Topic 4-L4 - Control of microbial growth Flashcards
Decontamination:
Neutralization or removal of microbes (general term)
Disinfection:
Eliminating or removing harmful organisms
Sterilization:
killing all microorganisms. If something is sterile, it is completely free of microbes. Sterility can be thought of as binary –it’s either sterile or it isn’t.
Decimal reduction time:
amount of time it takes to reduce number of microbes by a factor of 10 (a log)
Thermal death time:
Amount of time if takes to kill all cells at a given temperature. Depends on number of cells.
Both decimal reduction time and thermal death time depend on a number of factors:
pH, salt concentration, moisture levels, presence of fats/sugars/proteins in samples (can decrease heat penetration), etc.
small changes in temp can have a huge impact on
of microbes remaining after a given heating time.
Autoclaves are widely used to
sterilize by heat using steam.
- Endospores (highly resistant!) require
temperatures of 121oC for 15 minutes
How are autoclave 121C?
Water (steam) is therefore pressurized to increase boiling temp - allowing it to be heated to 121oC
- High pressure steam circulated throughout autoclave. Typical cycle length ~ 30 minutes to ensure materials reach 121oC for at least 15 minutes (varies - longer for large volumes of liquids, for example)
Pasteurization is a common methods to prevent
spoilage and protect against food-
borne pathogens with certain foods/beverages
How is pasteurization done?
Heated for a specific amount of time to eliminate pathogens and to reduce spoiling agents (non-pathogenic bacteria, enzymes)
Pasteurization For milk,
71oC for 15 seconds
- In milk, heat-resistant lactic acid bacteria survive pasteurization and are what cause the milk to spoil over time.
Pasteurization is NOT
Sterilization
Ultraviolet (UV) light (220-300 nm wavelength)
damages DNA and is lethal to microbes at high enough intensities
- used for sterilizing bio safety hood (poor penetrating power)
Ionizing radiation (such as gamma rays) have
improved penetrating power and also can be used to sterilize
- used for sterilizing surgical supplies,
labware, even food!
Heat is a great way to sterilize, heat can also cause
damage (e.g., inactivate proteins or other molecules)
Filter sterilization
Passing liquids through filters.
- very effective, but not as reliable as autoclaving
Sterilants:
Kill all microbes – e.g. formaldehyde
Disinfectants:
largely for surfaces, kill many/most microbes but not all (usually not endospores) – e.g. lysol
Sanitizers:
Unlike the above, less harsh to humans, but also generally less effective – e.g. soaps
Antiseptics:
Kill or inhibit growth of microbes, non-toxic enough to use on tissues (external – such as wounds). E.g. ethanol.
Antimicrobial agent:
Chemical that kills or inhibits the growth of microbes
“cidal” (bactericidal, fungicidal) –
Agents that kill cells. Irreversible.
“static” (bacteriostatic) –
Agents that do not kill, but that inhibit growth. Prevent growth, but microbes are not dead and can recover.
“lytic” (bacrteriolytic) –
Agents that not only kill cells, but that cause them to lyse (pop open – no dead cells can be observed)
The “MIC” of a compound against a particular microbe is the
minimal inhibitory concentration
minimal inhibitory concentration
Lowest concentration of a compound that fully inhibits microbial growth
How is minimal inhibitory concentration determined?
using serial dilutions of the compound (usually 2x dilutions) – measuring microbial growth.
Measuring toxicity of chemicals
- MIC
- solid media, zones of inhibition
Solid media, zones of inhibition
- Antimicrobial activity can also be assessed using solid media combined
with disks that contain the compound of interest - Disk placed on agar plate – diffuses
- Closer to disk = higher concentration
of antimicrobial agent - The size of the “zone of inhibition”
– area of no microbial growth –
indicates susceptibility to compound - “Kirby-Bauer test”
antibiotic typically refers to medicines (chemicals) that have
antimicrobial properties that are used to treat infections.
Antibiotics use
specific mechanisms to tangent microbes, rather then generic and non-specific toxicities
Topical antibiotics are antibiotics manufactured into
creams/ointments that are applied to the skin. Others are ingested and dont hard human cells
Antibiotics are Generally they are
small molecules that target a specific aspect of the biology of the microbe that is absent or very different in humans (e.g.
bacterial cell wall)
Fleming discovered
penicilin (not used for a decade after discovery) and lysozyme
First effective, ingested antibiotic used in clinics was
protonsil (discov. By Bayer by screening dyes)
Protonsil not effective in test tube, only
live animals – pro-drug that must be processed by the body to active form
How do antibiotics work
- specific targets, disrupt microbial processes
- target essential processes (cell wall biosynthesis, translation(protein synthesis), DNA replication, essential biosynthetic processes)
- need to get into cell, permeability is an issue
How do antibiotics work: penicillin
- binds and inhibits activity of penicillin binding proteins – which are proteins that catalyze transpeptidation rxn that crosslink the cell wall
- In growing bacteria, cell wall is constantly being remodeled (autolysin proteins). Digested cell wall can’t be repaired – cell wall loses integrity, cells lyse due to osmotic pressure.
Many antibiotics are
natural products (or chemically modified versions of natural products) isolated from microbes
Microbes that produce a lot of antibiotics
Certain Actinomycetes such as Streptomyceshave been a very rich source of antibiotics
Streptomyces are soil-dwelling bacteria that produce large numbers of
antibiotics –
large genomes, many interesting biosynthetic gene clusters
Antibiotics are not just for bacteria – some target
eukaryotes, such as fungal pathogens
polyene antibiotics from
Streptomyces
Polyenes bind to a
cholesterol-like molecule, ergosterol (not in humans), found in fungal
cytoplasmic membranes – permeabilize membrane, cell death.
For virtually all medically-important microbial pathogens,
strains resistant to commonly-used antibiotics can be found
Antibiotic-resistant bacteria are
altered genetically (mutation or acquiring resistance genes) in a way that reduces sensitivity to that antibiotic.
4 main mechanisms of resistance:
1) Modification of drug target (mutation of enzyme targeted by drug)
2) Enzymatic inactivation of the drug (degrade the drug)
3) Removal from cell by efflux pumps (pump drug out of cell)
4) Metabolic bypass (find another way to do what the drug is blocking)
For many antibiotics,
derivatives of a parent class have been developed
- improved activity, more broad spectrum, less toxicity in humans
derivatives are designed to
overcome known
resistance mechanisms
For penicillin, some derivatives are not
sensitive to an enzyme called
β-lactamase
β-lactamase is an
antibiotic-resistance gene – an enzyme that degrades penicillin
- β-lactamase-encoding strain can therefore
be treated with these derivatives
Examples of derivatives that are β-lactamase resistant
- methicillin
- oxacillin
β-lactamase sensitive derivatives
- ampicillin
- carbenicillin
Persistence is when antibiotic-sensitive bacteria includes
rare cells that are transientlytolerant to antibiotics
Most antibiotics depend on metabolic activity (& often growth) in
order to be effective – e.g. if you’re not building a cell wall,
inhibiting cell wall synthesis is not effective
Dormancy
(slowing or shutting off metabolism) is a common route to persistence
Unlike resistant bacteria,
persisters are
genetically unchanged – can be killed by antibiotics once again after they
emerge from their tolerant state.
