Microbial Control
Basic Principles of Microbial Control
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Action of Antimicrobial Agents
Alteration of cell walls and membranes
Cell wall maintains integrity of cell
Cells burst due to osmotic effects when damaged
Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell
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Cellular contents leak out when damaged
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Nonenveloped viruses have greater tolerance of harsh conditions
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Damage to proteins and nucleic acids
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Protein function depends on 3-D shape
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Extreme heat or certain chemicals denature proteins
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Chemicals, radiation, and heat can alter or destroy nucleic acids
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Can produce fatal mutants
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Can halt protein synthesis through action on RNA
The Selection of Microbial Control Methods
Ideally, agents should be:
• Inexpensive
• Fast-acting
• Stable during storage
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Capable of controlling microbial growth while being harmless to humans, animals, and objects
• Factors Affecting the Efficacy of Antimicrobial Methods
◦ Site to be treated
Harsh chemicals and extreme heat cannot be used on humans, animals, and fragile objects
Method of microbial control based on site of medical procedure
Relative susceptibilities of microbes to antimicrobial agents
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Factors Affecting the Efficacy of Antimicrobial Methods
Relative susceptibility of microorganisms
Germicides classified as high, intermediate, or low effectiveness
High-level kill all pathogens, including endospores
Intermediate-level kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria
Low-level kill vegetative bacteria, fungi, protozoa, and some viruses
◦ Methods for Evaluating Disinfectants and Antiseptics
▪ Phenol coefficient
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Evaluates efficacy of disinfectants and antiseptics by comparing an agent’s ability to control
microbes to phenol
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Greater than 1.0 indicates agent is more effective than phenol
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Has been replaced by newer methods
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Methods for Evaluating Disinfectants and Antiseptics
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Use-dilution test
Metal cylinders dipped into broth cultures of bacteria
Contaminated cylinder immersed into dilution of disinfectant
Cylinders removed and placed into tube of medium to see how much bacteria survived
Most effective agents entirely prevent growth at highest dilution
Current standard test in the U.S.
New standard procedure being developed
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Kelsey-Sykes capacity test
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Alternative assessment approved by the European Union
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Bacterial suspensions added to the chemical being tested
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Samples removed at predetermined times and incubated
Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective
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Physical Methods of Microbial Control
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Heat-Related Methods
Effects of high temperatures
Denature proteins
Interfere with integrity of cytoplasmic membrane and cell wall
Disrupt structure and function of nucleic acids
Thermal death point
Lowest temperature that kills all cells in broth in 10 min
Thermal death time
Time to sterilize volume of liquid at set temperature
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
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Moist heat
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Used to disinfect (remove organisms and spores), sanitize (kill organisms but not necessarily
their spores), and sterilize (kill all organisms and spores)
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Denatures proteins and destroys cytoplasmic membranes
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More effective than dry heat
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Methods of microbial control using moist heat
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Boiling
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Autoclaving
Pasteurization
Ultrahigh-temperature sterilization
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Boiling
Kills vegetative cells of bacteria and fungi, protozoan trophozoites, and most viruses
Boiling time is critical
Different elevations require different boiling times
Endospores, protozoan cysts, and some viruses can survive boiling
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Moist heat
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Autoclaving
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Pressure applied to boiling water prevents steam from escaping
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Boiling temperature increases as pressure increases
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Autoclave conditions – 121ºC, 15 psi, 15 min
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Pasteurization
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Used for milk, ice cream, yogurt, and fruit juices
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Not sterilization
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Heat-tolerant microbes survive
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Pasteurization of milk
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Batch method
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Flash pasteurization (High temp, short time)
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Ultrahigh-temperature pasteurization (very short time)
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◦ Pasteurization of milk Batch method
◦ The batch method uses a vat pasteurizer which consists of a jacketed vat surrounded by either
circulating water, steam or heating coils of water or steam.
◦ Pasteurization of milk Ultrahigh-temperature method
• Heating for 1-2 seconds at a temperature exceeding 135°C (275°F), which is the temperature
required to kill spores in milk.
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The most common UHT product is milk, but the process is also used for fruit juices, cream, soy
milk, yogurt, wine, soups, and stews.
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Can cause browning and change the taste and smell of dairy products.
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UHT milk has a typical shelf life of six to nine months, until opened
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Ultrahigh-temperature sterilization
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140ºC for 1 sec, then rapid cooling
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Treated liquids can be stored at room temperature
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Dry heat
Used for materials that cannot be sterilized with moist heat
Denatures proteins and oxidizes metabolic and structural chemicals
Requires higher temperatures for longer time than moist heat
Incineration is ultimate means of sterilization
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Refrigeration and Freezing
Decrease microbial metabolism, growth, and reproduction
Chemical reactions occur slower at low temperatures
Liquid water not available
Psychrophilic microbes can multiply in refrigerated foods
Refrigeration halts growth of most pathogens
Slow freezing more effective than quick freezing
Organisms vary in susceptibility to freezing
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Dessication and Lyophilization
Drying (98% of the water is removed) inhibits growth due to removal of water
Lyophilization (freeze-drying)
Substance is rapidly frozen and sealed in a vacuum
Substance may also be turned into a powder
Used for long-term preservation of microbial cultures
Prevents formation of damaging ice crystals
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Osmotic Pressure
High concentrations of salt or sugar in foods to inhibit growth
Cells in hypertonic solution of salt or sugar lose water
Fungi have greater ability than bacteria to survive hypertonic environments
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Radiation
Ionizing radiation
Wavelengths shorter than 1 nm
Electron beams, gamma rays
Ejects electrons from atoms to create ions
Ions disrupt hydrogen bonding, cause oxidation, and create hydroxide ions
Hydroxide ions denature other molecules (DNA)
Electron beams – effective at killing but do not penetrate well
Gamma rays – penetrate well but require hours to kill microbes
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Nonionizing radiation
Wavelengths greater than 1 nm
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Excites electrons, causing them to make new covalent bonds
Affects 3-D structure of proteins and nucleic acids
UV light causes pyrimidine dimers in DNA
UV light does not penetrate well
Suitable for disinfecting air, transparent fluids, and surfaces of objects
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Biosafety Levels
▪ Four levels of safety in labs dealing with pathogens
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Biosafety Level 1 (BSL-1)
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Handling pathogens that do not cause disease in healthy humans
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Biosafety Level 2 (BSL-2)
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Handling of moderately hazardous agents
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Biosafety Level 3 (BSL-3)
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Handling of microbes in safety cabinets
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Biosafety Level 4 (BSL-4)
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Handling of microbes that cause severe or fatal disease
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Chemical Methods of Microbial Control
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Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA
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Effect varies with differing environmental conditions
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Often more effective against enveloped viruses and vegetative cells of bacteria, fungi, and
protozoa
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Phenol and Phenolics
Intermediate- to low-level disinfectants
Denature proteins and disrupt cell membranes
Effective in presence of organic matter
Remain active for prolonged time
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Commonly used in health care settings, labs, and homes
Have disagreeable odor and possible side effects
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Alcohols
Intermediate-level disinfectants
Denature proteins and disrupt cytoplasmic membranes
More effective than soap in removing bacteria from hands
Swabbing of skin with 70% ethanol prior to injection
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Halogens
Intermediate-level antimicrobial chemicals
Believed to damage enzymes via oxidation or by denaturation
Widely used in numerous applications
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disinfection
Iodine tablets, iodophores, chlorine treatment, bleach, chloramines, and bromine
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Oxidizing Agents
◦ Peroxides, ozone, and peracetic acid
◦ Kill by oxidation of microbial enzymes
◦ High-level disinfectants and antiseptics
◦ Hydrogen peroxide (H2O2) can disinfect and sterilize surfaces
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Not useful for treating open wounds due to catalase activity: the tissues convert it into
H20 and 0ygen bubbles.
◦ Ozone treatment of drinking water
◦ Peracetic acid is an effective sporocide used to sterilize equipment
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Surfactants
◦ “Surface active” chemicals
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Reduce surface tension of solvents
◦ Soaps and detergents
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Soaps have hydrophilic and hydrophobic ends
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Good degerming agents but not antimicrobial
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Detergents are positively charged organic surfactants
◦ Quats (Quaternary ammonium cations)
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Low-level disinfectants; disrupts cell membranes
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Ideal for many medical and industrial application
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Good against fungi, amoeba, and enveloped viruses, but not endospores,
Mycobacterium tuberculosis and non-enveloped viruses.
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Heavy Metals
Heavy-metal ions denature proteins
Low-level bacteriostatic and fungistatic agents
1% silver nitrate to prevent blindness caused by N. gonorrhoeae
Thimerosal used to preserve vaccines
Copper inhibits algal growth
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Aldehydes
Compounds containing terminal –CHO groups
Cross-link functional groups to denature proteins and inactivate nucleic acids
Glutaraldehyde disinfects and sterilizes
Formalin used in embalming and disinfection of rooms and instruments
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Gaseous Agents
Microbicidal and sporicidal gases used in closed chambers to sterilize items
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Denature proteins and DNA by cross-linking functional groups
Used in hospitals and dental offices
Disadvantages
Can be hazardous to people
Often highly explosive
Extremely poisonous
Potentially carcinogenic
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Enzymes
Antimicrobial enzymes act against microorganisms
Human tears contain lysozyme
Digests peptidoglycan cell wall of bacteria
Enzymes to control microbes in the environment
Lysozyme used to reduce the number of bacteria in cheese
Prionzyme can remove prions on medical instruments
Antimicrobials
Antibiotics, semi-synthetic, and synthetic chemicals
Typically used for treatment of disease
Some used for antimicrobial control outside the body
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Development of Resistant Microbes
◦ Little evidence that products containing antiseptic and disinfecting chemicals is beneficial to
human or animal health
◦ Use of such products promotes development of resistant microbes
Antimicrobial Agents
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Chemicals that affect physiology in any manner
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Chemotherapeutic agents
◦ Drugs that act against diseases
◦ Drugs that treat infections
The History of Antimicrobial Agents
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Semi-synthetics
▪ Chemically altered antibiotics that are more effective than naturally occurring ones
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Synthetics
▪ Antimicrobials that are completely synthesized in a la
Mechanisms of Antimicrobial Action
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Key is selective toxicity
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Antibacterial drugs constitute largest number and diversity of antimicrobial agents
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Fewer drugs to treat eukaryotic infections (protozoa, fungi, helminthes)
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Even fewer antiviral drugs
Inhibition of Cell Wall Synthesis
◦ Inhibition of bacterial wall synthesis
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Most common agents prevent cross-linkage of NAM subunits
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Beta-lactams are most prominent in this group
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Functional groups are beta-lactam rings
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Beta-lactams bind to enzymes that cross-link NAM subunits
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Bacteria have weakened cell walls and eventually lyse
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Inhibition of synthesis of bacterial walls
Semi-synthetic derivatives of beta-lactams
More stable in acidic environments
More readily absorbed
Less susceptible to deactivation
More active against more types of bacteria
Simplest beta-lactams – effective only against aerobic Gram-negatives
Vancomycin and cycloserine
Interfere with particular bridges that link NAM subunits in many Gram-positives
Bacitracin
Blocks secretion of NAG and NAM from cytoplasm
Effective against Gram positives
Isoniazid and ethambutol
Disrupt mycolic acid formation in mycobacterial species
Prevent bacteria from increasing amount of peptidoglycan
Have no effect on existing peptidoglycan layer
Effective only for growing cells
Inhibition of Protein Synthesis
Prokaryotic ribosomes are 70S (30S and 50S)
Eukaryotic ribosomes are 80S (40S and 60S)
Drugs can selectively target translation
Mitochondria of animals and humans contain 70S ribosomes
Can be harmful
Aminoglycosides: excellent against Gram negatives, partially effective against Gram positives
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amikacin (Amikin®)
gentamicin (Garamycin®)
kanamycin (Kantrex®)
neomycin (Mycifradin®)
streptomycin
tobramycin (TOBI Solution®, TobraDex®)
Disruption of Cytoplasmic Membranes
Some drugs form channel through cytoplasmic membrane and damage its integrity
Amphotericin B attaches to ergosterol in fungal membranes
Humans somewhat susceptible because cholesterol similar to ergosterol
Bacteria lack sterols; not susceptible
Azoles and allyamines inhibit ergosterol synthesis
Polymyxin disrupts cytoplasmic membranes of Gram-negatives
Oral form is toxic to human kidneys, so only used topically
Some parasitic drugs act against cytoplasmic membranes
Which topical ointment is best?
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Neomycin is an aminoglycoside antibiotic (disrupts protein synthesis). It has excellent
activity against Gram-negative bacteria, and has partial activity against Gram-positive bacteria.
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Polymixin disrupts bacterial cell membranes by interacting with its phospholipids. They are
selectively toxic for Gram-negative bacteria.
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Bacitracin disrupts cell wall synthesis. Its action is on Gram-positive organisms. It can cause
contact dermatitis and cross-reacts with allergic sensitivity to sulfa-drugs.
Which topical ointment is best: Neomycin or Triple Antibiotic (contains all three)
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Mechanisms of Antimicrobial Action
Inhibition of Metabolic Pathways
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Antimetabolic agents can be effective when pathogen and host metabolic processes differ
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Quinolones interfere with the metabolism of malaria parasites
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Heavy metals inactivate enzymes
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Some agents disrupt glucose uptake by many protozoa and parasitic worms
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Some drugs block activation of viruses
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Antiviral agents can target unique aspects of viral metabolism
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Amantadine, rimantadine, and weak organic bases prevent viral uncoating
Protease inhibitors interfere with an enzyme that HIV needs in its replication cycle
Inhibition of Nucleic Acid Synthesis
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Several drugs block DNA replication or mRNA transcription
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Drugs often affect both eukaryotic and prokaryotic cells
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Not normally used to treat infections
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Used in research and perhaps to slow cancer cell replication
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Nucleotide analogs
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Interfere with function of nucleic acids
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Distort shapes of nucleic acid molecules and prevent further replication, transcription, or
translation
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Most often used against viruses
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Effective against rapidly dividing cancer cells
Acyclovir
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Acyclovir is used to decrease pain and speed the healing of herpes sores or blisters in
people who have varicella (chickenpox), herpes zoster (shingles; a rash that can occur in people who
have had chickenpox in the past), and first-time or repeat outbreaks of genital herpes (a herpes virus
infection that causes sores to form around the genitals and rectum from time to time).
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Acyclovir is also sometimes used to prevent outbreaks of herpes sores in people who are
infected with the virus.
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Acyclovir disrupts nucleic acid function. It works by stopping the spread of the herpes virus in
the body. Acyclovir will not cure herpes or protect others from catching it.
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Quinolones and fluoroquinolones
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Act against prokaryotic DNA gyrase (enzyme that is needed for DNA to unwind during
replication)
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Inhibitors of RNA polymerase (enzyme used during transcription)
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Reverse transcriptase inhibitors
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Act against an enzyme HIV uses in its replication cycle
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Does not harm people because humans lack reverse transcriptase
Prevention of Virus Attachment
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Attachment antagonists block viral attachment or receptor proteins
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New area of antimicrobial drug development
Clinical Considerations in Prescribing Antimicrobial Drugs
Ideal Antimicrobial Agent
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Readily available
Inexpensive
Chemically stable
Easily administered
Nontoxic and nonallergenic
Selectively toxic against wide range of pathogens
Spectrum of Action
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Number of different pathogens a drug acts against
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Narrow-spectrum effective against few organisms
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Broad-spectrum effective against many organisms
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May allow for secondary or superinfections to develop
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Killing of normal flora reduces microbial antagonism
Spectrum of action for selected antimicrobial agents
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Efficacy
Ascertained by
Diffusion susceptibility test
Minimum inhibitory concentration test
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Minimum bactericidal concentration test
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Routes of Administration
Topical application of drug for external infections
Oral route requires no needles and is self-administered
Intramuscular administration delivers drug via needle into muscle
Intravenous administration delivers drug directly to bloodstream
Must know how antimicrobial agent will be distributed to infected tissues
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Safety and Side Effects
Toxicity
Cause of many adverse reactions poorly understood
Drugs may be toxic to kidneys, liver, or nerves
Consideration needed when prescribing drugs to pregnant women
Allergies
Allergic reactions are rare but may be life threatening
Anaphylactic shock
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Disruption of normal microbiota
May result in secondary infections
Overgrowth of normal flora causing superinfections
Of greatest concern for hospitalized patients
Resistance to Antimicrobial Drugs
The Development of Resistance in Populations
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Some pathogens are naturally resistant
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Resistance by bacteria acquired in two ways
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New mutations of chromosomal genes
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Acquisition of resistance genes (R-plasmids) via transformation, transduction, and conjugation
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Mechanisms of Resistance
At least six mechanisms of microbial resistance
Production of enzyme that destroys or deactivates drug
Slow or prevent entry of drug into the cell
Alter target of drug so it binds less effectively
Alter their metabolic chemistry
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Pump antimicrobial drug out of the cell before it can act
Mycobacterium tuberculosis produces MfpA protein
Binds DNA gyrase preventing the binding of fluoroquinolone drugs
Multiple Resistance and Cross Resistance
Pathogen can acquire resistance to more than one drug
Common when R-plasmids exchanged
Develop in hospitals and nursing homes
Constant use of drugs eliminates sensitive cells
Superbugs
Cross resistance
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Retarding Resistance
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Maintain high concentration of drug in patient for sufficient time
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Kills all sensitive cells and inhibits others so immune system can destroy
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Use antimicrobial agents in combination
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Synergism vs. antagonism
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Use antimicrobials only when necessary
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Develop new variations of existing drugs
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Second-generation drugs
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Third-generation drugs
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Search for new antibiotics, semi-synthetics, and synthetics
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Bacteriocins
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Design drugs complementary to the shape of microbial proteins to inhibit them
Vaccination
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Vaccine – use the immune system to protect against infectious disease
Types of vaccines
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attenuated (weakened) microbe; virulence factors are removed
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heat-killed / chemically killed microbe
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toxoids
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Passive versus Adaptive vaccination
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passive – immune system products from another
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mothers milk (presence of IgA)
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gamma-globulin (anti-bee venom, anti-hepatitis A, etc)
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active – stimulate individuals immune system to produce memory cells
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