4. Control of microorganisms Flashcards
DEFINE:
STERILIZATION
INHIBITION
DECONTAMINATION
DISINFECTION
STERILIZATION:
- The killing or removal of all viable organisms (including endospores)
INHIBITION:
- Effectively limiting microbial growth.
- ie fridge!
DECONTAMINATION:
- treatment of an object to make it safe to handle.
- ie food setting –> wash dishes to decrease overall amount of microorgs
DISINFECTION:
- Directly targets the removal of all pathogens, not necessarily all microorganisms
- ie medical setting! intention = remove all pathogens!
what are physical methods (3) to do antimicrobial control? vs chemical methods (2 ish)
PHYSICAL
- heat
- radiation
- filtration
CHEMICAL:
- on surface: sterilants, disinfectants, sanitizers, antiseptics
- internally: antibiotics, antivirals, antifungals
- death rate of microbes is _____________ –> explain
- what is the decimal reduction time (D) ? 3 ways to explain ish
- time necessary to kil a define fraction is _________ of the initial cell concentration
- if D = 5min, and you start with 10^10 cells. how many cells will you have after 10min?
- constant/exponential! –> a fixed percentage of the population dies per unit of time
- amount of time required to reduce viability tenfold! ie decrease pop 10-fold OR kill 90% OR 10% are left
- independent!
- 0min = 10^10
- 5min = 10^9
- 10min = 10^8 (99% cells are killed compared to at 0min)
- what is the most widely used method for controlling microbial growth?
- what does it do to the cell?
- D is __________ correlated to this method.
- do mesophiles or thermophiles have smaller decimal reduction times?
HEAT!
- high temps DENATURE macromolecules
- decimal reduction time is INVERSELY correlated with temperature! –> increase temp = decrease D (faster it takes to kill 90% of pop)
- different microorgs have different decimal reduction times. Mesophiles have smaller! bc less tolerant to heat = are killed faster
- what is thermal death time?
- dependent on what?
- need to do what to compare sensitivity of different microorgs?
- are endospores easily killed? how to kill them?
- time needed to kill all cells at a given temperature!
- dependent on population size of microorg tested!
- need to standardize the starting number of cells to be able to compare!
- survive heat that would rapidly kill vegetative cells! –> need higher temp to kill endospores
- can resist boiling for a long time –> 5min for decimal reduction (ie 90% dead)
what is an autoclave?
- allows temp of water to get to what? HOW?
- sterilization is achieved in how long?
- what temp will the object being sterilized reach? consequence? (2)
- what kills the microrgs?
- sealed device that uses steam under pressure!
- get above 100°C! at 15 psi (pound/square inch), steam reaches 121°C
- in 10-15min! (even kills endospores)
- will reach 121°C! so not suitable method for heat-sensitive object/liquid + need to wait until object reaches that temperature! (usually takes about 15min to reach 121°C before starting the sterilization time)
- NOT the pressure that kills microorg but high temp!
what is pasteurization?
- is it a method of sterilization or decontamination?
- goal?
- reduces significantly population of which pathogens (5)
- describe flash vs bulk pasteurization
- process of using precisely controlled heat to reduce microbial load in heat sensitive liquids!
- NOT sterilization bc does not kill all organisms + not targeting pathogens –> it is DECONTAMINATION!: reduces microbial load
- goal = increase shelf-life of product + decrease transmission of pathogens
- listeriz monocytogenes, salmonella enterica, campylobacter, E.coli, mycobacterium
FLASH: 72°C for 15sec
BULK: 65°C for 30min
*15sec and 30min are the decimal reduction time!
*both have same result!
what are 2 main sources of radiation to control microbial growth?
first one:
- explain + pro + con
second one:
- explain + how?
- what is analogous to D value?
- soures?
- used for what?
UV!
- has sufficient E to cause modifications and breaks in DNA (produces thymine dimers) –> inhibits replication, transcription = causes death
- useful for decontamination of surfaces
- CANNOT penetrate solid, opaque or light-absorbing surfaces
IONIZING RADIATION:
- electromagnetic radiation produces ions and other reactive molecules
- generates e- and hydroxyl radicals causing damage to DNA and proteins
- amount of E (ie dose of radiation) required to reduce viability tenfold is analogous to D value
- sources: cathode ray tubes (electrons), x-rays, radioactive nuclides
- used for STERILIZATION in medical field and food industry (ie destroy microbes on surface of fresh foods)
do microorgs have high sensitivity to radiation compared to humans?
YES!
humans die at 10 Gy
vs some bacteria/virus can resist 200, 300, 2200, 3300, 13000, 20 000-50 000 Gy!
*ie some mold was growing after Chernobyl explosion….
FILTER STERILIZATION
- concept?
- what is a benefit?
- what are 2 types + explain
- can filtering lead to sterilization?
- pores of filters are too small for orgs to pass through = get stuck in filter VS liquid and gas can pass through pores
- BENEFIT: avoids use of heat on sensitive liquids and gases
DEPTH FILTERS
- fibrous sheet or mat made from array of fiber (paper or glass)
- used to sterilize liquid, air
- HEPA (high efficiency particulate air) filters
- high chance that microorg hits filter
MEMBRANE FILTERS
- function more like a sieve –> membrane with holes
- type of membrane filter is the nucleation track (nucleopore) filter
- filtration speed can increase by syringe, pump or vaccuum
- yes!
what are 3 classifications of antimicrobial agents?
*graphs
- effect depends on (2)
BACTERIOSTATIC
- inhibit growth of microorganism
- viable cell count and total cell count don’t decrease –> plateaus!
BACTERIOCIDAL
- kills microorganisms
- viable cell count decreases
- total cell count stays the same! bc cells are dead but skeleton of cell is still there so can still count them
BACTERIOLYTIC
- kill microorganism by inducing lysis
- decrease viable cell count AND total cell count (bc intact cells lyse/explode)
*effect depends on species of microorganisms and concentration of agent that you use
what are 2 ways to measure antimicrobial activity?
1) DILUTION + PLATING: using minimum inhibitory concentration and minimum bacteriocidal concentration
- preparing suspensions of microbe of interest + prepare a 2-fold serial dilution of test compounds –> incubate bacteria and test compounds –> check if there is growth or no –> plate control and no-growth wells in agar
2) DISC DIFFUSION ASSAY
- inoculate whole surface of agar with microbe to be tested –> antimicrobial agent added to filter paper disc + put in disk
- filter will create a zone of inhibition: area of no growth around the disk, where concentration of agent will decrease away from disk
- what is minimum inhibitory concentration (MIC)?
- what is minimum bacteriocidal concentration (MBC)
- how to state effect of antimicrobial activity? (2) (think of what’s written on a lysol spray)
- MIC: smallest amount of an agent needed to inhibit GROWTH of a microorg –> varies with organism used, inoculum size, temp, pH
- ie minimum concentration where there is no growth BUT when you put on a plate with nutrients, it grows bc microrgs are not dead
- MBC: lowest concentration of an agent that KILLS test bacterium –> if you put on plate, it won’t grow bc it’s dead
- decimal reduction time at concentration x (ie 30sec at concentration) OR decimal reduction concentration after x minutes (ie kills 99.9% of germs in 30sec)
what to measure after disc diffusion assay?
- benefits of disc diffusion assay?
- does an antimicrobial resistant bacterium grow close or far from the paper disc?
- measure the diameter of the zone of inhibition! MIC is reached at edge of that zone (ish) –> compare diameter with established table
- quicker! can set up in 10’ + test several substances at the same time
- antimicrobial resistant growth close to the disc! bc will resist treatment
VS susceptible/not resistant will grow far from the disc
which plate was invented by who to test for antimicrobial resistance?
Mueller-Hinton Agar!
by J. Howard Mueller and Jane Hinton (one of first 2 black females to get a PhD)
in 1949
- purpose of antimicrobial compounds (3)
- can be separated into 2 categories: describe
1) prevent spreading of pathogen in environment
2) prevent contamination of the host
3) cure superficial bacterial infections.
a) Products used to control microorganisms in commercial and industrial applications (or domestic). ie: chemicals in paper, air-conditioning, cooling towers, textile and paper products, fuel tanks, chlorine (pools)
b) Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces: Sterilants, disinfectants, sanitizers, and antiseptics.
define:
STERILANTS
DISINFECTANTS/SANITIZERS
ANTISEPTICS
ANTIMICROBIAL DRUGS
*kills endospores?
STERILANTS:
- destroy ALL forms of microorganisms (= sterilize), including endospores. Called COLD STERILIZATION (vs autoclave, heat)
DISINFECTANTS/SANITIZERS
- applied to nonliving objects or surface (can be toxic for animals/humans). Do not kill endospores.
- ie ethanol, alcohol gels, lysol
ANTISEPTICS:
- applied to the surface of living tissues or skin (ie wound) (must NOT be toxic for animals/humans). Do not kill endospores.
ANTIMICROBIAL DRUGS:
- Antibiotics, antifungals, antivirals: applied outside or inside the body of animals/humans (must NOT be toxic for animals/humans). Do not kill endospores.
*far more toxic for microorg than humans –> ie cannot use ethanol to kill microorg in your body bc would also kill you
what are 6 examples of antiseptics, disinfectants and sterilants?
- phenol/phenolics
- alcohols
- halogens –> chloride! (oxidizing agent: steals e- = kills microorgs bc oxidizes everything) ie sodium hypochlorite (KNOW THIS)
- heavy metals
- QUATERNARY AMMONIUM (know this) –> interact with phospholipids of cytoplasmic membrane
- can use on our hands bc have a layer of dead skin + keratin
- alkylating agent
antimicrobial drugs are classified on basis of (3)
- can be used in humans or animals? side effects?
- bacteriostatic, cidal or lytic?
- usually have what?
- good antimicrobial drug has (5)
- molecular structure, mechanism of action, spectrum of antimicrobial diversity (how many microbes it can target)
- yes! with minimal side effects (low toxicity)
- can be either bacteriostatic or bactericidal
- have a specific target (ie a specific enzyme that only microbes have)
a) NO severe side effects, must be far more toxic for the microorganism than mammalian
cells (might have effect on microbes in gut)
b) Low risk/benefit ratio.
c) Broad spectrum of activity to facilitate rapid medical intervention –> target everything! popular before but now, leads to antibiotic resistance…
d) Appropriate bioavailability and pharmacokinetics (must reach the site of infection).
e) Low cost to develop and manufacture.
what do antibacterials target/mode of action? (5 + sub)
- cell wall (b-lactams, glycopeptides…)
- plasma membrane
- ribosomes: 30S and 50S subunits –> 70S
- metabolic pathways: folic acid synthesis…
- DNA and RNA synthesis
*mostly target stuff that only bacteria have!
*humans also have 70S ribosome in mitochondria –> so antibacterials that target 70S need to be at low concentrations to kill microbes but not humans
what is selective toxicity?
- who studied it in what year?
- what was one of the first antimicrobial drugs? used to treat what?
- ability to inhibit or kill pathogen WITHOUT affecting the host
- Paul Ehrlich in early 1900s
- Salvarsan (salt of arsenic) –> used to treat syphilis (treponema pallidum) –> early 1920-30s
what are 3 types (+ 2 sub in the first one) of SYNTHETIC antimicrobial drugs?
GROWTH FACTOR ANALOGS:
- structurally similar to growth factors but do NOT function in the cell
- block pathways that synthesize important compounds by FOOLING the enzyme
- Analogs similar to vitamins, aa, and other compounds.
a) SULFONAMIDE:
- discovered by Gerhard Domagk in the 1930’s
- analogue of p- aminobenzoic acid (PABA) (precursor/growth factor to make tetrahydrofolic acid)
b) TRIMETHOPRIM:
- analogue of dihydrofolic acid (intermediate btw PABA and THFA) –> once blocks 2nd enzyme, cannot produce THFA anymore
NUCLEIC ACID BASE ANALOGS
- formed by the addition of bromine or fluorine.
- Stop DNA replication, translation.
QUINOLONES:
- antibacterial compounds that interfere with DNA gyrase (control DNA supercoiling/untwists DNA so enzymes can read it)
what are antibiotics?
- less than ____% of known antibiotics are clinically useful
- can be modified? for what? called what?
- what is a major factor that affects bacteria sensitivity to antibiotics?
- what are one of the most important groups of antibiotics of all time? –> produced by what?
- many antibiotics effective against bacteria are produced by _________
- antimicrobial agents NATURALLY produced by bacteria and fungi to inhibit or kill other microorganisms –> for competitive purposes!
- Less than 1% (bc some also kill humans)
- Can be modified to enhance efficacy (semisynthetic antibiotics)
- Gram-positive and Gram-negative bacteria vary in their sensitivity to antibiotics. The cell wall is a major factor. (outer membrane blocks antibiotics)
- b-Lactams –> produced by molds. Include penicillins, cephalosporins, and cephamycins.
- produced by Bacteria (e.g. Streptomyces)
what is the most famous ish b-lactam antibiotic?
- discovered by who? isolated from what?
- primarily affective against gram-pos or neg bacteria?
- inhibits what?
- oral or parenteral?
- what are its different variations ish? what changes?
- Alexander Fleming, isolated from Penicillium chrysogenum
(mold) (mold that was growing on his plate of staphylococci) - Primarily effective against Gram-positive bacteria BUT Some synthetic forms are effective against some Gram-negative bacteria
- Inhibit cell wall synthesis
- parenteral! (needle injection) –> NOT oral
- R group changes! penicillin G, penicillin V, ampicillin, amoxicillin (banana syrup), methicillin)
- what is super important of b-lactam antibiotics? –> explain mode of action
- bacterialstatic, cidal or lytic?
- b-lactam ring! –> inhibits synthesis of peptidoglycan/transpeptidation = bacteria cannot grow/make new peptidoglycan again
- usually transpeptidase recognizes a structure similar to b-lactam ring (attachment btw D-Ala and D-Ala) –> and attaches them together
- SO when b-lactams are there, transpeptidase bind penicillin instead –> which inhibits enzyme
- β-lactams are bactericidal or bacteriolytic (depending
on the species, growth phase, etc). - Can be bacteriostatic (in isotonic solutions: ie blood, lymph)
antibiotics from bacteria
- describe the 3 major classes of protein synthesis-inhibiting antibacterials
*name
*function/mode of action
1) chloramphenicol, macrolides, lincosamides
- bind 50S ribosomal unit
- prevent peptide bond formation –> stops protein synthesis = stops growth!
- first bacteriostatic, than bacteriocidal
2) aminoglycosides:
- bind 30S ribosomal subunit –> impairs proofreading –> results in production of fault proteins –> starts making mistakes (ie wrong codons)
- bacteriocidal bc faulty proteins are toxic
3) tetracyclines
- bind 30S ribosomal subunit
- block binding of tRNAs –> inhibits prot syntehsis bc stops feeding ribosome with aa
what are
- DAPTOMYCIN
- VANCOMYCIN
- what ish?
- mode of action
- treat gram-pos or neg?
*both are more exotic antibiotics from bacteria
DAPTOMYCIN
- also produced by streptomyces
- used to treat gram-pos bacterial infections
- forms pores in cytoplasmic membrane (bc has a ring like structure)
VANCOMYCIN
- glycopeptide
- block transpeptidation by binding to tip of peptide side chain (D-ala-D-ala)
- used to treat gram-positive bacterial infections
- what is antimicrobial drug resistance?
- is it increasing or decreasing in the world? correlated with what? but also what?
- ACQUIRED ability of a microorganism to resist the effects of a chemotherapeutic agent to which it is normally sensitive
- change in how pathogens respond to antibiotics
- increasing! correlated with how much antibiotics we’re using. but also with public health, infection rates, sanitation
- antibiotic producers are ____________ –> explain + 3 mechanisms
- TOLERANT to the antibiotic it produces! ie microorg (mold, eukaryote) that produces penicillin will not be killed by penicillin (bc mold = not a bacteria)
1) lack target sites (ie no peptigodlygan)
2) modify target sites (ie modification to ribosome so that its not affected by antibiotic)
3) lack of uptake mechanism (ie final step needed to activate the compound and transport to right place)
how to bacteria acquired resistance to some antibiotics?
- Most drug-resistant bacteria isolated from patients contain WHAT located WHERE. explain
- Usually mediated by the acquisition of a new gene (or groups of genes) that provide the cells with a new function (i.e. antimicrobial resistance).
- Most drug-resistant bacteria isolated from patients contain drug-resistance genes located on R plasmids –> R plasmids can be transferred between bacteria of the same species or related species.
- R plasmids are spread from microorg to microorg
what are 3 mechanisms for antibiotic resistance mechanisms + examples
1) DESTRUCTION of antibiotic (ie b-lactam degradation by b-latamase –> canno’t bind to transpeptidase) OR MODIFICATION of antibiotic (ie acetylation of chloramphenicol –> cannot bind target anymore)
2) PROTECTION of target site by modification (ie 23S rRNA modification (part of 50S ribosome) –> macrolides, blocks binding to antibiotic)
3) REDUCING intracellular concentration with efflux pumps –> ie tetracycline efflux pump: pumps antibiotic out as soon as it comes into cell so no concentration can accumulate
- use of antibiotics in medicine, veterinary medicine, and agriculture selects for WHAT
- do most pathogenic microbes have acquired resistance to some chemotherapeutic agent?
- which pathogens have developed resistance to all known microbial agents? problem for what?
- how can resistance be minimized?
- the spread of R plasmids.
- yes! almost all pathogenic microbes have acquired resistance
- Methicillin-resistant S. aureus (MRSA)
- Vancomycin-resistant Enterobacter (VRE)
- Extended-spectrum cephalosporins resistant E. coli
*problem in hospitals! especially for immunocompromised patients - Resistance can be minimized by using antibiotics correctly and only when needed (reduce selection).
*ie decrease antibiotic use in livestock –> not used to growth promotion
- why do fungi pose problems for chemotherapy? explain
- what are 2 drug targets of antifungal drugs?
- bc they are eukaryotes! much of cellular machinery is the same as that of animals and humans!
*target unique metabolic processes not found in mammals
1) ergosterol synthesis (nystatin, fluconzaole –> both inhibit ergosterol synthesis)
2) cell wall synthesis (ie inhibit chitin synthesis)
