Microbial Control 1. Basic Principles of Microbial Control 2.

Microbial Control

1

Basic Principles of Microbial Control

2

The Selection of Microbial Control Methods

•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

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Relative susceptibilities of microbes to antimicrobial agents

Figure 9.2

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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

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Effect of temperature on the efficacy of an antimicrobial chemical

Figure 9.3

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•Methods for Evaluating Disinfectants and Antiseptics▫Phenol coefficient

Evaluates efficacy of disinfectants and antiseptics by comparing an agent’s ability to control microbes to phenol

Greater than 1.0 indicates agent is more effective than phenol

Has been replaced by newer methods

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• Methods for Evaluating Disinfectants and Antiseptics– 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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•Methods for Evaluating Disinfectants and Antiseptics▫Kelsey-Sykes capacity test

Alternative assessment approved by the European Union

Bacterial suspensions added to the chemical being tested

Samples removed at predetermined times and incubated

Lack of bacterial reproduction reveals minimum time required for the disinfectant to be effective

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•Methods for Evaluating Disinfectants and Antiseptics▫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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Physical Methods of Microbial Control

•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

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• Heat-Related Methods▫Moist heat

Used to disinfect (remove organisms and spores), sanitize (kill organisms but not necessarily their spores), and sterilize (kill all organisms and spores)

Denatures proteins and destroys cytoplasmic membranes

More effective than dry heat Methods of microbial control using moist heat

Boiling Autoclaving Pasteurization Ultrahigh-temperature sterilization

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•Heat-Related Methods▫Moist heat

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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Autoclaving Pressure applied to boiling water prevents steam

from escaping Boiling temperature increases as pressure

increases Autoclave conditions – 121ºC, 15 psi, 15 min

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The relationship between temperature and pressure

Figure 9.5

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Sterility indicator

Figure 9.7

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Pasteurization Used for milk, ice cream, yogurt, and fruit

juices Not sterilization

▫Heat-tolerant microbes survive Pasteurization of milk

▫Batch method▫Flash pasteurization (High temp, short time)▫Ultrahigh-temperature pasteurization (very high

temp, very short time)

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Pasteurization of milkBatch 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.

In the vat the milk is heated and held throughout the holding period while being agitated. The milk may be cooled in the vat or removed hot after the holding time is completed for every particle.

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Pasteurization of milkFlash method• High Temperature Short Time (HTST)• Milk is heated to 72°C (161.6°F) for at least 15

seconds. • Used for perishable beverages like fruit and

vegetable juices, beer, and some dairy products. Compared to other pasteurization processes, it maintains color and flavor better.

• It is done prior to filling into containers in order to kill spoilage microorganisms, to make the products safer and extend their shelf life. Flash pasteurization must be used in conjunction with sterile fill technology.

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Pasteurization of milkUltrahigh-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.

• The most common UHT product is milk, but the process is also used for fruit juices, cream, soy milk, yogurt, wine, soups, and stews.

• Can cause browning and change the taste and smell of dairy products.

• UHT canned milk has a typical shelf life of six to nine months, until opened.

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Ultrahigh-temperature sterilization 140ºC for 1 sec, then rapid cooling Treated liquids can be stored at room

temperature

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•Heat-Related Methods▫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▫Dessication is 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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The use of dessication as a means of preserving apricots

Figure 9.8

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Filtration equipment used for microbial control

Figure 9.9

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The role of HEPA filters in biological safety cabinets

Figure 9.10

High-Efficiency Particulate Arresting (HEPA) air filters are used in medical facilities, automobiles, aircraft, and homes. The filter must remove 99.97% of all particles greater than 0.3 micrometer from the air that passes through.

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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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Increased shelf life of food achieved by ionizing radiation

Figure 9.11

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•Radiation▫Nonionizing radiation

Wavelengths greater than 1 nm 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 Biosafety Level 1 (BSL-1)

Handling pathogens that do not cause disease in healthy humans

Biosafety Level 2 (BSL-2) Handling of moderately hazardous agents

Biosafety Level 3 (BSL-3) Handling of microbes in safety cabinets

Biosafety Level 4 (BSL-4) Handling of microbes that cause severe or fatal

disease

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A BSL-4 worker carries Ebola virus cultures

Figure 9.12

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Chemical Methods of Microbial Control

•Affect microbes’ cell walls, cytoplasmic membranes, proteins, or DNA

•Effect varies with differing environmental conditions

•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▫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

Iodine tablets, iodophores, chlorine treatment, bleach, chloramines, and bromine disinfection

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Pre-op preparation for hand surgery

Figure 9.14

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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 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

Reduce surface tension of solvents▫Soaps and detergents

Soaps have hydrophilic and hydrophobic ends Good degerming agents but not antimicrobial

Detergents are positively charged organic surfactants▫Quats (Quaternary ammonium cations)

Low-level disinfectants; disrupts cell membranes Ideal for many medical and industrial applications 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▫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

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•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

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Antimicrobial Agents

•Chemicals that affect physiology in any manner

•Chemotherapeutic agents▫Drugs that act against diseases

•Antimicrobial agents ▫Drugs that treat infections

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The History of Antimicrobial Agents

•Semi-synthetics▫Chemically altered antibiotics that are

more effective than naturally occurring ones

•Synthetics▫Antimicrobials that are completely

synthesized in a lab

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Mechanisms of Antimicrobial Action

•Key is selective toxicity•Antibacterial drugs constitute largest

number and diversity of antimicrobial agents

•Fewer drugs to treat eukaryotic infections (protozoa, fungi, helminthes)

•Even fewer antiviral drugs

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• Inhibition of bacterial wall synthesis• Disruption of existing cytoplasmic

membranes• Inhibition of Protein Synthesis• Inhibition of Nucleic Acid Synthesis• Inhibition of Metabolic Pathways• Prevention of Virus Attachment

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Basic Principles of Microbial Control•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 controls passage of chemicals into and out of cell Cellular contents leak out when damaged

Non-enveloped viruses have greater tolerance of harsh conditions

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Mechanisms of Antimicrobial Action•Inhibition of Cell Wall Synthesis

▫Inhibition of bacterial wall synthesis Most common agents prevent cross-linkage of

NAM-NAG subunits Beta-lactams are most prominent in this

group Functional groups are beta-lactam rings Beta-lactams bind to enzymes that cross-link

NAM-NAG subunits Bacteria have weakened cell walls and

eventually lyse

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•Inhibition of Cell Wall Synthesis▫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

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▫Inhibition of synthesis of bacterial walls 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

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▫Inhibition of synthesis of bacterial walls Prevent bacteria from increasing amount of

peptidoglycan Have no effect on existing peptidoglycan layer Effective only for growing cells

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Mechanisms of Antimicrobial Action•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

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•Disruption of Cytoplasmic Membranes▫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

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Which topical ointment is best?• Neomycin is an aminoglycoside antibiotic (disrupts protein

synthesis). It has excellent activity against Gram-negative bacteria, and has partial activity against Gram-positive bacteria.

• Polymixin disrupts bacterial cell membranes by interacting with its phospholipids. They are selectively toxic for Gram-negative bacteria.

• 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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Basic Principles of Microbial Control•Action of Antimicrobial Agents

▫Damage to proteins and nucleic acids Protein function depends on 3-D shape

Extreme heat or certain chemicals denature proteins

Chemicals, radiation, and heat can alter or destroy nucleic acids Can produce fatal mutants Can halt protein synthesis through action on

RNA

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•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

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•Inhibition of Protein Synthesis▫Aminoglycosides: excellent against Gram

negatives, partially effective against Gram positives amikacin (Amikin®) gentamicin (Garamycin®) kanamycin (Kantrex®) neomycin (Mycifradin®) streptomycin tobramycin (TOBI Solution®, TobraDex®)

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Antimicrobial inhibition of protein synthesis

Figure 10.4

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•Inhibition of Nucleic Acid Synthesis▫Several drugs block DNA replication or

mRNA transcription▫Drugs often affect both eukaryotic and

prokaryotic cells▫Not normally used to treat infections ▫Used in research and perhaps to slow

cancer cell replication

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Nucleotides and some of their antimicrobial analogs

Figure 10.7

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Nucleotides

Acyclovir• 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).

• Acyclovir is also sometimes used to prevent outbreaks of herpes sores in people who are infected with the virus.

• 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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Mechanisms of Antimicrobial Action•Inhibition of Nucleic Acid Synthesis

▫Quinolones and fluoroquinolones Act against prokaryotic DNA gyrase (enzyme

that is needed for DNA to unwind during replication)

▫Inhibitors of RNA polymerase (enzyme used during transcription)

▫Reverse transcriptase inhibitors Act against an enzyme HIV uses in its

replication cycle Does not harm people because humans lack

reverse transcriptase

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•Inhibition of Nucleic Acid Synthesis▫Nucleotide analogs

Interfere with function of nucleic acids Distort shapes of nucleic acid molecules and

prevent further replication, transcription, or translation

Most often used against viruses Effective against rapidly dividing cancer cells

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Nucleotides and some of their antimicrobial analogs

Figure 10.7

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Nucleotides

Mechanisms of Antimicrobial Action•Inhibition of Metabolic Pathways

▫Antimetabolic agents can be effective when pathogen and host metabolic processes differ

▫Quinolones interfere with the metabolism of malaria parasites

▫Heavy metals inactivate enzymes▫Some agents disrupt glucose uptake by

many protozoa and parasitic worms▫Some drugs block activation of viruses

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Mechanisms of Antimicrobial Action•Inhibition of Metabolic Pathways

▫Antiviral agents can target unique aspects of viral metabolism Amantadine, rimantadine, and weak organic

bases prevent viral uncoating▫Protease inhibitors interfere with an enzyme

that HIV needs in its replication cycle

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•Prevention of Virus Attachment▫Attachment antagonists block viral

attachment or receptor proteins▫New area of antimicrobial drug

development

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Clinical Considerations in Prescribing Antimicrobial Drugs

• Ideal Antimicrobial Agent▫ Readily available▫ Inexpensive▫Fast-acting▫ Chemically stable during storage▫ Easily administered▫ Nontoxic and nonallergenic▫ Selectively toxic against wide range of pathogens▫ Capable of controlling microbial growth while being

harmless to humans, animals, and objects

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•Spectrum of Action▫Number of different pathogens a drug acts

against Narrow-spectrum effective against few organisms

(Gram positive bacteria only) Broad-spectrum effective against many organisms

(Gram positive and Gram negative bacteria) May allow for secondary or superinfections to develop Killing of normal flora reduces microbial antagonism

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Spectrum of action for selected antimicrobial agents

Figure 10.8

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•Efficacy▫Ascertained by

Diffusion susceptibility test Minimum inhibitory concentration test Minimum bactericidal concentration test

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Diffusion Susceptibility Test:Zone of inhibition

Figure 10.9

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Minimum inhibitory concentration test

Figure 10.10

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A minimum bactericidal concentration test

Figure 10.12

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•Routes of Administration▫Topical application of drug for external

infections▫Oral route requires no needles and is self-

administered▫Intramuscular (IM) administration delivers

drug via needle into muscle▫Intravenous (IV) administration delivers

drug directly to bloodstream▫Must know how antimicrobial agent will be

distributed to infected tissues

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Effect of route of administration on chemotherapeutic agent

Figure 10.13

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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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Side effects resulting from toxicity of antimicrobial agents

Figure 10.14

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•Safety and Side Effects▫Disruption of normal microbiota

May result in secondary infections Overgrowth of normal flora causing

superinfections Of greatest concern for hospitalized patients

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Resistance to Antimicrobial Drugs•The Development of Resistance in

Populations▫Some pathogens are naturally resistant ▫Resistance by bacteria acquired in two

ways New mutations of chromosomal genes Acquisition of resistance genes (R-plasmids)

via transformation, transduction, and conjugation

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The development of a resistant strain of bacteria

Figure 10.15

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Resistance to Antimicrobial Drugs•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 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

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How -lactamase renders penicillin inactive

Figure 10.16

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Resistance to Antimicrobial 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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Resistance to Antimicrobial Drugs•Retarding Resistance

▫Maintain high concentration of drug in patient for sufficient time Kills all sensitive cells and inhibits others so

immune system can destroy▫Use antimicrobial agents in combination

Synergism vs. antagonism

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Example of synergism between two antimicrobial agents

Figure 10.17

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Resistance to Antimicrobial Drugs•Retarding Resistance

▫Use antimicrobials only when necessary▫Develop new variations of existing drugs

Second-generation drugs Third-generation drugs

▫Search for new antibiotics, semi-synthetics, and synthetics Bacteriocins Design drugs complementary to the shape of

microbial proteins to inhibit them

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Vaccination•Vaccine – use the immune system to protect

against infectious disease•Types of vaccines

▫attenuated (weakened) microbe; virulence factors are removed

▫heat-killed / chemically killed microbe▫toxoids

•Passive versus Adaptive vaccination▫passive – immune system products from another

mothers milk (presence of IgA) gamma-globulin (anti-bee venom, anti-hepatitis A, etc)

▫active – stimulate individuals immune system to produce memory cells

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Effect of smallpox vaccine

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Initial “modern” vaccine: 1796.

U.S. cases against diseases for which there are vaccines.

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SSPE: sub-acute sclerosingpanencephalitis (late stagemeasles)

•Why Your Cellphone Has More Bacteria Than a Toilet Seat

•By Susan E. Matthews, MyHealthNewsDaily Staff Writer | LiveScience.com – 3 hrs ago

•http://news.yahoo.com/why-cellphone-mor

e-bacteria-toilet-seat-124147769.html

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http://news.yahoo.com/why-cellphone-more-bacteria-toilet-seat-124147769.html

http://news.yahoo.com/why-cellphone-more-bacteria-toilet-seat-124147769.html

•Cellphones carry 10 times more bacteria than most toilet seats, so it shouldn't be surprising that a man in Uganda reportedly contracted Ebola after stealing one.

•He stole the phone from a quarantined ward of a hospital, near the site of a recent Ebola outbreak.

•While toilets tend to get cleaned frequently, because people associate the bathroom with germs, cellphones and other commonly handled objects — like remote controls— are often left out of the cleaning routine.

•Cellphones pick up germs all the time; some people talk on their phone on toilets.

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•However, the amount of germs on a phone isn't a problem — it’s the sharing of phones between people. Without sharing, each phone carries just one set of germs, and won't get its owner sick.

•The problem with phones is that we're in constant contact with them, and they spend a lot of time in close proximity to our faces and mouths.

•And, because it's an electronic device, most people are hesitant about cleaning them.

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•This is also this case with remote controls, which, are also often used by people when they're sick.

•Remotes are more frequently shared, too, so they're usually even worse than phones for spreading germs.

•Other common culprits that are hotspots of unseen disease include office phones, shopping carts and the first-floor buttons of elevators.

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Microbial Control 1. Basic Principles of Microbial Control 2.

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Transcript of Microbial Control 1. Basic Principles of Microbial Control 2.