Micro Lecture Chapter 5

Controlling Microbial Growth in the Body: Antimicrobial Drugs

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Surfactants

• or Surface-active agents, act to reduce the surface tension of solvents such as water by decreasing the attraction among solvent molecules. • One result of this reduction in surface tension is that the solvent becomes more effective at dissolving solute molecules. • Two common surfactants involved in microbial control are soaps and detergents. • The mechanical removal of microorganisms uses soaps and detergents to remove transient flora from the skin and allow them to be rinsed off. Soaps are made up of hydrophobic and hydrophilic ends. •The hydrophobic ends break down oily deposits, a process called emulsification, and the hydrophilic ends attract the water molecules. • Hence the oily film with any bacteria they harbor is easily dissolved and washed away. • Synthetic detergents are organic surfactants that are more soluble in water than soaps. • The most popular detergent for microbial control is quaternary ammonium compounds or quats. • Quats are not only antimicrobial; they are harmless to humans making them ideal for many industrial and medical applications.

Efficacy of an Antimicrobial Drug

•To effectively treat infectious diseases, physicians must know which chemotherapeutic agent is most effective against a specific pathogen. • To ascertain the efficacy of an antimicrobial drug, microbiologists conduct a variety of tests, including diffusion susceptibility tests, the minimum inhibitory concentration test, and the minimum bacteriocidal concentration test.

Mechanisms of Antimicrobial Action

Antimicrobial drugs can be categorized into several groups according to their mechanisms of action. A. Drugs that inhibit cell wall synthesis B. Drugs that inhibit protein synthesis C. Drugs that disrupt the cytoplasmic membrane D. Drugs that inhibit general metabolic pathways E. Drugs that inhibit nucleic acid synthesis F. Drugs that block a pathogen attaching to its host

Inhibition of Metabolic Pathways

Antiviral agents can target unique aspects of viral metabolism. Several antiviral drugs are analogs of nucleosides and nucleotides. • The most commonly used nucleoside analog,Acyclovir,is used to treat genital herpes. • Ribavirin is a guanosine analog used to stop viral RNA synthesis and is primarily used to treat hepatitis C and viral hemorrhagic fevers. • The nucleoside analog, lamivudine, is used to treat chronic hepatitis B. • Another nucleoside analog, cidofovir, is currently used in the treatment of cytomegalovirus infections of the eye. • Analogs of nucleotides and nucleosides are often the basis for drugs to block the enzyme reverse transcriptase in the human immunodeficiency virus (HIV). • Zidovudine or azidothymidine (AZT) is a nucleoside analog reverse transcriptase inhibitor. • Zanamivir(Relenza) and Oseltamivirphosphate(Tamiflu) have been recently introduced as inhibitors of the enzyme neurominidase for the treatment of influenza. • Another approach to treating viral infections is to inhibit enzymes that control viral replication and assembly. • Protease inhibitors prevent viral replication by selectively binding to viral proteases. • Saquinavir and indinavir are effective when combined with inhibitors of reverse transcriptase.

Factors Influencing the Effectiveness of Antimicrobial Agents

Concentration of the Chemical Antimicrobial Agent: Some organisms are sensitive to a low concentration of the antimicrobial agent, while others are sensitive to high concentrations of the same agent. • Contact time: Some microorganisms are killed or inhibited with shorter exposure times. Dormant forms, such as bacterial endospores, are very resistant to the action of chemical agents. Actively growing microorganisms are the most sensitive to the action of these agents. Viruses are metabolically dormant when not within a host cell and are therefore more resistant to the action of chemical agents. • Environmental Conditions: Temperature and pH affect not only the efficacy of antimicrobial methods but most significantly the death rates of microorganisms. •Certain disinfectants at high temperatures generally are more effective than disinfectants at low temperatures. Acidic conditions enhance the disinfection efficiency of an antimicrobial agent. For example, household chorine bleach is more effective at lower pH values.

Antisepsis

Is the inhibition or killing of microorganisms on living tissue by the use of a chemical. The chemical is called an antiseptic.

Heavy Metals and their Compounds

Several heavy metals, such as silver, mercury, arsenic, zinc and copper are biocidal. • In the past physicians were required to put a drop of 1% silver nitrate in the eyes of newborns to prevent blindness caused by Neisseria gonorrhea while it passed through the birth canal. • Today, silver nitrate has largely been displaced by antibiotic ointments, because they are less irritating and are also effective against Chlamydia trachomatis and other pathogens. • Medical personnel incorporate silver in surgical dressings, burn creams, and as an antibiotic coating on catheters. • Due to the potential toxicity and corrosiveness of silver-impregnated devices, its use is now limited. • Currently, the use of mercurials is to control mildew in paints. • Copper, which interferes with chlorophyll, is used to control algal growth in reservoirs, fish tanks, swimming pools, and water storage tanks. • In addition, copper, zinc and mercury are used to control mildew in paint.

Evaluating an Antimicrobial Drug

The effectiveness of disinfectants and antiseptics can be tested by using the disc-diffusion assay. •Filter paper discs that have been soaked with various commercially available antiseptics and disinfectants are placed on the agar that has been inoculated with the pathogen . •After incubation, if the antimicrobial agent is effective, a zone of inhibition will appear as a clear area in which there is no microbial growth around the filter disc. • The test is based on the diffusion rate of the agent and the antibacterial effect of the disinfectant or antiseptic.

Sanitization

The process of reducing microbiological contamination to a level that is acceptable to local health regulations

Degerming

The removal of transient microbes from the skin by mechanical cleansing

Cidal Agent

a chemical or physical agent that destroys or permanently inactivates a particular type of microbe; a bacteriocidal agent kills bacteria.

Static Agent

a chemical or physical agent that inhibits microbial metabolism and growth but doesn't necessarily kill microbes. Thus, refrigeration is bacteriostatic for most bacterial species; it inhibits their growth, but they can resume metabolism when the optimal temperature is restored.

Pasteurization

involves gentle heating to kill microbes associated with food spoilage. Milk, wine, fruit juices, beer, and wine are routinely pasteurized.

Pasteurization

is used to kill pathogens in milk, ice cream, yogurt, and fruit juices. • is not sterilization. • Thermoduric (heat-tolerant) and thermophilic (heat-loving) prokaryotes survive pasteurization, but they do not cause spoilage over the relatively short times during which properly refrigerated and pasteurized foods are stored before consumption. • Brucella melitensis, Coxiella burnetti, Escherichia coli, Mycobacterium bovis, and Salmonella are controlled in this manner.

Sterilization

refers to the removal of living organisms including viruses from a growth medium

Disinfection

refers to the use of physical or chemical agents such as bleach, heat and ammonia to kill or inhibit the growth of pathogenic microorganisms. Most disinfectants are too harsh to be applied to living tissue, and are applied to only inanimate surfaces

Autoclave

• A basic principle of chemistry states that the pressure of a gas is propotional to the temperature of that gas. • Since steam is a gas, increasing its pressure in a sealed device subsequently raises its temperature. • a large vessel through which steam is circulated to sterilize culture media, instruments, solutions, dressings, and intravenous equipment. • Scientists have determined that a temperature of 121°C, which requires the addition of 15 pounds per square inch (psi) of pressure, destroys all microbes in a small volume in about 15 minutes. • A biological indicator of sterility uses ampules containing endospores of the bacterium Bacillus stearothermophilus. • The sealed vial contains growth media and a pH indicator. • If sterilization was effective, there will be no growth in the vial since the spores are killed. However, if sterilization was ineffective, the spores will germinate into vegetative cells that will grow, producing acid that will change the pH indicator

Resistance to Antimicrobial Drugs

• A population may contain a few organisms that are resistant. Resistance in bacteria can be acquired through mutations in the chromosome or by acquiring R-plasmids via the mechanisms of genetic exchange.

Inhibition of Metabolic Pathways

• Antimetabolic agents are drugs that interfere with enzymes or their reactions that are important to the metabolism of the cell. • Sulfanilamides act as antimetabolic drugs because they are structural analogs of para- aminobenzoic acid (PABA), a compound that is required for the synthesis of purine and pyrimidine nucleotides. • Para-aminobenzoic acid in microorganisms is the substrate for an enzymatic reaction leading to the synthesis of the vitamin, folic acid. • Folic acid also functions as a coenzyme for the synthesis of purine and pyrimidine bases of DNA and RNA. • Sulfonamides compete with para-aminobenzoic acid for the active site of the enzyme that converts para-aminobenzoic acid to folic acid. • This combination prevents the synthesis of folic acid which leads to a decrease in the production of DNA and RNA, leading to the cessation of cell metabolism and ultimately cell death. • Because, humans obtain folic acid from their diet, sulfonamides exhibit selective toxicity by affecting the microorganism but does not harm the human host. • Another antimetabolic agent that also interferes with nucleic acid synthesis is trimethoprim. • Trimethoprim and sulfanilamides can be used synergistically to interfere with nucleic acid synthesis in microorganisms.

Salting

• High concentrations of salts and sugars in foods inhibit microbial growth by osmotic pressure. • The high solute concentration is hypertonic to bacterial cells causing water to flow out of the cell. • The removal of water inhibits cellular metabolism because enzymes are fully functional only in aqueous environments. • This premise is used in the preservation of foods such as honey, jerky, jams, jellies, and salted fish. • Molds and yeasts have a greater capability than bacteria of tolerating hypertonic environments with little moisture. • This characteristic of fungi along with its ability to tolerate acidic conditions is the major cause of fruit spoilage. • Another issue is the formation of mildew on damp walls.

Inhibition of Nucleic Acid Synthesis

• Antimicrobial drugs target nucleic acid, either DNA or RNA, synthesis. • The antimicrobial actions of these agents are a result of differences in prokaryotic and eukaryotic enzymes involved in nucleic acid synthesis. • Quinolones and their derivatives, fluoroquinolones, are antibiotics that interfere with DNA replication. • Drugs in this family such as ciprofloxacin and norfloxacin, inhibit topoisomerases, most frequently gyrase (topoisomerase II) required for the supercoiling of the DNA. • Fluoroquinolones are often used to treat genitourinary infections and are widely used in the treatment of infections associated with catheters. • Rifampin blocks the initiation of RNA synthesis by inhibiting the bacterial RNA polymerase. • It is used to treat infections caused by Mycobacterium tuberculosis and Mycobacterium lepae as well as methicillin-resistant Staphylococcus aureus. • Some antibiotics such as actinomycin bind DNA at the transcription initiation complex and prevent the elongation of RNA by RNA polymerase. • It affects not only the bacterial pathogen but the mammalian cells. • Hence, actinomycin is often used as an antitumor drug, attacking both the malignant cells as well as the normal cells.

Ionizing Radiation

• As electron beams, X-rays, and gamma rays pass through microbial molecules, they create ions by ejecting electrons from atoms. • Such ions disrupt hydrogen bonding, oxidize double covalent bonds, and create highly reactive hydroxide ions. • These ions denature other molecules, particularly DNA, causing fatal mutations and cell death. • Electron beam radiation is a form of ionizing energy characterized by highly energetic electron beams. • They are used to sterilize disposable dental and medical supplies, such as surgical gloves, plastic syringes, catheters, and suturing materials. • To mitigate bioterrorism attacks, the United States Postal Service uses ionizing radiation to sanitize certain classes of mail.

Minimum Inhibitory Concentration Test (MIC)

• As the name suggests, the Minimum Inhibitory Concentration (MIC), is the smallest amount of the antimicrobial drug that will inhibit growth of the pathogen. • The MIC is often determined by a broth dilution test, in which a standardized amount of bacteria is added to serial dilutions of antimicrobial drugs in tubes or wells containing broth. • Growth is assessed after incubation for a defined period of time. • The lowest concentration of the drug that inhibits the growth of the pathogen is the Minimum Inhibitory Concentration.

Chemical Methods of Controlling Microbial Growth (stops at gaseous agents)

• Chemical agents act to adversely affect the cell walls, cytoplasmic membranes, proteins, or DNA of microorganisms. • The effect of a chemical agent varies with temperature, pH, concentration, and length of exposure

Chemical Preservatives

• Chemical preservatives make our food appear fresher, more attractive or last longer on the shelf. • Common additives are calcium propionate, sodium nitrate, sodium nitrite, sodium benzoate, and sorbic acid. • Calcium propionate is incorporated into bakery products where it inhibits the growth of molds and the ropiness due to the Bacillus species. • Propionate interfers with cellular metabolism or damages the cytoplasmic membrane of fungi. • Sorbic acid and sodium benzoate inhibit the growth of molds in acidified foods such as fruit juices, jams and carbonated drinks. • Sodium nitrate and sodium nitrite are curing agents added to meat products such as bacon, hot dogs, sausage and ham. • These additives prevent the germination of Clostridium botulinum endospores and preserve the pleasing red color of meat. • When added to processed foods, both nitrates and nitrites can form carcinogens such as nitrosamines. • Hence, recent trends in the food industy involve minimal processing and reducing the amount of chemical preservatives.

Dessication and Lysophilization

• Dessication inhibits microbial growth because the evaporation of water inhibits metabolism. • Growth is resumed when water is made available. • Foods such as fruits, peas, beans, cereals, nuts, and grains are preserved by desiccation or drying. • Scientists use a technique combining freezing and drying, called lyophilization, to preserve microbes for several years. • In this process, a culture of microbes or other cells is frozen in liquid nitrogen or frozen carbon dioxide (dry ice); then subjected to a vacuum that removes frozen water through a process called sublimation, in which water is transformed directly from a solid to a gas. •Lyophilization prevents the formation of large ice crystals that otherwise would damage the cells. Although not all cells survive, enough cells are viable to enable the culture to be reconstituted several years later.

Routes of Administration

• For internal infections, drugs can be administered orally, intramuscularly (IM) or intravenously (IV). • The oral route is the most common route of drug administration. It is cheap, easy to use and convenient. • It may be in the form of tablets, capsules, syrup, emulsions, and powders. • However, the drug concentrations it achieves are the lowest and the drug can be potentially destroyed by gastric juices in the stomach. • Some drugs can cause gastrointestinal irritation. • Parenteral routes (intramuscular and intravenous) offer the direct and reliable method of administering medications while also providing the most rapid absorption. • These routes are used when the gastrointestinal tract would absorb the drug ineffectively. • The disadvantage of using a parenteral route includes risk of infection, tissue damage, and pain and anxiety for the patient. • Intramuscularly (IM) via a hypodermic needle allows a drug to diffuse slowly into the blood vessels within the muscle tissue, but the concentration of the drug in the blood is never as high as that achieved by intravenous (IV) administration, which delivers the drug directly into the bloodstream through either a needle or a catheter

Heat Related Methods

• High temperatures distrupt the function and structure of nucleic acids, denature proteins, and interfere with the integrity of cytoplasmic membranes and cell walls. • Thermal death time (TDT) is the length of time required to kill a bacterial population at a given temperature while thermal death point (TDP) is the lowest temperature required to kill a bacterial population in ten minutes. • Decimal reduction time (D), is the time required to destroy 90% of the viable organisms at a given temperature. • This concept is especially useful in food processing because foods must be heated to eliminate the endospores of Clostridium botulinum, which could germinate and produce the botulinum toxin inside sealed cans.

Inhibition of Cell Wall Synthesis - continued

• However, there are two major drawbacks to the use of penicillin: • The first is anaphylaxis occurring in allergic individuals. • Hives, itching, difficulty breathing, dizziness, and loss of consciousness are symptoms of sensitivity. • The second disadvantage is the evolution of penicillin-resistant bacteria. These organisms produce an enzyme called beta-lactamase (penicillinase) that converts penicillin into penicilloic acid that is harmless. •Giuseppe Brotzu, an Italian scientist, discovered that a fungus Cepahosporium acremonium was producing cephalosporins. • They are used as alternatives to penicillin where allergic reactions occur or where resistance is encountered. • Vancomycin obtained from Streptomyces orientalis prevents NAG and NAM from being incorporated into the peptidoglycan matrix. • Bacitracin blocks the secretion of NAG and NAM from the cytoplasm. • Isoniazid and ethambutol disrupts the synthesis of mycolic acid in the cell walls of Mycobacterium tuberculosis and Mycobacterium leprae.

The History of Antimicrobial Drugs

• In 1638, the Countess of Chinchon, the wife of the Spanish ambassador to Peru, developed malaria. She was treated by the native healers with the bark of "the fever tree." After a few days, the Countess was cured of malaria and the use of the tree was introduced into European medicine. A century later, Linnaeus named it Cinchona after the Countess. Although the use of the bark has been superseded by more effective pharmaceutical drugs, cinchona is the only economically source of quinine, an effective treatment for malaria. The term "antibiosis" meaning against life was first used by a French researcher, Paul Vuillemin, in 1889, to describe his discovery of a substance from Pseudomonas aeruginosa. The substance, called pyocyanin, was effective against bacteria but too toxic to be used for the therapy of diseases. In 1910, Paul Ehrlich developed an arsenic-based drug that was effective against syphilis called Salvarsan. He proposed the term "chemotherapy" to describe using chemicals to selectively kill pathogens without harming the host. In 1929, Alexander Fleming, reported the antibacterial action of pencillin produced by a mold identified as Penicillium notatum. He coined the term antibiotics to describe antimicrobial drugs that are produced naturally by an organism. Other microorganisms, such as the fungus Cepahlosporium and bacteria in the genera Bacillus and Streptomyces, are sources of antimicrobics. Gerhard Domagk discovered sulfanilamides in 1932. Sulfonamides inhibit bacterial growth by inhibiting the synthesis of folic acid, a compound required by all cells for the biosynthesis of nucleic acids.

Boiling

• In general, moist heat is more effective in microbial control than dry heat as water is a better conductor of heat than air. • In contrast to dry heat, shorter exposure times and lower temperatures are therefore required. • Moist heat acts by coagulation and denaturation of proteins. • Denaturation involves the destruction of hydrogen and disulfide bonds between the amino acids, disrupting its three- dimensional shape. •kills most vegetative bacteria, trophozoites of protozoa, fungi, and most viruses within ten minutes at sea level. • Bacterial endospores, protozoan cysts, and some viruses (such as hepatitis viruses) can survive boiling at sea level for many minutes or even hours. • In fact, bacterial endospores can withstand boiling for more than twenty hours.

Diffusion Susceptibilty Test

• In the disc diffusion method, filter paper discs impregnated with specific concentrations of antimicrobial drugs are placed on the surface of agar plates that are inoculated with a standardized amount of pathogen in question. • During incubation, the antimicrobial drug will diffuse from the disc into the medium. • If the organism is susceptible to the particular antibiotic, a zone of inhibition will appear as a clear area in which there is no microbial growth around the filter disc. • The size of the zone is a function of both the antibacterial effect of the antimicrobial drug and the diffusion rate of the antimicrobial drug in the medium. • The minimum inhibitory concentration is the amount of the antimicrobial drug present at the outer edge of the zone of inhibition. • To determine if the antimicrobial drug is effective for treating the infection, the diameter of the zone of inhibition must be compared to a standard table for that particular drug and concentration. • The Kirby-Bauer method, named after W.M. Kirby and A.W. Bauer, is a highly standardized disc diffusion method that is widely used in clinical laboratories. • Categories of sensitive, intermediate and resistant are then determined for that antimicrobial drug.

Refrigeration and Freezing

• Low temperatures found in the refrigerator and freezer retard spoilage by lowering the metabolic rate of microorganisms and thereby reducing their rate of growth. • Refrigeration retards the growth of most pathogens, which are predominantly mesophiles. • However, psychrophilic microbes can multiply in refrigerated food and spoil its taste and suitability for consumption

Gaseous Agents

• Many items, such as heart-lung machine components, surtures, plastic laboratory ware, mattresses, pillows, artificial heart valves, catheters, electronic equipment, and dried or powdered foods can be sterilized by highly reactive biocidal gases. • Gaseous chemosterilizers include ethylene oxide, propylene oxide and beta- propiolactone. • Ethylene oxide is frequently used as a gaseous sterilizing agent in hospitals and dental offices, and NASA uses the gas to sterilize spacecraft. • Large hospitals often use ethylene oxide chambers to sterilize instruments and equipment sensitive to heat. • Gaseous agents can be extremely hazardous to the people using them. • Because they are often highly explosive, they must be administered by combining them with 10% to 20% nitrogen gas or carbon dioxide. • They are extremely poisonous, so workers must extensively flush sterilized objects with air to remove every trace of the gas.

Clinical Considerations in Prescribing Antimicrobial Drugs

• Most of the pharmaceutical drugs used to treat diseases are ineffective because they lack potency, are expensive and have potential side effects. • The ideal antimicrobial drug to treat an infection would be one that is easily administered, inexpensive, readily available, chemically stable, and has no side effects. • Side effects are categorized into three groups: toxicity, allergies and disruption of normal flora. • Physicians must be cautious in prescribing drugs to pregnant women, as many drugs have adverse effects when absorbed by the fetus. • In addition to toxicity, some drugs trigger allergic immune reactions in sensitive patients. • Although very rare, some allergies can result in life-threatening reactions called anaphylasis. • Some drugs disrupt normal microbiota allowing the growth of pathogenic fungi and bacteria leading to superinfections. • Long-term use of broad-spectrum antimicrobials results in the overgrowth of Candida albicans in the oral cavity (thrush) or vagina (vaginitis). • Pseudomembranous colitis is a potentially fatal condition, caused by the overgrowth of Clostridium difficle, which forms membranous lesions in the intestinal tract. • These secondary infections are of greatest concern to hospitalized patients who are exposed to pathogens that are resistant to antimicrobial drugs.

Mechanisms of Resistance

• Pathogens are resistant to antimicrobial drugs by the following mechanisms: 1. Resistant bacteria produce an enzyme called β-lacatmase (penicillinase) that breaks the β-lactam rings of penicillin and its derivatives, rendering them inactive. 2. Pathogens acquire resistance to tetracycline and penicillin by altering the structure of the proteins in the cytoplasmic membrane that constitute channels. In gram-negative bacteria, the proteins in the outer membrane are called porins. These mutations in the proteins prevent the entry of the drug into the cell. 3. Erythromycin and Sulfonamides alter the receptor of the drug so that it cannot attach to the host cell. 4. Resistant cells can modify metabolic pathways to circumvent the antibiotic effect. 5. Efflux pumps increase antibiotic resistance in bacteria.

Multiple Resistance

• Pathogens can acquire resistance to several antibiotics at one time, especially when resistance is conferred by R- plasmids, which are exchanged among bacterial cells. • These microorganisms are called superbugs. Common types of superbugs include strains of Staphylococcus, Streptococcus and Pseudomonas. • The World Health Organization report released in April 2014 states that antibiotic resistance is a global health risk.

Peroxygens (Oxidizing Agents)

• Peroxygens exert antimicrobial activity by oxidizing cellular components of microorganisms. • Examples include hydrogen peroxide, peracetic acid and ozone. • Peroxygens are particularly effective against anaerobic microorganisms contaminating deep wounds by releasing hydroxyl radicals. • Hence, health care workers use oxidizing agents to kill anaerobes in deep puncture wounds. • Hydrogen peroxide is a common household chemical that is used to disinfect and sterilize the surface of inanimate objects but is mistakenly used to treat open wounds. It is not a good antiseptic as the enzyme catalase in the tissue breaks down hydrogen peroxide into water and oxygen. • Though aerobes and facultative anaerobes on inanimate surfaces also contain catalase, the volume of peroxide used as a disinfectant overwhelms the enzyme, making hydrogen peroxide a useful disinfectant. • Peracetic acid is an extremely effective sporicide that can be used to sterilize surfaces. • Since it is not adversely affected by organic contaminants, and it leaves no toxic residue, food processors and medical personnel use peracetic acid to sterilize equipment. • Ozone is a reactive form of oxygen that is used by municipalities to treat their drinking water. • Although it is more effective than chlorine, it is expensive and difficult to maintain a residual capable of continuing to kill microrganisms throughout the distribution system.

Inhibition of Protein Synthesis

• Prokaryotes have 70s ribosomes, each consisting of a large 50s subunit and a small 30s subunit. • The antimicrobials that target the 30S ribosomal subunit are aminoglycosides and tetracyclines. • Aminoglycosides, such as amikacin, gentamycin, kanamycin, streptomycin and tobramycin, bind to the 30s subunit and cause misreading of the genetic code. • This results in high levels of translational errors which can be lethal. • The major side effects of aminoglycosides are renal failure and damage to the auditory nerve affecting hearing. • Tetracyclines inhibit tRNA binding to the ribosomal A site, preventing the incorporation of additional amino acids into a growing polypeptide. • Tetracyclines may alter the normal intestinal microbiota causing gastrointestinal irritations. • Continuous usage often leads to superinfections caused by the fungus Candida albicans. • Consuming large quantities of yogurt and probiotic supplements can be used to replace gastrointestinal normal flora during an antibiotic regimen.

Disruption of Cytoplasmic Membranes

• Some antimicrobial drugs are incorporated into the cytoplasmic membrane and damage its integrity. • This group of drugs is called polyenes. • Amphotericin B, isolated from Streptomyces nodosus, is used to treat systemic fungal infections. • It targets the sterol, ergosterol, in the plasma membrane, disrupting the membrane leading to fungal cell death. • While amphotericin B has a higher affinity to ergosterol, it can also bind to the cholesterol component of mammalian cells leading to cytotoxicity. • Amphotericin B can form pores in both the host membrane as well as the fungal membrane which can be lethal. • There are other agents that disrupt bacterial membranes. • Polymyxin, produced by Bacillus polymyxa, is effective against Gram-negative bacteria, particularly Pseudomonas. • It causes disruption of the plasma membrane by attaching to the phospholipids of the membrane. • Polymyxin is reserved for topical treatment of superficial infections as it is toxic to the human kidneys.

Non Ionizing Radiation

• Ultraviolet (UV) light, visible light, infrared radiation, and radio waves are examples of nonionizing radiation. • Of these, UV light has sufficient energy to be an antimicrobial drug. • UV light with a wavelength of 260 nm is specifically absorbed by adjacent thymine nucleotide bases in DNA, causing them to form covalent bonds with each other rather than forming hydrogen bonds with adenine bases in the complementary DNA strand. • These thymine dimers, distort the shape of the DNA making it impossible to replicate its genetic material. • A UV, or "germicidal," lamp is commonly found in newborn nurseries, operating rooms, hospital rooms, and cafeterias. • Many biological materials, such as antisera and vaccines, are sterilized by U.V. radiation. • Since U.V. light has a longer wavelength than ionizing radiation, it penetrates poorly and hence it has limited use in eliminating microorganisms. • Organisms protected by dust or other coverings such as textiles, glass or paper may not be killed. Prolonged exposure to U.V. light, however, may result in sun burns, and in serious cases cataracts and skin cancer.

Inhibition of Protein Synthesis - continued

• Tetracycline should be used with caution with young children and pregnant women as it causes brown discoloration of the teeth and stunted bones in children. • Chloramphenicol blocks the enzymatic activity of the 23srRNA of the 50s subunit. • Hematological side effects due to bone marrow suppression are the most serious adverse effects of chloramphenicol. • Fatal cases of aplastic anemia, a condition that prevents hemoglobin incorporation into red blood cells, have been reported. • In newborns, intravenous administration of chloramphenicol accumulates in the blood causing a poisonous reaction and collapse of the cardiovascular system known as gray syndrome. • For these reasons, physicians are advised not to use the drug for minor infections or use suitable derivatives. • Erthromycin is a macrolide antibiotic that interferes with translocation by preventing the transfer of the tRNA bound to the A site of the ribosome to the P site. • Hence the addition of the incoming aminoacylated tRNA is blocked as the A site remains occupied. • Erythromycin is a product of Streptomyces and is recommended for patients with penicillin allergic reactions. • It is also effective against both Neisseria and Chlamydia species that can affect the eyes of newborns. • Common side effects of erythromycin are mild diarrhea, vomiting, loss of appetite, and stomach pain.

Minimum Bactericidal Concentration Test (MBC)

• The Minimum Bactericidal Concentration (MBC) test is an extension of the Minimum Inhibitory Concentration test (MIC). • This method determines whether the antimicrobial drug is bactericidal or bacteriostatic. • In the Minimum Bactericidal Concentration (MBC), samples taken from clear MIC tubes are transferred to broth free of the antimicrobial drug. • If growth occurs in the broth, the drug is not bactericidal, and the MBC can be determined. • The lowest concentration of the antibacterial agent for which no viability occurs in the subcultures is the Minimum Bactericidal Concentration (MBC).

Phenol

• The antiseptic properties of phenol (carbolic acid) were used by Joseph Lister, a physician at the University of Glasgow, to reduce infections during surgery. • Phenol is highly irritating to the skin, eyes and mucous membranes and has a disagreeable odor. • Phenolics have greater antimicrobial efficacy as they are chemically modified by the addition of organic functional groups or halogens. • Phenolics affect the integrity of the cytoplasmic membrane causing the leakage of cellular contents. • The cell walls of Mycobacterium tuberculosis and Mycobacterium leprae, are rich in a lipid component called mycolic acid, which make them susceptible to phenol derivatives. • They are effective even in the presence of contaminating organic material such as vomit, pus, saliva, and feces, and they remain active on surfaces for a prolonged time. • For these reasons, phenolics are commonly used in health care settings, laboratories, and households. • Bisphenols are composed of two covalently linked phenolic groups. • One bisphenol, hexachlorophene, is commonly used in surgical and hospital procedures. • Skin infections in newborns caused by Staphylococci and Streptococci are particularly susceptible to hexachlorophene. • However, excessive use of this bisphenol can cause brain damage in infants. • Another widely used bisphenol is triclosan which has been incorporated into numerous consumer products, including garbage bags, utensils, diapers, and cutting boards. • There is a growing concern that the overuse of triclosan is leading to the potential emergence of drug resistant bacteria.

Prevention of Attachment

• The entry of many pathogens, particularly viruses, involves the chemical interaction between the viral attachment proteins and the complementary receptor proteins on a host. • These protein-protein interactions can be inhibited by sugar and peptide analogs of viral proteins or receptor proteins called attachment anatagonists. • Thus, the virus can neither attach nor enter their host cells. • Research of these drugs is very expensive and is still in the developmental stage. • The antiviral effect of a new drug, arildone, inhibits uncoating of the poliovirus and has a potent effect on the replication of the virus. • Enfuvitide is a biomimetic peptide analog that blocks the CD4 and a chemokine receptor preventing the entry of HIV into the cell.

Hot Oven

• The hot air oven utilizes radiating dry heat for sterilization. •This type of energy does not penetrate materials easily, and hence exposure to high temperatures for a long length of time is necessary. • A temperature of 160°C (320°F), maintained for a period of two hours ensures sterilization. • This hot-air method is useful for sterilizing pipettes, glassware and syringes.

Radiation

• The severity of radiation on microbial cells depends on the wavelength, intensity and duration. • The shorter the wavelength of an electromagnetic wave, the more energy it carries and the farther it can penetrate. •Therefore, shorter wavelength radiation is more suitable for microbial control than longer wavelength radiation, which carries less energy and is less penetrating. • There are two types of radiation: ionizing and non ionizing

Spectrum of Action

• The spectrum of action is the range of microorganisms that an antibiotic inhibits or kills. • A broad-spectrum drug acts against a larger group of pathogens whereas a narrow-spectrum antibiotic is effective against a few kinds of pathogens. • For example, erythromycin is a broad-spectrum drug that acts against protein synthesis in numerous pathogens. • In contrast, penicillin is largely limited to gram-positive bacteria; thus penicillin has a narrower spectrum of action than erythromycin.

Inhibition of Cell Wall Synthesis

• The structural component of a bacterial cell wall is the peptidoglycan layer. Peptidoglycan is composed of two alternating sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are cross-linked by short peptide chains. • The most common antibacterial agents act by preventing the cross-linkages of the N- acetylmuramic acid subunits. • Consequently, the cell wall is weakened and the cell ruptures. • The most prominent among these drugs are beta-lactams, which are antimicrobial drugs that contain a beta-lactam (β-lacatam) ring. • These include penicilins, cephalosporins and monobactams. Penicillin G is currently the most popular penicillin. Other types are penicillin F and penicillin V. • The penicillins are active against a variety of gram-positive bacteria, including Staphylococcus, Streptococcus, Pneumococci, and Clostridia. • Pharmaceutical companies have created semisynthetic derivatives, such as amoxicillin and cephalothin, which are not inactivated by gastric acid in the stomach and are more readily absorbed in the gastrointestinal tract.

Aldehydes

• are among the most effective antimicrobial drugs. • Glutaraldehye, which is a liquid, and formaldehyde, which is a gas, are highly reactive chemicals. • Hospital personnel and scientists use 2% solutions of glutaradehyde to sterilize medical and dental equipment. • Formaldehyde is an excellent disinfectant. • Morticians and scientists use commercial solutions of formaldehyde in water, commonly called formalin, for embalming and preserving biological specimens. • Formaldehyde must be handled with care because it irritates mucous membranes and is carcinogenic.

Alcohols

• are effective against bacteria, fungi and enveloped viruses. • However they are not effective against noneneveloped viruses, fungal and bacterial endospores. • It denatures proteins and dissolves lipids, an action that may lead to cell membrane disintegration. • tendency to evaporate rapidly leaving no residue on surfaces is an advantage. • Swabbing the skin with alcohol prior to an injection by wiping away dirt-laden with microorganisms is more effective than other abrasive chemicals. • Two of the most commonly used alcohols are ethanol and isopropanol. • Pure alcohol is not an effective antimicrobial drug because the denaturation of proteins requires water; therefore, 70% to 90% alcohol is typically used to control microorganisms. • evaporate rapidly, which is advantageous in that they leave no residue, but disadvantageous in that they may not contact microbes long enough to be effective.

Halogens

• consist of a group of highly reactive, nonmetallic chemical elements: bromine, chlorine, fluorine and iodine. • It is effective against many viruses, protozoan cysts, vegetative bacterial and fungal cells as well as bacterial and fungal spores. • Iodine is one of the oldest and most effective antiseptic. • Iodine denatures proteins and damages the integrity of the cytoplasmic membrane by attaching to the phospholipids of the membrane. • Health professionals use iodine either as a tincture or as an iodophor, which is iodine complexed with a surfactant that gradually releases iodine. • Iodophors have the advantage of being long-lasting, nonstaining, and nonirritating to the skin. • Municipalities commonly use chlorine to treat drinking water supplies, swimming pools, and wastewater from sewage treatment plants. • Another chlorine compound, sodium hypochlorite, is used as a disinfectant in hemodialysis systems, food-processing establishments, dairies, and household bleach (Clorox). • Chlorine dioxide is a gas that can be used to disinfect large spaces such as federal office buildings contaminated with anthrax spores following the bioterrorism attacks. •Chemical combinations of chlorine and ammonia called chloramines are used in wound dressings and skin antiseptics. • Bromine is an effective disinfectant in hot tubs as it evaporates less readily than chlorine at high temperatures.

Direct Flaming

• is one of the simplest procedures of dry heat sterilization. • In the microbiology laboratory, incinerating inoculating loops and top of test-tubes are part of the standard aseptic procedure. • Health care workers incinerate disposable hospital gowns and contaminated dressings; field epidemiologists incinerate the carcasses of animals that have diseases such as anthrax or bovine spongiform encephalopathy (mad cow disease).

Filtration

• is the process of passing a liquid or gas through a filter with pores small enough to trap microorganisms. • A vacuum is often created to assist the movement of the liquid through the filter. • This method is used to sterilize heat-sensitive liquids and gases, such as antibiotic solutions, vaccines, liquid vitamins, culture media, and enzymes. • Healthcare and laboratory workers routinely use filtration to prevent airborne contamination by microbes. • The use of surgical masks prevents contamination of the environment by microbes exhaled by medical personnel, and cotton plugs in culture flasks prevents contamination of cultures by airborne diseases.