Module 4

Question 1

Explain the differences between disinfectants, antiseptics, and sterilants

Answer 1

• Sterilants: Effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores (chemicals). Reserved for labs, medical, manufacturing and food industry settings.
• Disinfectants: Inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Does not lead to sterilization because endospores tend to survive even when all vegetative cells have been killed. Should be fast acting, stable, easy to prepare, inexpensive, and easy to use. Used in clinical or non-clinincal settings.
• Antiseptics: Safe on living skin or tissues unlike disinfectants. In addition to having characteristics of a disinfectant must also be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage.

Question 2

Describe how sterilization and disinfection control the presence of microorganisms

Answer 2

• Sterilization can be accomplished through physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter. Or by chemical means through sterilants.
• Disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or head.

Question 3

How many biological safety levels are there?

Answer 3

• Four classification levels by NIH

Question 4

Give examples of how microbes are handles at different levels

Question 5

BSL-1

Answer 5

• Agents that generally do not cause infection in healthy human adults
• Bacteria and viruses known to infect animals other than humans, such as baculoviruses (insect viruses)
• Few precautions necessary
• Lab workers use standard aseptic technique, may work with agents at an open lab bench or table, wearing PPE (lab coat, gloves, goggles)
• Other than a sink for handwashing and doors to separate lab from rest of building, no additional modifications needed, may also have autoclave.
• Nonpathogenic strains of E. coli

Question 6

BSL-2

Answer 6

• Pose moderate risk to lab workers and community
• Indigenous, meaning commonly found in geographical area
• Restricted access, required PPE (including face shield at times), use of biological safety cabinets for procedures that may disperse agents through air (called aerosolization)
• Labs equiped with self-closing doors, eyewash station, autoclave
• Staphylococcus aureus

Question 7

BSL-3

Answer 7

• Have potential to cause lethal infections by inhalation
• May be indigenous or exotic, meaning they are derived from a foreign location
• include pathogens
• Require restricted access
• Lab workers are under medical surveillance, possibly receiving vaccinations for the microbes they work with
• Need PPE, must wear a respirator and work with microbes and infectious agents in biological safety cabinet at all times
• Require a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors at the entrance
• Labs equipped with directional airflow, meaning that clean air is pulled through lab from clean areas to potentially contaminated areas
• Air cannot be recirculated, so a constant supply of clean air is required.
• Ex. Mycobacterium tuberculosis

Question 8

BSL-4

Answer 8

• Most dangerous and fatal
• Typically exotic
• Easily transmitted by inhalation
• Cause infections for which there are no treatments or vaccinations
• Have same things as BSL-3 + more
• Must change clothing on entering the lab, shower on exiting, and decontaminate all material on exiting
• In lab must wear full-body protective suit with a designated air supply or conduct all work within a biological safety cabinet with a high-efficiency particulate air (HEPA) filtered air supply and a doubly HEPA-filtered exhuast
• If wearing a suit, air pressure within suit must be higher than that outside the suit, so that if a leak in the suit occurs, laboratory air that may be contaminated cannot be drawn into the suit
• Lab must be located either in a separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own decontamination system.
• Ex. Ebola and marburg viruses

Question 9

Compare how different physical methods affect or limit microbial growth, including:
heating, refrigeration, freezing, high pressure, desiccation, lyophilization, irradiation and
filtration

Question 10

How does heat limit or affect microbial growth?

Answer 10

• Kills microbes by altering membranes and denaturing proteins
• Thermal death point: lowest temp at which all microbes are killed in a 10-minute exposure
• Thermal death time: length of time needed to kill all microorganisms in a sample at a given temp
• Two protocols: dry-heat sterilization and moist-heat sterilization (better because penetrates cells better than dry heat)

Question 11

How does Refrigeration and Freezing limit or affect microbial growth?

Answer 11

• Psychrophiles are an exception
• Refrigerators maintain temp between 0 and 7. This temp inhibits microbial metabolism, slowing growth of microogranisms
• Freezing below -2 may stop microbial growth and even kill susceptible organisms

Question 12

How does pressure limit or affect microbial growth?

Answer 12

• Exposure to high pressure kills many microbes
• Food industry uses high-pressure processing (pascalization)
• Application of high pressure between 100 and 800 MPa is sufficient to kill vegetative cells by denaturation, but endospores may survive these pressures.
• High pressure processing is not commonly used for disinfection or sterilization of fomites. Although the application of pressure and steam in an autoclave is effective for killing endospores, it is the high temperature achieved, and not the pressure directly, that results in endospore death.

Question 13

How does desiccation affect or limit microbial growth

Answer 13

• Drying or dehydration
• Works because all cells, including microbes require water for their metabolism and survival
• Controls microbial growth
• might not kill all microbes or their endospores, which may start to regrow when conditions are more favorable and water content is restored

Question 14

How does lyophilization affect or limit microbial growth

Answer 14

• Freeze-drying
• Another method of desiccation
• Item is rapidly frozen (“snap-frozen”) and placed under vacuum so that water is lost by sublimation
• Combine both exposure to cold temp and desiccation
• Causes less damage to an item than desiccation and better preserves the item’s original qualities

Question 15

How does radiation affect or limit microbial growth

Answer 15

• Can be used to kill microbes
• From high-energy to sunlight can kill
• Ionizing radiation includes X-rays, gamma rays, and high-energy electron beams. Strong enough to pass into the cell, where it alters molecular structures and damages cell components
• Ex. Introduces double-strand beaks in DNA molecules
• May directly cause DNA mutations to occur, or mutations may be introduced when the cell attempts repair the DNA damage
• Mutations accumulate and eventually lead to cell death
• nonionizing radiation, is commonly used for disinfection and uses less energy than ionizing radiation. It does not penetrate cells or packaging. Ultraviolet (UV) light is one example; it causes thymine dimers to form between adjacent thymines within a single strand of DNA (Figure 13.13). When DNA polymerase encounters the thymine dimer, it does not always incorporate the appropriate complementary nucleotides (two adenines), and this leads to formation of mutations that can ultimately kill microorganisms.

Question 16

Sonication

Answer 16

Use of high-frequency ultrasound waves to disrupt cell structures

Question 17

How does Filtration affect or limit microbial growth

Answer 17

• Method of physically separating microbes from samples
• Air is filtered through HEPA filters. Physically removes microbes from air
• HEPA filter have effective pore sizes of 0.3um, small enough to capture microbes
• Membrane filters: can also be used to remove microbes from liquid samples. Use of membrane filter with 0.2um or smaller pore size. Physically removes microbes from liquid solutions.

Question 18

Understand and compare commonly-used chemicals for the control of microbial growth

Question 19

List modes of action and advantages and disadvantages of chemicals used to control microbial growth

Question 20

Phenolics

Answer 20

• Denature proteins and disrupt membranes
• ex. disinfectant in Lysol prevent contamination of crops (citrus) Antibacterial soap pHisoHex for handwashing in hospitals
• Types: Cresols, o-Phenylphenol, Hexachlorophene, Triclosan

Question 21

Metals

Answer 21

• Types: Mercury, silver, copper, nickel, zinc
• Bind to proteins and inhibit enzyme activity
• Ex. topical antiseptic, treatment of wounds and burns, prevention of eye infections in newborns, antibacterial in catheters and bandages, mouthwash, algicide for pools and fish tanks, containers for long-term water storage

Question 22

Halogens

Answer 22

• Types: Iodine, chlorine, fluorine
• Oxidation and destabilization of cellular macromolecules
• Ex. Topical antiseptic, hand scrub for medical personnel, water disinfectant, water treatment plants, household bleach, food processing, prevention of dental carries

Question 23

Alcohols

Answer 23

• Types: Ethanol, isopropanol
• Denature proteins and disrupt membranes
• Ex. Disfectant antiseptic

Question 24

Surfactants

Answer 24

• Types: Quaternary, ammonium salts
• Lowers surface tension of water to help with washing away of microbes, and disruption of cell membranes
• Ex. Soaps and detergent, disinfectant, antiseptic mouthwash

Question 25

Bisbiguanides

Answer 25

• Types: Chlorhexidine, Alexidine
• Disruption of cell membranes
• Oral rinse, Hand scrub for medical personnel

Question 26

Alkylating Agents

Answer 26

• Types: Formaldehyde, glutaraldehyde, o-Phthaladehyde, Ethylene oxide, beta-propionolactone
• Inactivation of enzymes and nucleic acid
• Disinfectant, Tissue specimen storage, embalming, sterilization of medical equipment, vaccine component for sterility

Question 27

Peroxygens

Answer 27

• Types: Hydrogen peroxide, peracetic acid, benzoyl peroxide, benzoyl peroxide, carbamide, peroxide, ozone gas
• Oxidation and destabilization of cellular macromolecules
• Ex. Antiseptic, disinfectant acne medication, toothpaste ingredient

Question 28

Supercritical gases

Answer 28

• Carbon dioxide
• Penetrates cells, forms carbonic acid, lowers intracellular pH
• Food preservation, disinfection of medical devices, disinfection of transplant tissues

Question 29

Chemical Food Preservatives

Answer 29

• Types: sorbic acid, benzoic acid, propionic acid, potassium sorbate, sodium benzoate, calcium propionate, sulfur dioxide, nitrites
• Decrease pH and inhibit enzymatic function
• Preservation of food products

Question 30

Natural Food Preservatives

Answer 30

• Types: Nisin Natamycin
• Inhibition of cell wall synthesis (Nisin)
• Preservation of dairy products, meats, and beverages

Question 31

What does a phenol coefficient of 1.0 mean?

Answer 31

• Chemical agent have about the same level of effectiveness as phenol

Question 32

Why is the phenol coefficient used

Answer 32

To determine the effectiveness of a disinfectant or antiseptic compared to phenol

• No longer commonly used because the conditions and organisms were arbitrarily chosen

Question 33

Compare and contrast the disk-diffusion and use-dilution tests for antiseptics and disinfectants

Question 34

What does a phenol coefficient less than 1.0 tell us

Answer 34

The chemical agent is less effective than phenol

Question 35

What does a chemical agent with a phenol coefficient greater than 1.0 mean

Answer 35

• The chemical agent is more effective than phenol

Question 36

Disk-Diffusion method

Answer 36

• Involves applying different chemicals to separate, sterile filter paper disks
• Disks placed on inoculated agar plate with target bacterium, and chemicals diffuse out of the disks into the agar where the bacteria have been inoculated
• “lawn” of bacteria grows, zones of inhibition of microbial growth are observed as clear areas around the disks
• Although there are other factors that contribute to the sizes of zones of inhibition (e.g., whether the agent is water soluble and able to diffuse in the agar), larger zones typically correlate to increased inhibition effectiveness of the chemical agent. The diameter across each zone is measured in millimeters.
• Used for measuring the effectiveness of a chemical agent in clinical settings

Question 37

In-Use Test

Answer 37

• Used for measuring the effectiveness of a chemical agent in clinical settings
• Commonly used to determine a chemical’s disinfection effectiveness on an inanimate surface
• Cylinder of stainless steel is dipped in a culture of the targeted microorganisms and then dried
• Cylinder then dipped in solutions of disinfectant at various concentrations for a specified amount of time
• Cylinder is transferred to a new test tube containing fresh sterile medium that does not contain disinfectant, and this test tube is incubated
• Bacterial survival is demonstrated by the presence of turbidity in the medium, whereas killing of the target organism on the cylinder by the disinfectant will produce no turbidity

Question 38

Prove examples of how antimicrobials were used in ancient societies

Answer 38

• Beer used to treat variety of ailments in adults and childing, including gum disease and wounds
• Used antimicrobial properties of fungi and used moldy bread or other mold-containing products to treat wounds
• Early 20th century compound 606 cured syphilis in rabbits then marketed to humans
• Josef Klarer, Frits Mietzsch, and Gerhard Domagk discovered the antibacterial activity of a synthetic dye, prontosil, that could treat streptococcal and staphylococcal infections in mice
• Sulfanilamide first synthetic antimicrobial (developed from chemical not found in nature), sulfa drugs
• Alexandar Flemming - penicillin (first natural antibiotic)

Question 39

Distinguish between “bacteriostatic” and “bactericidal”

Answer 39

• Drugs can be either one in their interactions with target bacteria
• Bacteriostatic drugs cause a reversible inhibition of growth with bacterial growth restarting after elimination of the drug
• Bactericidal drugs kill their target bacteria.
• Decision on which one to use depends on infection and immune status of patient

Question 40

Contrast broad-spectrum drugs versus narrow-spectrum drugs

Answer 40

• Narrow-spectrum antimicrobial target only specific subsets of bacterial pathogens
  
  • ex. only gram-positive or only gram-negative
  • If pathogen causing infection has been identified best to use a narrow-spectrum antimicrobial and minimize collateral damage to the normal microbiota
• Broad-spectrum antimicrobial - targets a wide variety of bacterial pathogens
  
  • ex. Gram positive and Gram negative
  • Used as empiric therapy to cover wide range of potential pathogens while waiting on the laboratory indentification of the infecting pathogen
  • Also used for polymicrobial infections (multiple) or as prophylactic prevention of infections with surgery/invasive procedures
  • May be selected to treat infection when narrow-spectrum drug fails because of development of drug resistance by the target pathogen
  • Risk is that they will also target a broad spectrum of the normal microbiota, increasing risk of super infection (secondary infection in a patient with a preexisting infection)

Question 41

Explain why side effects and drug interactions can affect the efficacy of drugs

Answer 41

For the optimum treatment of some infections, two antibacterial drugs may be administered together to provide a synergistic interaction that is better than the efficacy of either drug alone. A classic example of synergistic combinations is trimethoprim and sulfamethoxazole (Bactrim). Individually, these two drugs provide only bacteriostatic inhibition of bacterial growth, but combined, the drugs are bactericidal.

Whereas synergistic drug interactions provide a benefit to the patient, antagonistic interactions produce harmful effects. Antagonism can occur between two antimicrobials or between antimicrobials and nonantimicrobials being used to treat other conditions. The effects vary depending on the drugs involved, but antagonistic interactions may cause loss of drug activity, decreased therapeutic levels due to increased metabolism and elimination, or increased potential for toxicity due to decreased metabolism and elimination. As an example, some antibacterials are absorbed most effectively from the acidic environment of the stomach. If a patient takes antacids, however, this increases the pH of the stomach and negatively impacts the absorption of these antimicrobials, decreasing their effectiveness in treating an infection. Studies have also shown an association between use of some antimicrobials and failure of oral contraceptives.

The amount of medication given during a certain time interval is the dosage, and it must be determined carefully to ensure that optimum therapeutic drug levels are achieved at the site of infection without causing significant toxicity (side effects) to the patient. Each drug class is associated with a variety of potential side effects, and some of these are described for specific drugs later in this chapter. Despite best efforts to optimize dosing, allergic reactions and other potentially serious side effects do occur. Therefore, the goal is to select the optimum dosage that will minimize the risk of side effects while still achieving clinical cure, and there are important factors to consider when selecting the best dose and dosage interval. For example, in children, dose is based upon the patient’s mass. However, the same is not true for adults and children 12 years of age and older, for which there is typically a single standard dose regardless of the patient’s mass. With the great variability in adult body mass, some experts have argued that mass should be considered for all patients when determining appropriate dosage.7 An additional consideration is how drugs are metabolized and eliminated from the body. In general, patients with a history of liver or kidney dysfunction may experience reduced drug metabolism or clearance from the body, resulting in increased drug levels that may lead to toxicity and make them more prone to side effects.

There are also some factors specific to the drugs themselves that influence appropriate dose and time interval between doses. For example, the half-life, or rate at which 50% of a drug is eliminated from the plasma, can vary significantly between drugs. Some drugs have a short half-life of only 1 hour and must be given multiple times a day, whereas other drugs have half-lives exceeding 12 hours and can be given as a single dose every 24 hours. Although a longer half-life can be considered an advantage for an antibacterial when it comes to convenient dosing intervals, the longer half-life can also be a concern for a drug that has serious side effects because drug levels may remain toxic for a longer time. Last, some drugs are dose dependent, meaning they are more effective when administered in large doses to provide high levels for a short time at the site of infection. Others are time dependent, meaning they are more effective when lower optimum levels are maintained over a longer period of time.

Question 42

Describe structures/functions of bacteria that can be targeted by drugs

Question 43

Explain the mechanisms of activity of drugs that inhibit cell wall biosynthesis or protein synthesis.

Answer 43

The β-lactam antibacterials block the crosslinking of peptide chains during the biosynthesis of new peptidoglycan in the bacterial cell wall. They are able to block this process because the β-lactam structure is similar to the structure of the peptidoglycan subunit component that is recognized by the crosslinking transpeptidase enzyme, also known as a penicillin-binding protein (PBP). Although the β-lactam ring must remain unchanged for these drugs to retain their antibacterial activity, strategic chemical changes to the R groups have allowed for development of a wide variety of semisynthetic β-lactam drugs with increased potency, expanded spectrum of activity, and longer half-lives for better dosing, among other characteristics.

• Interact directly with PBPs (membrane-associated proteins involved in the biosynthesis of peptidoglycan) and inhibit transpeptidase activity

Question 44

Describe structures/functions of bacteria that can be targeted by drugs: Lipopolysaccharide, inner and outer membranes

Answer 44

• Mode of action: Disrupt membranes
• Drug class: Polymyxin B, colistin, daptomycin

Question 45

Describe structures/functions of bacteria that can be targeted by drugs: RNA, DNA

Answer 45

• Mode of action: Inhibit nucleic acid synthesis
• Drug class: RNA, Rifamycin
• Drug class: DNA, fluoroquinolones

Question 46

Describe structures/functions of bacteria that can be targeted by drugs: Folic acid synthesis enzyme

Answer 46

• Mode of action: Antimetabolites
• Drug class: Sulfonamides, trimethoprim

Question 47

Describe structures/functions of bacteria that can be targeted by drugs: Mycolic acid synthesis enzyme

Answer 47

• Mode of action: Antimetabolites
• Drug class: Isonicotinic acid hydrazide

Question 48

Describe structures/functions of bacteria that can be targeted by drugs: Mycobacterial ATP synthase

Answer 48

• Mode of Action: Mycobacterial adenosine triphosphate (ATP) synthase inhibitor
• Drug Class: Diarylquinoline

Question 49

Provide examples of drugs that target eukaryotic pathogens, and describe their modes
of action

Question 50

Explain the concept of drug resistance

Answer 50

• Microbes constantly evolving in order to overcome the antimicrobial compounds produced by other microorganisms.
• Human development of antimicrobial drugs and their widespread clinical use has simply provided another selective pressure that promotes further evolution

Question 51

Describe how a microbe might develop drug resistance

Answer 51

• overuse and misuse of antimicrobials, inappropriate use of antimicrobials, subtherapeutic dosing, and patient noncompliance with the recommended course of treatment.
• Exposure of a pathogen to an antimicrobial compound can select for chromosomal mutations conferring resistance, which can be transferred vertically to subsequent microbial generations and eventually become predominant in a microbial population that is repeatedly exposed to the antimicrobial. Alternatively, many genes responsible for drug resistance are found on plasmids or in transposons that can be transferred easily between microbes through horizontal gene transfer (see How Asexual Prokaryotes Achieve Genetic Diversity). Transposons also have the ability to move resistance genes between plasmids and chromosomes to further promote the spread of resistance.

Question 52

Give example of mechanisms of antimicrobial drug resistance

Answer 52

• Enzymatic modification of the drug
• Modification of the antimicrobial target
• Prevention of drug penetration or accumulation

Question 53

Explain how the Kirby-Bauer disk diffusion test determines susceptibility or resistance of
a microbe to a drug

Answer 53

The Kirby-Bauer disk diffusion test has long been used as a starting point for determining the susceptibility of specific microbes to various antimicrobial drugs. The Kirby-Bauer assay starts with a Mueller-Hinton agar plate on which a confluent lawn is inoculated with a patient’s isolated bacterial pathogen. Filter paper disks impregnated with known amounts of antibacterial drugs to be tested are then placed on the agar plate. As the bacterial inoculum grows, antibiotic diffuses from the circular disk into the agar and interacts with the growing bacteria. Antibacterial activity is observed as a clear circular zone of inhibition around the drug-impregnated disk, similar to the disk-diffusion assay depicted in Figure 13.31. The diameter of the zone of inhibition, measured in millimeters and compared to a standardized chart, determines the susceptibility or resistance of the bacterial pathogen to the drug.

Question 54

Distinguish between minimum inhibitory concentration (MIC) and minimal bactericidal
concentration for an antimicrobial drug

Answer 54

• Minimum inhibitory concentration: The lowest concentration of drug that inhibits visible bacterial growth
• Minimal bactericidal concentration: The lowest drug concentration that kills >/= 99.9% of the starting inoculum