells is the aim, less stringent measures can be taken. If, on the other hand, endospores must be eliminated more rigorous measures will be required.

D. Temperature, pH and osmolarity Many antimicrobial agents lose their effectiveness under certain environmental conditions and become more effective under others. Generally speaking, higher temperatures lead to increased rates of antimicrobial affect. No such broad statement can be made for the relative effectiveness of agents under differing conditions of pH and osmolarity. For some agents, decreases in pH make them more effective while other agents become inactive as the pH drops. It becomes important that the affect which pH and osmolarity exert on the efficacy of an antimicrobial agent be understood and taken into account when using that agent.

E. Concentration or intensity of agent Usually for an agent to be effective it must be present at or above a certain concentration or intensity.

F. Milieu This term refers to other substances (proteins, solvents, etc.) that are present in the environment that you are trying to disinfect. These other substances may interfere with the action of the chemical or physical agent you intend to use to kill the bacteria. This is especially true of proteins. High levels of protein will interfere with the action of many chemical agents and will reduce the effectiveness of some physical agents.

III.  Many chemical agents are available that are said to be effective at reducing of eliminating bacteria from the environment or from body surfaces. Disinfectants are chemical compounds that are designed to kill bacteria and are to be used only on inanimate objects.  Antiseptics are compounds designed to kill or inhibit the growth of bacteria on external body surfaces or certain mucus membranes. In clinical settings these agents, when used properly, are an important part of aseptic technique. But overuse of these products, especially outside of clinical settings, carries several risks. First, the inappropriate reduction of nonpathogenic normal flora on external body surfaces and mucus membranes can lead to infection by pathogenic organisms. (i.e. Some yeast infections can be traced to the inappropriate use of antiseptic douche.)  Secondly, genes for resistance to antimicrobial drugs have been shown to be found on the same plasmids as genes for resistance to certain antiseptics and disinfectants. Thus, inappropriate use of antiseptics and disinfectants selects for those organisms that carry these plasmids.  As a consequence of this overuse of antiseptics and disinfectants, the level of drug resistance increases in those bacterial populations that are found in the environment and on the body.  It is important that you are aware of the appropriate usage of antiseptics and disinfectants. It is equally important that you are not pulled in by the current media driven hyperbole regarding the need to kill every bacterium that is found on the body or in the environment.

 

IV.  The actual manner in which a physical or chemical agent affects bacteria is referred to as its mode of action. Generally speaking, if the mode of action of a chemical or physical agent interferes with a process or destroys a structure that is common to both the target microorganisms and our cells, high levels of side effects can be expected. For many drugs, the mode of action entails interference with a process that is unique to the target microorganism, thus minimizing the impact the agent has on our cells.

A. The cell wall is a common target of antimicrobial action. Most bacteria and all fungi have cell walls while our cells lack them. Thus agents that interfere with the synthesis of or specifically destroy the cell wall can be used at high concentration with little chance of affecting our cells.

1. As we will see later, many antimicrobial drugs exert their effect by interfering with the processes that lead to the synthesis of the cell wall.

2. In the case of the gram-negative cell wall, destruction of the outer membrane by solvents and detergents can be easily accomplished.

3. Many bodily secretions contain the enzyme lysozyme. This enzyme digests the peptidoglycan of the gram-positive cell wall.

B. Many disinfectants damage the cell membrane. This can be accomplished by disrupting the phospholipid bilayer or altering the transmembrane proteins. Remember, the membrane is a structure shared by both our cells and the cells of microorganisms, most agents with this mode of action can not be used internally or on mucus membranes as they will harm our cells also.

1. Organic solvents and strong surfactants both act by dissolving the phospholipid bilayer. This destroys the barrier that usually limits movement of ions and other chemicals into or out of the cell.

2. Agents that alter transmembrane proteins destroy the ability of a cell to selectively import or export substances and, in the bacterial cell, can lead to the inactivation of cytochromes and ATP synthase. Inactivation of these proteins destroys the ability of the cell to generate ATP.

C. As previously discussed, microorganisms contain many different types of large biochemicals including proteins, DNA, RNA, and lipids. Agents that will damage or inhibit the synthesis of these biological polymers will have an adverse effect on the microorganism.

1. Damage to a cell’s DNA will inhibit that cell from properly reproducing and stop the use of the DNA as a guide to make RNA. This, in turn, will keep the cell from making proteins that were coded for by the damaged DNA. It appears that all living organisms have the ability to repair DNA. This repair mechanism involves enzymes that will remove the damaged DNA and replace it with functioning DNA. This process is very error prone and thus results in high levels of mutations in the DNA. It also takes time to carry out the repair process, so rapidly growing cells that divide before they have the time to fix the damaged DNA are more adversely effected than slow growing cells.

a. Bombardment of cells with radiation will lead to DNA damage.

b. Certain drugs bind to the enzymes needed to make DNA or RNA and interfere with the functioning of these enzymes.

c. Nucleotide analogs are chemicals that have considerable similarities to the nucleotides used in the synthesis of DNA. Often the enzymes that make DNA cannot distinguish between a real nucleotide and a nucleotide analog. When the analog is added to a growing DNA strand during replication, the synthesis of the DNA strand immediately stops. This keeps cells from copying their DNA completely. Cells that receive only partial copies of the DNA are usually not viable and immediately die.

2. It should be clear at this point the central role played by proteins in metabolic process. Without the action of those proteins known as enzymes, life could not continue. Thus by blocking the synthesis of proteins or inactivation of existing enzymes an organism can be killed. Many antimicrobials work solely or in part by altering the tertiary structure (shape) of a protein or by blocking the active site of an enzyme.

a. The osmolarity of a solution and hydrophilic attraction between amino acids in the protein and water help determine the tertiary structure of a protein. Under normal conditions the proper shape is taken on. But altering the osmolarity or adding substances to the environment which alter hydrophilic bonds, will lead to the protein losing it proper shape.

b. Proteins assume their proper shape and are stable in that configuration only at a very limited temperature range. Outside of that range (either hotter or colder) the protein will take on a different shape and its functionality will be decreased or eliminated. Alteration of the shape of protein through chemical or physical means is referred to as denaturing.

c. Reactive chemicals will often covalently bind to proteins. This changes the shape of a protein in ways that leave it unable to function properly.

d. Many antimicrobial drugs bind to the ribosome or active site of RNA polymerase. By blocking the action of these two enzymes protein synthesis can be effectively shut down.

V. Chemical agents do not have equal levels of disinfection. Certain agents are very effective and will inactive even endospores.  These agents are said to have a high level of activity. Agents with an intermediate level of activity will kill vegetative cells of the most resistant organism (TB, naked viruses), sexual fungal spores. Agents with a low level of activity kill vegetative cells of less resistant organisms and enveloped viruses. 

A.  Halogens react with proteins in such a way that secondary and tertiary structure is altered. Most halogens exert an intermediate level of activity. Examples of halogen based agents are bleach, chlorine and bromine gas (water purification for drinking and swimming pools), iodine and iodophores (Betadine, providone).  

B. Phenolic agents disrupt membranes and alter secondary and tertiary structure of proteins. Most of these agents exhibit intermediate to low level of activity.  Examples of common phenolic compounds include Hibiclens, creosote (a wood preservative) and amphyl.  

C.  Alcohols    At 50-95% concentration are effective in disrupting membranes and alter protein tertiary structure. At 95-100% concentrations alcohols mainly serve to dehydrate cells. Alcohols exhibit an intermediate level of activity.

D.  Hydrogen peroxide produces reactive hydroxyl radicals that oxidize proteins and other organic molecules. This chemical alteration leads to changes in the tertiary structure of proteins, which leads to reduced function by these proteins.  Hydrogen peroxide exhibits a high level of activity.

E.  Detergents mainly disrupt membranes but also will alter the tertiary structure of some proteins.  Most detergents exhibit a low level of activity.

F.  Ethylene oxide is the gas used to sterilize instruments that can not be autoclaved (referred to as gas sterilization). It chemically alters proteins, DNA and RNA. It exhibits a high level of activity.

 

VI. Sterilization, sanitization or simply affecting microbiostasis of inanimate substances can be accomplished through several physical means.

A. Heat is widely used to sterilize and sanitize objects and solutions. The goal (whether you hope to render the substances sterile or simply reduce the bacterial load) and the possible target organisms must be considered. Most vegetative cells are easily destroyed by heat while endospores are much more resistant.

1. The thermal energy of heat has a greater effect in the form of moist heat. This involves exposing the solutions or items to be sterilized to boiling or steam. Boiling occurs at the 100 C and the steam produced by boiling is usually at that temperature. Though this temperature is effective against vegetative cells, it is not very effective against endospores. By allowing the boiling to occur in a pressurized chamber, the boiling point and the steam produced by this boiling is hotter. One of the most common types of medical sterilizers is the autoclave. The autoclave usually is pressurized so that the boiling point is pushed to 121 C by raising the pressure to 15 pounds per square inch.

2. Many solutions do not hold up well to the high heats and pressures of the autoclave. Gentler means of decontamination are needed. These means usually do not produce sterile solutions but reduce the bacterial count so that the solutions spoil more slowly. These methods utilize lower temperatures and target the vegetative cells.

a. Tradition pasteurization methods (known as batch pasteurization) involve heating the solution to 63 -65 C for 30min. These methods kill most of the vegetative cells and decrease the rate of spoilage.

b. Flash pasteurization involves heating the solutions to 71.6 C for 15 seconds. These have similar effects as batch pasteurization.

c. Ultrahigh temperature (or ultrapasteurization) involves superheating the solution to 134 C for 1-2 seconds. This usually produces a sterile or nearly sterile solution.

3. To have the same decontaminating effect as moist heat, dry heat temperatures must be much higher. Commonly in laboratories, flaming of instruments (placing them in a flame and heating them to very high temperatures) is a common means for rapidly decontaminating an instrument. On a larger scale, hospitals incinerate (burn) contaminated wastes to kill any and all microorganisms contaminating these wastes.

B. Sanatizing surfaces often involves the use of surfactants. In this setting the surfactant is designed to loosen the bacteria from the surface by binding to the charged materials on the surface of the microorganism.  These charged proteins and polysaccharides help the microorganism attach to surfaces.  The surfactant binds to these charges and thus interferes with the ability of the microorganism to attach to the surface. The microorganism can then be simply wiped away.  

C.  In the home and in the medical setting, control of microbial growth is affected by keeping materials cold (freezing or refrigerating). It should be noted that this does not kill the microorganisms, cold simply stops or slows the rate of growth of the microbes. Desiccation involves removal of water from the material that you wish to preserve. Desiccation stops the activity of the enzymes of the microbes contaminating these materials which, in turn, stops growth. When the materials are rehydrated, the microbes often continue their growth.

D. Several types of radiation are commonly used to kill contaminating organisms.

1. Ionizing radiation penetrates organic matter very easily and when it strikes a molecule it will often cause the molecule to breakdown into highly reactive ions. If the radiation hits a DNA strand it will cause alteration of breakage of the strand. Other molecules in the vicinity of the DNA that are hit by the radiation can give rise to highly reactive ions. These ions then react with DNA, leading to breaks in the "backbone" of the DNA strand. In either case the DNA is damaged. The most commonly used form of ionizing radiation is gamma radiation. It is used to sterilize drugs and medical supplies that are sensitive to heat. Increasingly, gamma radiation is being used to treat foods. Recently, poultry and beef producers received approval from the FDA to allow gamma irradiation of these meats to reduce the chances of transmission of several common pathogens.

2. Ultraviolet radiation (UV light) reacts with the pyrimidine bases of DNA (thymine and cytosine). When UV radiation hits DNA it imparts the pyrimidine bases with substantial amounts of energy. This energy allows the pyrimidines to form inappropriate covalent bonds with adjacent pyrimidine bases. This covalent linkage between the pyrimidine bases is known as a pyrimidine dimer. These dimers interfere with the ability of the effected pyrimidines to complementary base pair. This destroys the ability of the damaged DNA to carry out transcription or replication. Repair of this damage can occur but this process is error prone and thus introduces mutations into the repaired DNA.

E. For many solutions the most effective means of decontamination is to force the solution through a filter. Filtration is especially useful in sterilizing extremely sensitive drugs that would be adversely effected by any of the aforementioned means of decontamination. The size of the openings in the filter (pore size) will determine which pathogens are removed from the solution. Extremely small pores are necessary to remove viral pathogens.

 

 

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