Control of Microorqanisms

Methods used to control the growth of microorganisms and theirtransmission of infectious disease involve stopping the growth ofthe microorganism for a period of time, reducing the number ofmicroorganisms to a safe level, or destroying the microorganisms.The degree of effectiveness depends on the number ofmicroorganisms, the type of microorganism, their physiologicalstate, such as the stage of growth or formation of endospores, andthe ~nvironment in which they are growing (glassware, instruments,tissue, food).

Terms Related to Destruction of Microorqanisms

I. sterilization - the destruction of all microorganisms,including endospores, on an object or in a material.2. disinfection the destruction of pathogens, but notendospores, on an object or in a material. The number of pathogensis reduced or growth is inhibited to a level that does not producedisease.

3. antisepsis - chemical disinfection of the skin, mucosalmembranes, or other living tissues.

4. germicide ("cide" = kill) - a chemicalagent thatkills microorganisms.

Specific germicides include:

rapidly

a. sporicide - kills sporesb. bactericide - kills bacteriac. viricide - kills virusesd. funqicide - kills fungi

Terms Related to Suppression of Microorqanisms

i. asepsis ("without infection") the absence of pathogens froman object or area. Aseptic techniques prevent the entry ofpathogens into the body.

There are two types of asepsis:

a. surqical asepsis - techniques designed to exclude allmicroorganisms. It prevents infectious agents from reaching awound. Prevention begins before the patient enters the operatingroom and includes removal of hair and cleansing and disinfectingthe skin with an antiseptic that removes skin oils andmicroorganisms from the area of the skin to be opened. Substancesinclude Betadine, Isodine, Ioprep, and Surgidine.

b. medical asepsis - techniques designed to excludemicroorganisms associated with communicable diseases. It includesdust control, hand-washing, use of individualized equipment andinstruments, waste disposal, care of instruments, syringes,needles,~ thermometers, and dressings.

2. sanitization - the reduction or removal of pathogens oninanimate objects by chemical or mechanical cleansing.3. bacteriostasis -("static" = halt) - bacterial growth andmultiplication are inhibited, but the bacteria are not killed.

Terms for Destruction or Suppression o__f Microorqanisms

i. antimicrobialmicroorganism.

agents that destroy or suppress any

2. antibiotic - a product formed by microorganisms that destroy orsuppress microorganisms.

Physical Aqents

I. Heat

Heat is the most common, inexpensive, simplest, reliable, andeffective method used to destroy microorganisms. Heat denaturesthe proteins and enzymes of the microorganisms. Most pathogenswill be destroyed at temperatures between 50 and 70C for aduration of i0 minutes, except endospores which may survive 1 to 2hours at 100C.

The heat used in sterilization is either moist or dry heat.

a. moist heat

(i). boilinq - Bacteria, fungi, and many viruses aredestroyed by boiling at 100C for I0 to 30 minutes. Some virusesand endospores may require boiling for up to 20 hours.Sterilization is accomplished by denaturing the proteins andenzymes of the microorganisms.

(2). steam under pressure - Water is heated underpressure which raises the temperature above 100C which denaturesproteins and amino acids. The most common device used is anautoclave for sterilizing surgical bandages, instruments, media,and contaminated material. At a pressure of 15 pounds/square inchand a temperature of 121C for 15 to 20 minutes it will destroymicroorganisms and endospores.

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(3). pasteurization - It is mainly used in the food anddairy industries and it involves raising the temperature highenough to destroy pathogens or inhibit their growth withoutaffecting the quality of the product.

b. dz-Z heat

Dry heat penetrates substances more slowly than moist heat.

(I). incineration - The burning of disposable substancesin a chamber.

(2). direct flaminq - Sterilization of inoculatingloops, needles, and rims of test tubes with a bunsen burner. Wireheated to a red glow is 100% effective.

(3). hot-air oven - Sterilization of glassware, testtubes, petri dishes, instruments, syringes, and needles requireshigher temperatures for a longer period of time than othermethods. Most endospores will be destroyed at 160-165"C for aperiod of 2 hours.

2. cold

The effect of low temperature on microorganisms depends on thetype of microorganism and the intensity of the application.Temperature in a refrigerator ranges from 0-8"C and has abacteriostatic effect which reduces the metabolic rate of mostorganisms so that they cannot reproduce or synthesize toxins.

Freezing at -20"C kills most bacteria, but some may survive ina frozen state.

3. dryinq or desiccation

To grow and multiple, microorganisms require water. Theremoval of water by evaporation or freeze-drying (solid to gas)inhibits growth and reproduction of microorganisms by inhibitingenzymes. They may be viable for years so when water is madeavailable they resume growth and reproduction.

ultra-violet radiation and ionizinq radiation (x-rays and~amma rays)

Both types of radiation damage the DNA of microorganisms anddenatures their proteins. U-V radiation cannot penetratematerials such as glass, clothing, dirt, paper, or pus and is usedto kill microorganisms on surfaces. Germicidal lamps in operatingrooms, nurseries, and communicable disease wards reduce the numberof bacteria in the air, but they do not sterilize.

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5. filtration - Filtration is the passage of material or liquidsthrough a filter containing small pores that retain microorganisms.Membrane filters of cellulose acetate are the most common, butgauze, cotton, and paper serve as filters for air. Vaccines thatrequire the presence of live viruses, such as polio, are passedthrough filters which prevent bacteria from going through thepores. Air filters are used in operating rooms to lower the numberof airborne microorganisms.

Chemical Aqents

Chemical agents are used to control the growth ofmicroorganisms on inanimate objects and on living tissue. Mostchemical agents reduce the number of microorganisms (disinfect),but do not achieve sterility. Selection of a disinfectant dependson the mode of action, concentration necessary, the type ofmicroorganism, the number of microorganisms present, the type ofmaterial to be disinfected, temperature, and pH.

Disinfectants are classified based on their chemical structureand activity.

I. phenols (carbolic acid)

Phenol and derivatives called phenolics disrupt the plasmamembrane, denature proteins, and inactivate enzymes. Phenol israrely used any more because it causes skin irritation and has adisagreeable odor.

Common phenolics are cresols such as that foLuud in L_~I, andhexachlorophene which is used as an antiseptic.

2. alcohols

Ethanol and isopropanol are widely used as skin antiseptics,but they are not effective against enveloped viruses or bacterialendospores. They effectively destroy bacteria and fungi bydisrupting the lipids in the plasma membrane resulting in lysis,and denatures proteins.

3. surface-active agents (surfactants)

They include soaps and detergents that lower the surface-tension of liquid molecules and make microorganisms accessible toother agents. The pH of soaps is usually alkaline which destroyssome bacteria. Detergents are more effective against gram-positivethan gram-negative bacteria and they disrupt the plasma membrane.

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4. haloqens

The major halogens are iodine and chlorine. Iodine is aneffective antiseptic for the skin and superficial wounds. Itdestroys many kinds of bacteria, some endospores, fungi, and someviruses. Iodine inhibits protein function in the microorganism.Chlorine is used as a disinfectant and it is a strong oxidizingagent which affects functions of the microorganismss enzymes.

5. io___ns of heav~ metals

The major metal ions are mercury and silver, and they functionin denaturing proteins and enzymes. Both are used in combinationwith other substances as antiseptics.

6. chlorhexidine

Chlorhexidine is used as an antiseptic on skin and mucosalmembranes and functions in disruption of the plasma membrane ofgram-positive and gram-negative bacteria resulting in lysis.

7. alkylatinq awents

Alky!ating agents disrupt the structure of proteins andnucleic acids. Formaldehyde (.2 - .4%) is used to inactivateviruses and toxins used in vaccines, it also destroys spores inbacteria and fungi. Glutaraldehyde is less irritating and moreeffective than formaldehyde. It kills many microorganisms,viruses, and endospores. Ethylene oxide is a gas used in a closedchamber to sterilize materials. It kills all bacteria, includingendospores. Betapropiolactone kills spores in concentrations notmuch more than required for killing cells. The effect is rapid andit disappears in a few hours. It is used to sterilize bone,cartilage, and artery grafts.

Antimicrobial Chemotherapy

Chemotherapy is the use of chemical substances to treatvarious aspects of disease.

Chemotherapeutic aqents (drugs) are any chemical substancesused in medical practice to treat disease.

Antimicrobial aqents are a special group of chemotherapeuticagents used to treat infection caused by microorganisms. Many areproduced by microorganismsand are specifically called antibiotics.Some antimicrobial agents are synthetic druq~which are produced ina laboratory. Others may be semis~nthetic dru~s which aresynthesized by chemically modifying a substance from amicroorganism.

Properties of Antimicrobial Aqents

!. solubility in bod~ fluids - They must dissolve in body fluidsto be transported and reach the area of infection ormicroorganisms.

2. selective toxicit~ - They must harm the microorganisms withoutcausing significant damage to the host.

3. spectrum of activity - The range of different microorganisms onwhich an antimicrobial agent acts is its spectrum of activity.

There are two major types of spectrum activity:a. broad-spectrum activity - Agents are effective against a

great number and types of microorganisms, including gram-positiveand gram-negative bacteria. They are useful when an infection iscaused by an unidentified microorganism and it increases thechances that the microorganism will be susceptible to the drug.

A disadvantage is that many normal flora of the host aredestroyed. Normal flora compete with and help destroy pathogensand other microorganisms. Some normal flora may flourish when thecompetition is diminished and they become opportunistic pathogens.

b. narrow-spectrum activity - Agents are effective againstonly a small number or type of microorganism. They reduce thechance of normal flora being destroyed and decrease the chance themicroorganisms will develop drug resistance.

4. nonallerqenic - The drug should not cause an allergic reaction,hypersensitivity, or anaphy!actic shock in the host.

5. stability - Toxicity should not be altered by food, otherdrugs, or conditions of the host and the concentration in the bloodand tissue fluids should be stable over a period of hours (degradedand excreted slowly).

6. resistance - Few microorganisms should be resistant or have theability to develop resistance to the effects of the drug. Twodrugs with different modes of action on the microorganism may beadministered. The drugs do not interfere with each other, have anadditive or synerqistic effect and reduce the likelihood ofresistance.

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Most antimicrobial agents are antibacterial agents that arebactericidal (killing) or bacteriostatic (growth-inhibiting) whilehaving minimal effects on host cells. Antimicrobial agents may becategorized by their mode of action.

There are five modes of action:

I. inhibition of cell wall s~nthesis - Peptidoglycan synthesis inthe cell wall of growing bacteria is affected. The cell wall isweakened and lysis occurs. Human cells are not affected becausethey do not have cell walls or peptidoglycan in their plasmamembrane. Drugs such as vancomTcin and bacitracin interfere withthe synthesis of peptidoglycan chains. Penicillin andcephalosporin prevent the linking betweenthe peptidoglycanchains.

2. disruption of the plasma membrane - The selective permeabilityof the plasma membrane is affected and important substances used inmetabolic activities leave the bacterial cell. The drugs used areselectively toxic. PolymTxin B causes damage by attaching to thephospholipids of the membrane. It allows dissolved substances topass freely into and out of the cell eventually resulting in lysis.Antifungal drugs, such as nTstatin, amphotericin ~, and miconazole,combine with sterols in the funga! membrane causing disruption andleakage of cytoplasmic contents.

3. inhibition of protein synthesis - Antimicrobial drugs affectthe bacterial ribosome which is made up of a large and smallprotein subunit. When ribosomal function is disturbed, thesynthesis of protein may slow down so that normal growth cannotoccur or protein synthesis may stop. Ribosomal function may beaffected in different ways Chloramphenicol and erythromycin reactwith the large ribosomal subunit and prevent peptide bonds fromforming between the amino acids. Streptomycin, neomycin,kanamycin, and qentamicin bond with the small subunit and cause thegenetic code on mRNA to be improperly read and inhibit initiationof protein synthesis by preventing the ribosome from moving.Tetracycline bonds with the small subunit and interferes with theattachment of the tRNA carrying the amino acid to the ribosome.

4. inhibition of nucleic acid sTnthesis - DNA replication and thecopying of the information from DNA by mRNA is affected(transcription). Quinolones (ciprofloxacin) are synthetic drugsthat block the enzyme which unwinds the DNA double helix duringpreparation for replication. Rifampin is a semisynthetic drug thatinhibits the enzyme RNA polymerase which is necessary fortranscription.

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5. inhibition o__f s~nthesis of essential metabolites - Thesynthesis of folic acid (B vitamin), which is needed to make thenitrogenous bases of DNA is inhibited. Sulfonamides (sulfa drugs)are antimetabolites which function as competitive inhibitors of ~enzyme in the pathway that forms folic acid. Sulfonamides resemblethe substrate para-aminobenzoic acid (PABA). The enzyme thatnormally converts PABA to fo!ic acid combines with the sulfonamideinstead. The combination prevents fo!ic acid synthesis and stopsthe growth of the microorganism.

Antiviral Aqents

Chemical agents that inhibit the growth of viruses are not ascommon as antibiotics. A few inhibit a wide variety of viruses,while most are restricted to a single genus or group of viruses.

There are four modes of action:

i. antiviral proteins sT~thesized bv cells infected with a virus -Interferons are small proteins that are produced in most cells whenthey are infected by a virus. Once produced, interferon preventsthe subsequent infection of noninfect~d cells.

2. interfere with viral DNA synthesis - Most antiviral drugs arestructural analogues of the normal purines and pyrimidines thatform nucleic acids. Because they are structurally similar to thenormal nucleotides, they prevent viral reproduction by inhibitingDNA synthesis. Iodoxuridine, Vidarabine, and AcTclovir are used totreat herpesvirus eye infections, herpesvirus encephalitis,cytomegalovirus infections in newborns, and herpes zoster.

3. acted on b~ viral enzTmes or that block the activit7 of viralenzTmes - Retrovirus replication requires that the viral RNA firstbe transcribed into DNA. The viral enzyme that catalyzes this stepis reverse transcriptase. Retroviruses cause leukemias and AIDS.AzidothTmidine (AZT) inhibits the action of reverse transcriptase.

4. prevent the release o__f the viral nucleic acid into thecytoplasm of the infected cell - Amantadine prevents the release ofviral RNA into the cytoplasm and is effective against influenzavirus type A. Ribavirin inhibits nucleic acid synthesis byinterfering with the formation of guanine nucleotides.