week 3 material Flashcards
What is a gram stain used for?
To differentiate between 2 types of bacteria
What is the cell membrane structure of gram positive + gram negative
Gram positive
- Has multiple peptidoglycan layers
- Is thick and rigid
Gram negative
- Has one peptidoglycan layer in between his outer and cytoplasmic membrane
- It is flexible, porous, and helps with regulating osmosis of the cell
Where do gram positive and gram negative bacteria live
GP: lives in external environments such as the skin
GN: lives in watery + moist environments such as the GI tract or GU tract, catheters or IV lines
which bacteria can NOT be gram stained
- bacteria with NO cell wall
- bacteria that exists inside of a host cell
- bacteria not large enough to be seen with a light microscope
what methods can we use to identify the organism that has caused a disease?
- mass spectroscopy using MALDI-TOF
- PCR - molecular based methods
- biochemicals/table top tests
what is MALDI-TOF
- a type of mass spectroscopy method
- laser is used to ionize protein molecules which come from bacterial isolate (pure culture of the bacteria)
- the now ionized molecules get put through a vacuum + the speed they travel at through the vacuum depends on its protein’s mass to charge ratio
- the different speeds of the molecules are presented on to a screen
- the speeds are unique to a species which can help identify the organism or the species of the organism
what are the limitations of MALDI-TOF + what is another method we can use
some species have a similar or the same spectra (they travel at the same speed) - this also means the species are very closely related and MALDI-TOF can not differentiate between the different species
another method to be used is the biochemicals or molecular based tests
what does the biochemical method look for
bile solubility
- is the bacteria soluble in the bile or not
when do we used molecular based methods or PCR
when we’re looking for a specific bacteria
- can be SINGLEPLEX (1 pathogen) or MULTIPLEX (more than 1 pathogen that can cause the disease)
what is susceptibility testing and what do they look for
it’s a test to see if a drug works against the pathogen
done in vitro - they put a bug and a specific concentration in a tube and look at the growth of the bug
they are looking for the lowest concentration of drug that can stop the pathogen from growing - this is call the MINIMUM INHIBITORY CONCENTRATION (MIC)
what are the 3 AST (antimicrobial susceptibility testing) methods in the clinical micro lab
- broth microdilution
- gradient strip diffusion
- disk diffusion
what is broth microdilution
tests the pathogen against serial dilutions of the drug over time
serial dilution = a series of dilutions with a gradual decrease in concentration (in this case the concentration of the drug)
this is the most used AST method
it can be either a manual or automated method
the MIC is shown where the concentration has visibly stopped the bug’s growth - dark gray in this case or clear tube
what is gradient diffusion
uses antimicrobial-impregnated strip (strip contains the antimicrobial drug) with a graded concentration (high to low concentration of drug) the strip then gets placed on a agar plate w/ bacteria
the different concentrations cause a symmetrical elliptical growth
the MIC is seen where the ellipse meets the strip
aka E-test
a manual method
what is disk diffusion
uses a circular disk with a fixed concentration of the drug
gets put with a fixed amount of the bug overnight
measures the diameter of the area of growth inhibition
large enough = the bacteria is susceptible to this drug/drug works against the bacteria
too small = the bacteria is resistant to the drug
manual method
aka kirby-bauer (KB) method
how are results interpreted
they look at a a lot of clinical data on the treatment outcomes of drugs that have been against pathogens
the bug-drug combination determines if the pathogen is susceptible or resistant to the drug
when the outcome is good for that combo at physiological dosing (the amount your body would naturally or normally produce) then the bacteria is susceptible
when the outcome is bad for the combo at physiological dosing, then the bacteria is resistant to the drug
what is selective reporting
when the most relevant antibiotics are reported to providers
it would be the safest + most effective drug for the patient, sample/body, and space
depends on the availability of the drug in the hospital, province, or country
it’s a way to implement safer practice - although there are exceptions in individual cases where they may need to use something more intense
when are some antibiotics not reported
if it is not used during pregnancy - they do not get reported in samples from the BC Women’s hospital patients
some antibiotics are last resort when dealing with pathogens that are resistant to multiple drugs - if a pathogen is susceptible to other drugs, it does not get reported
some drugs do not penetrate or get into the specific tissue or site the sample is from
how do you apply koch’s postulates to a virus
- you have to identify a gene that causes for the virus to be pathogenic or virulent
- this gene can encode for a virulence factor
Example
- Hemagglutinin is a gene that is present in the pathogenic strain of influenza and codes for surface proteins
- it helps the influenza virus to attach to the host cell and enter it
- it gets attacked by the immune system
- if the influenza is mutated, it’s HA gene is not functional which makes the influenza not as virulent
- if we restore the HA gene’s function, then the influenza is virulent again and pathogenic
how do antibiotics lose effectiveness
when bacteria goes through RANDOM mutations which gives them resistance and is then naturally selected for because they’re the ones who survive
what are the requirements for a bacteria to grow
- needs a source of energy
Example: sugars + fatty acids - metal ions
Example: iron - optimal environment
temperature
pH
oxygen or a lack of oxygen
what are extremophiles
microorganisms such as bacteria that can survive in harsh conditions (extreme pH or temperature)
what conditions do most microorganisms grow in
In moderate conditions
Temperature = mid-range temperatures (called mesophilics)
pH = neutral pH
Oxygen or lack of oxygen:
- some microorganisms are obligate aerobes - they require oxygen + perform aerobic respiration
- some are obligate anaerobes - can’t survive in oxygen + perform fermentation
- other microorganisms are aerotolerant anaerobes + facultative anaerobes - they’re somewhere in between the two
what is the bacterial growth curve and which phase is the best for antibiotics to attack
1. Lag phase = NO increase in number of living bacteria
2. Log phase = exponential increase in number of living bacteria
- actively dividing via binary fission
- this phase is the best for antibiotics to target b/ this is where a lot of the bacteria is in the middle of being created + antibiotics can ruin this process - specifically antibiotics that target the synthesis of DNA, cell wall, and proteins
3. Stationary phase = number of living and dead bacteria is the same, there is no increase or decrease to the amount of living bacteria, the amount is staying the same
- nutrients get used up and waste products accumulate
- the synthesis of nucleic acids, proteins, and peptidoglycans slow down which also makes them less susceptible to antibiotics
- in this phase - bacteria may go into survival mode by creating virulence factors or endospores
4. Death or Decline phase = exponential decrease in number of living bacteria
examples of physical methods of microbial control
microwaving food/heat
salt for drying out food
smoking food
fermentation of food
copper cookware
preserve bodies
pasteurization
refrigeration
altering the acidity
desiccation (removal of moisture)
- add solutes (salt or sugar)
- solutes draw water from area of low solute concentration (inside the cell) to high concentration (outside of cell)
- cells can NOT tolerate the high osmotic pressure conditions and will die
what does antimicrobial drugs target
non-human parts of cell replication (non-host cell components)
it can target components of the bacteria itself - called selective toxicity
what are the differences between prokaryotes + eukaryotes
Prokaryotes
- single chromosome
- no nucleus
- no membrane-bound organelles
- contains peptidoglycan in cell wall
- replicates via binary fission
- ribosomes is made up of 70S (30S + 50S subunits)
Eukaryotes
- multiple chromosomes inside nucleus
- contains membrane-bound organelles
- does not have cell wall + fungi has chitin
- most ribosomes are made up of 80S in the cytoplasm (free ribosome)
- 70S in mitochondria of plants
- replicates via meiosis + mitosis
what is selective toxicity
selectively kills or stops the growth of pathogens which causes little to no harm to hosts
antibiotics’ goal is to have selective toxicity
what are the 2 types of antibacterial drugs and examples
1. Bactericidal: kills the target bacteria
Example
Penicillin = targets cell wall synthesis + causes it to have a faulty cell wall - it can’t maintain the osmotic pressure causing it to lyse
2. Bacteriostatic: stops the growth of the bacteria but doesn’t kill it - causes reversible inhibition which allows for bacteria to grow again once drug is eliminated
Example
Aminoglycoside = targets protein synthesis by causing for mRNA to be misread (wrong amino acids get brought in?)
what is the spectrum of antibiotics/antibacterial drugs + spectrum of activity
Spectrum of activity: the range or diversity of bacteria that the antibiotic targets
Narrow spectrum: targets only a certain group of pathogens (e.g. ONLY gram-positive)
Broad spectrum: targets a wide group of pathogens (e.g. both gp + gn)
what are the advantages + disadvantages of using broad spectrum antibiotics
Advantages
- good for empiric therapy - when you don’t know the bacteria you’re targeting
- good for polymicrobial infections - when there is more than 1 type of bacteria involved
- good for bacteria that is resistant to antimicrobial
Disadvantages
- kills microbiome (the good bacteria) - can bring on side effects, select for the bacteria that is resistant to the antibiotic, and can lead to superinfections/opportunistic infections
superinfections/opportunistic infections = an additional secondary infection in addition to the pre-existing infection
what are the advantages of narrow spectrum antibiotics/antimicrobial drugs
- less impact on microbiome
- lower chance of superinfection
- less selection for bacteria that is resistant to the drug
what is the mechanism of penicillin
- peptidoglycan strands are needed to cross-link to make a strong cell wall
- transpeptidation: PBP (penicillin-binding protein) crosslinks the strands
- penicillin binds to PBP - prevents it from forming the peptidoglycan crosslinks - results in weak cell wall
- the weak cell wall is unable to withstand the osmotic pressure - results in cell lysis + death
the penicillin kills the bacteria by targeting the peptidoglycan cell wall
penicillin only targets certain types of bacteria
what are beta-lactam antibiotics
- group/class of antibiotics that have a beta-lactam ring
- the ring blocks the crosslinking of the peptidoglycan strands
- the R-group of this antibiotic allows for various characteristics and activities to occur within this group antibiotics
- this class of antibiotics includes:
cephalosporins - variable spectrum
carbapenems- broad spectrum - has R-groups that make it a strong antibiotic
which type of bacteria is penicillin most effective at killing
gram-positive
what was the first antibiotic found to kill gram-negative bacteria
Streptomycin
- comes from streptomyces griseus
- inhibits prokaryotic ribosomes - results in inhibition of protein synthesis
- does NOT affect eukaryotic ribosomes
what are the 5 bacterial targets for antimicrobial drugs + the drugs used there
1. Cell Wall
- Beta-lactams: monobactams
- Glycopeptides: vancomycin
2. DNA Synthesis
- Fluoroquinolones: ciprofloxacin + levofloxacin
3. Ribosomes - 30S, 50S
- 30S: Tetracyclines
- 50S: Macrolides
4. Cell Membrane
- Polymyxins
- Lipopeptide
5. Metabolic Pathways
- Folic Acid Synthesis
- Sulfones
principles of antimicrobial drug resistance
- microbes are constantly evolving to overcome natural antimicrobial compounds
- humans have increased selection pressure by using a variety of antimicrobials
- drug resistance occurs when microbes go through random mutations and are selected for - that gene then gets passed down to further generations
- drug resistance genes can be in plasmids of transposons and passed via horizontal gene transfer
How does drug resistance occur + what happens when you take antibiotics for a short amount of time and your immune is impaired
A large number of bacteria:
- some have mutated + became a antibiotic resistant strain
- antibiotic added: the sensitive strain gets killed + antibiotic resistant strain does not get killed
- antibiotic strain grows and multiples
- they can transfer the drug resistance gene to the sensitive strain
Taking antibiotics for a short amount of time:
- less time to kill majority of the bacteria allowing for drug resistant strain to multiply
Weak immune system: can’t fight against the drug resistant bacteria - allowing for it to grow and multiply
what are the mechanisms for drug resistance bacteria to get rid of the antibiotics
Efflux Pump: pumps antibiotics out
- part of the cell wall, DNA synthesis, and ribosomes
- example: tetracyclines
Inactivation by Enzymes: inactivates antibiotics
- part of cell wall + ribosomes
- example: macrolides
Target Modification: changes the target’s (of the antibiotic) structure
- part of cell membrane, cell wall, DNA synthesis, metabolic pathways, and ribosomes
- example: vancomycin
Blocked Penetration: blocks antibiotics from coming in
- example: tetracyclines
examples of all: beta-lactam
what does drug modification or inactivation do
the resistance gene codes for enzymes that modifies the drug to not function
how does drug modification /inactivation affect beta-lactam antibiotics
- Beta-lactamases (enzymes) hydrolyze the beta-lactam ring of the antibiotic
- this causes for the antibiotic to be unable to bind to the PBP to prevent the crosslinking
How can the beta-lactam antibiotic overcome beta-lactamase + examples
-Antibiotics can contain an inhibitor, beta-lactamase inhibitor (BLI)
- the BLI blocks beta-lactamase and prevents the hydrolysis of the beta-lactam ring
Examples
- amoxicillin (beta-lactam) - clavulanic acid (BLI)
- piperacillin (beta-lactam) n- tazobactam (BLI)
what does efflux pumps do
- porins = transporters on the outer membrane that allows nutrients + antibiotics to come in
- bacteria decreases the amount of porins
- bacteria actively transports antibiotics out of cell via efflux pumps
- 1 efflux pump can pump out multiple types of antibiotics
what does target modification do
changes the structure of the target that the antibiotic is trying to attack
changes the…
- ribosome - blocks the active site
- DNA gyrase
- Metabolic enzyme
- PBP
what is a multidrug resistant bacteria
- aka superbugs
- they have more than 1 resistance mechanism
-resistant to multiple drugs - what makes it multidrug resistant is it has a efflux pump that pumps out different type of drugs
how can we overcome the trend of antimicrobial resistance
1. invest in prevention of infections - vaccines
2. minimize inappropriate or unnecessary antibiotic use in humans + animals
- not using antibiotics for viral infections
- use narrow spectrum + short course drugs
- avoid not completing antibiotics or therapy
- decrease the use for livestock
3. research new drugs to combat the resistance
- most antibiotics we use today have been used for the past 40 years
- bacteriophage (virus) are being used to treat people with infections that drug resistant - has been successful in infections that have no antibiotics
