I.
Terminology
All of these processes are aimed at reducing microbial populations (i.e., microbial load)
Sterilization
Elimination of all viable cells, spores and acellular entities (viruses, viroids, prions)
The viable cells, spores and acellular entities are killed, removed or inactivated
Sterilant: chemical agent used to sterilize an object
Disinfection
Killing, inhibition or removal of microorganisms that may cause disease
A treatment with the primary goal of destroying potential pathogens
May not eliminate all microorganisms but usually substantially reduces microbial numbers
Disinfectants: agents, usually chemical, used in disinfection and are normally used on
inanimate objects.
Sanitization
Related to disinfection but reduces microbial population to levels considered safe by public
health standards also minimizes risk of disease transmission
An inanimate object is usually cleaned and partially disinfected with a sanitizer
Antisepsis
Prevention of infection or sepsis
Antiseptics: chemical agents applied to living tissues to prevent infection by killing or
inhibiting pathogen growth
Antiseptics are generally less toxic than disinfectants because it is important that they do not
destroy too much tissue
Chemotherapy
The use of chemical agents to kill or inhibit the growth of microorganisms within host tissue
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A variety of chemicals can be used to eliminate or reduce the numbers of microbes these are
often referred collectively as antimicrobial agents. Specific suffixes are used to indicate how
these agents work.
Bactericidal - "cidal" - to kill
Bacteriostatic - "static" - to inhibit; if the agents are removed then growth will resume.
Bacteriolytic lytic to lyse
suffixes can be applied to all types of agents
e.g.,
II.
1.
Direct Assessment
i)
Bacterial killing curves
Plot log %survival vs a measure of the sterilizing agent
e.g., log % viable Salmonella cells vs [singlet oxygen]
ii) Time-dose-rate relationship
The effect of the treatment depends both on concentration used and exposure time.
In order to kill all the cells in a particular culture, one can hold the time constant and vary
the dose or keep the dose constant and vary the time.
e.g., A high dose for a short time will have the same effect as a low dose for a longer period
of time
iii) Death point
Treatment dose necessary to sterilize the system in a given amount of time
e.g., Thermal death point = temperature necessary to sterilize a culture in 10 min.
iv) Death time
Time necessary to sterilize a system with a particular treatment
e.g., Time in min. necessary to sterilize the culture when a particular temperature is applied
(Thermal death time)
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Indirect Assessment
i)
Sterility indicators
Use certain bacterial endospores
The most durable life forms known.
e.g., Geobacillus stearothermophilus spores are capable of surviving 5 min in an autoclave
(121C; 15 psi)
III
IV.
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V.
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o High temperature - short time (HTST, also known as flash pasteurization) 71.7C for 15s.
This method is preferable for milk because it alters the taste less, kills heat resistant
microbes more effectively and can be done on a continuous flow bases
d. Ultra High Temperature sterilization (UHT)
Sterilizes food and other products
141C for 4 - 15 s; allows for a continuous flow system
e.
Dry Heat
often requires much higher temperatures and longer exposure times (e.g., 170C for 2 h)
Extreme temperature may damage materials that can resist lower temperature (121C)
Other examples include the flaming of an inoculating loop and incineration of combustible
materials
Low Temperature
Refrigeration (0 to 7C) and freezing
Does not kill all microbes but inhibits growth
3.
Radiation
Forms of electromagnetic radiation
i)
Microwaves
works partially due to heating
ii)
Ultraviolet radiation
220 to 300 nm in wavelength
This radiation is energetic enough to cause modifications or strand breaks in DNA that may
result in disruption of genetic material and death of the organism
low energy, low penetrating power - surface treatment
iii)
Ionizing radiation
high energy, high penetrating power that produces electrons, hydoxyl radicals, and hydride
radicals breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, degrade
and alter biopolymers
Standard for biological applications is absorbed radiation dose measured as
Rad = 100 erg/g (where 1 joule = 10,000,000 erg)
Gray (Gy) = 100 rad
A dose providing a 10 fold reduction in numbers is referred to as a D10. A standard killing
dose is 12 D10 equivalents
Sensitivity to radiation differs for different microbes
Application of radiation
60Co and 137Cs sources
medical supplies and food industry (spices and fresh meat)
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4.
Filtration
Applicable to liquids and gases
Remove microbes - does not kill them
Types include depth, membrane and nucleopore (Fig 7.4)
e.g., Biocontrol hoods & laminar flow hoods - High efficiency particulate air (HEPA) filters are a
type of depth filter remove 99.97% of 0.3m particles
5. Desiccation
Removal of water - microbes can't grow
Dried fruit, meat and fish
lyophilization - freeze dried food
VI.
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Phenol coefficient = reciprocal for the appropriate dilution of the disinfectant being tested
reciprocal for the appropriate dilution of phenol
2. Use-dilution test cylinders of metal or glass are dipped in standardized cultures of the test
organism, removed and allowed to dry. The dried cultures are placed in a solution of the
disinfectant and left for 10 minutes at 20C. The cylinder is then transferred to a suitable growth
medium and monitored for growth.
3. Disk diffusion method (Agar-diffusion method) See below for details
B. Inhibition of microbial growth in vivo
Chemotherapeutic Agents
Chemical compounds that can be used internally for control of microbes
Synthetic agents and Antibiotics = Antimicrobial agents
An antibiotic is a chemotherapeutic compound produced by microorganisms.
To be useful in therapy, a chemotherapeutic agent must have selective toxicity
e.g., high toxicity towards the microbe but low toxicity towards the host.
Selection of Chemotherapeutic Agents
Sensitivities of target microorganism
Side effects of antibiotic
Longevity of antibiotic (e.g., biotransformations)
Chemical properties of antibiotic- affect targeting of antibiotic in the body
Selectivity - broad spectrum vs narrow spectrum of activity (Table 20.2)
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Size of zone depends on a number of factors including amount of antimicrobial agent added to
the disc, solubility, and effectiveness of agent
Conclusion - whether an antimicrobial agent has the potential to control a particular
microorganism
This test is limited to use with rapidly growing bacteria
It is a standardized test not designed for assessing the effectiveness of antibiotics against
filamentous fungi, anaerobes or slow growing bacteria, although it is possible to modify test in
order to assess these organisms.
ii) Minimum Inhibitory Concentration (MIC, also known as tube dilution technique)
Dilutions of antibiotics or other chemical agents are tested for ability to inhibit growth of a
microbe in liquid culture
MIC is the lowest concentration of an agent that completely inhibits the growth of the test
organism
Determines the minimum amount of antimicrobial agent that must be present at the site of
infection to inhibit growth of the pathogen.
Affected by the nature of the test organism, inoculum size, medium composition and other
growth parameters (incubation time, temperature, pH, aeration)
aids in selection of antibiotic and dosage (required to know how antibiotic distributes).
Physicians generally add in a safety margin - 10 times MIC
Medium from tubes that do not show growth (i.e., have higher antimicrobial concentration
than the MIC) can be used to inoculate broth or on agar plates free of the antimicrobial agent.
If growth occurs then the drug was not bactericidal and the minimum bactericidal
concentration (MBC) can be determined.
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A.
1.
2.
Quinolones
Not growth factor analogs - interact with DNA gyrase and topoisomerase II (involved in DNA
replication packaging DNA)
Effective against both gram-positive and gram-negative bacteria
e.g.,
B.
Nalidixic acid
Fluoroquinolone derivatives norfloxacin and ciprofloxacin are more soluble than
Nalidixic acid
Antibacterial Antibiotics
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DNA scission
Phleomycin/bleomycin/zeocon
Broad spectrum antibiotics that are affectcive against prokaryotes and eukaryotes
Cleave DNA
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C.
Antifungal Antimicrobial Agents
Problems associated with host toxicity
1) Ergesterol inhibitors
Polyene - affinity for membrane sterols such as ergesterol - disrupt membrane structure
e.g., Nystatin, Amphotericin B (Fig 20.14)
2) Ergesterol synthesis inhibitors
Azoles (Fig 20.15) and allyamines
Inhibit ergesterol biosynthesis - alter membrane permeability
e.g., Ketoconazole
3) Cell wall synthesis
Polyoxins - interfere with chitin biosynthesis
Echinocandins interfere with 1,3 -D glucan synthase results in incomplete cell wall
4) Inhibit microtubule assembly
Griseofulvin is produced by Penicillium spp.
Prevents microtubule assembly, interfering with mitosis and thereby inhibiting fungal
reproduction
D.
Antiviral Agents
Problems associated with host toxicity
1) Nucleoside/nucleotide analogs (Fig 20.16)
Acyclovir - Herpes simplex - blocks viral DNA replication
Zidovudine (nucleoside analog) and tenofovir (nucleotide analog) - blocks DNA production
(reverse transcription) effective against retroviruses (HIV) RNA virus that converts
2) Non-nucleoside reverse transcriptase inhibitors
Bind directly to the reverse transcriptase and inhibit activity
e.g., nevirapine
3) Protease inhibitors
HIV requires protease activity in the maturation process
Research has identified peptides that bind in the protease active site and inhibit protease
activity
Atazanavir and indinavir and saquinavir are protease inhibitors of
4) RNA polymerase inhibitors
e.g., Rifamycin
5) Neuraminidase inhibitors
Neuraminidase is an enzyme on viral surface that is important in releasing virions from host cell
e.g., Tamiflu (oseltamivir) inhibitor of influenza neuraminidase
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1.
Antibiotic producers must protect themselves from the antibiotics that they produce
Mechanisms of Antibiotic resistance (Figure 20.20)
Impermeable to antibiotic
2.
Genetic Basis of Antibiotic Resistance
Antibiotic resistance is coded at the chromosomal or the plasmid level (R-plasmids)
Resistance at the chromosomal level almost always arises due to modification of the target of
the antibiotic
Plasmid resistance often results from the production of enzymes that inactivate, prevent uptake
or actively pump out the antibiotic
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3.
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