Antimicrobial Agents & Microbial Resistance

Question 1

What is an antibiotic?

Answer 1

Anything made naturally that attacks bacteria.

Question 2

What is an antibacterial?

Answer 2

Anything made synthetically that attacks bacteria.

Question 3

What are the four classes of antibacterial drugs?

Answer 3

Antibacterials can target DNA synthesis, protein synthesis, cell wall synthesis, or the cell membrane structure. DNA synthesis can be targeted through DNA replication or Folate synthesis (folate is used as a basis for nucleotides)

Question 4

What is minimum inhibitory concentration?

Answer 4

The lowest possible concentration to visibly decrease the presence of bacterial growth. Inhibition does not mean bacterial death, it just means that you’ve stopped proliferation from reaching the point that a vial becomes turbid (murky).

Question 5

How would an antibiotic target protein synthesis in bacteria?

Answer 5

The bacterial ribosome is smaller than the eukaryotic ribosomes (60s vs. 80s). You could target the 50s or 30s subunits of the 60s ribosome without risking any impact on human cells.

Question 6

What is the difference between an inhibitory and a bactericidal antibiotic?

Answer 6

Inhibitory just stops proliferation, bactericidal kills.

Question 7

What is a bacteriostatic drug?

Answer 7

An inhibitory drug, does not kill but halts proliferation.

Question 8

What is minimum bactericidal concentration?

Answer 8

The minimum drug concentration that leads to cell death (as seen on a culture plate, seeded from a vial with the bacteria, media, and drug mixed up)

Question 9

What is a disk diffusion test?

Answer 9

You get a bunch of different drug pills and put them on an agar plate. Incubate and see if any of the drugs caused a zone of clearance (cell death). Any zone, no matter how small, proves that this bacteria is susceptible to the drug. No zone would mean the bacteria is resistant to the drug.

Question 10

What is an E test?

Answer 10

The E test is a strip of paper with a known gradient of drug (going from high to low). The strip of paper is put in a well plate and incubated. The drug, if bacteria is susceptible to it, will cause a zone of clearance. BUT at some concentration the drug will no longer be effective, and the bacteria will no longer be cleared. This is where the bacteria will intersect with the paper. Any concentration above this will have some level of inhibitory/bactericidal effect. This is the minimum inhibitory concentration.

Question 11

What is MRSA?

Answer 11

Methicilin Staph Aureus (also resistant to oxacillin)

Question 12

What is efficacy of a drug (in a macro sense, not in pharmacology sense)?

Answer 12

The ability of a drug to address the target issue. Impacted by mechanism of action, binding affinity with target, cost, side effects.

Question 13

What is cMax is a drug availability curve?

Answer 13

cMax is the maximum concentration reached by a drug when it enters the body (on the bioavailability curve)

Question 14

What period within the drug availability curve represents time where the bacteria is being effectively acted upon?

Answer 14

Only in the timeframe where the concentration is greater than the minimum inhibitory concentration

Question 15

What is time-dependent killing in regards to drugs?

Answer 15

Time-dependent killing is when a drug requires some amount of time to impact cell function. It takes extended exposure to do a lot of damage. What matters most is the time your drug is in the plasma at a concentration at or above the minimum inhibitory concentration (MIC)

Question 16

What is concentration-dependent killing with regards to drugs?

Answer 16

Concentration-dependent killing is when a drug acts immediately on the cells but requires a higher concentration to do the most damage. What matters most here is how high you can get the concentration in the plasma.

Question 17

What is the post-antibiotic effect? When is it most relevant?

Answer 17

Post-antibiotic effect is a continuation/delayed effect of antibiotic administration. Sometimes, even when the drug concentration drops beneath the MIC you get continued damage to bacterial cells. It is defined as the time it takes for bacteria to return to logarithmic growth after drug concentration passes under the MIC. It’s usually seen more significantly in Concentration versus time-dependent killing.

Question 18

What do you want to optimize in a drug that shows time-dependent killing characteristics?

Answer 18

Try to optimize the amount of time a drug’s concentration is above the MIC. Ideally the drug would be above MIC for at least 50% of “dosed time”

Question 19

What do you want to optimize in a drug that shows concentration-dependent killing traits?

Answer 19

Try to optimize the maximum concentration reached. Maybe fast-release drugs would be more effective here.

Question 20

Is it easier to get drug into a gram + or - bacteria? Why?

Answer 20

Gram + is easier. Even though the peptidoglycan wall is way thicker, it’s like a drywall (super easy to break through, as per Hannah & the recliner) and stuff can get through it pretty easily. Gram + only has one lipid bilayer to get through after the peptidoglycan wall. Gram - has two lipid bilayers and a layer of lipopolysaccharides. The second bilayer is thin but almost like a bulletproof vest. Very little gets through

Question 21

What is the bacterial cell envelope?

Answer 21

Anything including/past the cell membrane but connected to the cell (excluding a polysaccharide (sugar) capsule if there’s one). In gram + this is the peptidoglycan layer and cell membrane. in gram - this is the lipopolysaccharide layer, the outer lipid bilayer, the thin peptidoglycan layer, and the inner cell membrane.

Question 22

What is different about mycobacteria? How does it affect drug choice?

Answer 22

Mycobacteria don’t have any peptidoglycan. Instead they have mycolic acid and some other structures (arabinogalactan). Mycolic acids are long, branched, greasy hydrocarbon chains.

Question 23

What do b-lactams do?

Answer 23

B-lactams inhibit the cross linking of peptidoglycan chains. B-lactams kill bacteria by messing with the peptidoglycan layer in the cell wall of gram + and sort of in gram - cells. The first b-lactam discovered was penicillin. B-lactams bind to Penicillin Binding Protein (which are normally catalyzing the transpeptidase reaction bringing strands of peptidoglycan together) and inhibit their action. PLP normally binds to D-Ala-D-Ala connections. Penicillin looks remarkably similar to D-ala-d-ala and is a competitive inhibitor.

Question 24

What do glycopeptides do?

Answer 24

glycopeptides inhibit the elongation of peptidoglycan chains (additions of sugars to chain)

Question 25

What is peptidoglycan?

Answer 25

Peptidoglycan is a key component of the cell wall in gram + cells and a minor component of the cell wall in gram - cells. It is a buffer from the external environment.

Question 26

What is d-ala-d-ala?

Answer 26

D-ala-d-ala is a peptide branch connected to the sugar-chain of peptidoglycan. D-ala-d-ala undergoes a transpeptidase reaction, combing two from different peptidoglycan chains (remove one d-ala-d-ala from each compound), and crosslinks the chains.

Question 27

How is peptidoglycan formed?

Answer 27

Peptidoglycan is formed by penicillin binding proteins. Chains of n-acetylmuramic acid and n-acetylglucosamine are added together through glycosylation reactions. Some of the n-acetylmuramic acids and n-acetylglucosamines have a branch made of d-ala-d-ala. d-ala-d-ala is a peptide chain that is used to form crosslinks between petpidoglycan strands. d-ala’s react with each other through penicillin binding proteins. Peptidoglycan is stuck into the lipid membrane by lipid linkages that are anchored in the cell membrane.

Question 28

What is a gram + cell?

Answer 28

A gram + cell stains purple! It has a thick petpidoglycan layer in the cell wall and a cell membrane underneath. Gram + cells can be targeted through their cell wall, since so much of it is the peptidoglycan layer.

Question 29

What is a gram - cell?

Answer 29

A gram - cell stains pink! A gram - cell has a thick lipopolysaccharide layer, followed by a lipid bilayer, and then periplasm (with some proteins and a thin peptidoglycan layer) and then another cell membrane. Gram - cells are much harder to kill because they’re more impenetrable.

Question 30

What is the major difference between gram + and gram - cells? Does this affect drug choice?

Answer 30

Gram + has a thicker peptidoglycan layer. Gram - has a second lipid bilayer. The lipid bilayer is much harder to penetrate so these cells are harder to target. Many drugs that work on gram + will not work on gram -.

Question 31

What does penicillin do?

Answer 31

Penicillin is a b-lactam. It binds to PBPs and disrupts peptidoglycan layer formation.

Question 32

What are PBPs in bacteria cells?

Answer 32

Penicillin binding proteins are enzymes used to form crosslinks between strands of peptidoglycan. They are crucial in forming a thick peptidoglycan layer and are the target of penicillin and other b-lactams.

Question 33

Why does penicillin interfere with d-ala-d-ala interactions with other peptide chains?

Answer 33

Penicillin looks almost identical (structurally and chemically) to d-ala-d-ala, so it’s able to bind as an alternative substrate/competitive inhibitor for d-ala-d-ala in PBPs.

Question 34

What are common mechanisms of antibiotic resistance in bacteria?

Answer 34

Modification of drug (b-lactamase or other enzymes are able to inactivate the drug)
Decreased intake of drug, increased efflux of drug
Altered drug target (you can change the shape of the drug target so the drug no longer binds, but the target cellular structure is still fully functional. This occurs in PBP alterations. PBP is no longer shaped to bind with b-lactams but can bind d-ala-d-ala perfectly fine. Another example is a change in d-ala-d-ala to d-ala-d-lac, a common target of glycopeptides. D-lac can still bind, and it’s knocked off in the transpeptidase reaction so the functionality of the peptidoglycan layer is not affected, but the glycopeptides can’t find/inhibit d-lac at all. With ribosomal RNA, another common target, a simple methylation stops the drug from working but the ribosome is still functional.)

Question 35

What is a b-lactamase? How does it work?

Answer 35

B-lactamase is an antibiotic resistance mechanism in bacteria. B-lactamase degrades b-lactams in the cell, keeping them from disrupting cell wall formation. It breaks the b-lactam ring in penicillin (and oxacillin and other b-lactams, they all have a 4 and 5 carbon ring). By breaking down b-lactam structure, they change the binding affinity of b-lactams with PBPs. The binding affinity for d-ala-d-ala is much greater, and the cell will be able to function as it would have without b-lactams.

Question 36

What are some important classes of b-lactamases?

Answer 36

Extended spectrum b-lactamases (ESBLs)
Metal dependent (new dehli metallo b-lactamase)
These classes of b-lactamases have a broad range of targets, where other b-lactamases could only inactivate a single b-lactam molecule type.

Question 37

What can we do to defeat b-lactamase-based antiobiotic resistance?

Answer 37

If we send in a b-lactam drug AND another drug that inhibits the production of b-lactamase proteins. Clavulanic acid is an extremely effective b-lactamase inhibitor

Question 38

Why haven’t antibiotic resistant bacteria taken over the world?

Answer 38

Developing antibiotic resistant features has a fitness cost for bacteria. Studies have shown that resistant bacteria grow slower than normal bacteria. It takes energy to create all the additional enzymes, or you might have a less stable genome or something. Antibiotic resistant bacteria are not evolutionarily ideal in the normal world, just in special environments like the hospital where other bacteria is killed off.

Question 39

How can antibiotic resistance develop in bacteria?

Answer 39

It can be passed vertically (to genetic offspring) or horizontally (phage transduction, transformation, conjugation). Antibiotic resistance is usually in vectors outside of the normal bacterial genome, but they can still be passed to offspring.

Question 40

What are penicillin resistant PBPs?

Answer 40

PBPs that are still able to bind d-ala-d-ala links on peptidoglycans and create the peptidoglycan crosslinks, but are changed in shape enough that they have a low affinity for b-lactams. This is a costly mutation in terms of evolutionary fitness, because the original PBP was optimized for function, and is a key enzyme in the cell.

Question 41

What forms of antibiotic resistance would have the lowest cost on fitness?

Answer 41

Making an enzyme that adds or modifies b-lactams or other antibiotics would be a pretty low cost adaptation. You wouldn’t need to make many, and a single enzyme could come from a pretty simple piece of genetic data.

An efflux pump would require a lot of ATP and would take a large genome to make (multiple proteins go into one pump), but it wouldn’t be taking away from the function of the rest of the cell, so this would be relatively low cost.

Question 42

What is induced resistance?

Answer 42

Induced resistance is when resistance mechanisms are activated by exposure to one drug, and they carry over to cause resistance against another drug. Could be activated by environmental factors as well. Induced resistance is a permanent change in an individual cell. Once it becomes resistant it will not lose that defense. So treating with two drugs is potentially risky! If one drug was normally potent/effective but the other was null in this bacteria, the bacteria’s defense to the first drug could actually help it become resistant to the second. Then, even if you treat with just the effective/potent drug, you’d still have a resistant bacteria population

Question 43

What is selective pressure? What is reversibility?

Answer 43

Pressure to optimize fitness in population. If there’s an antibiotic killing off cells, the few cells that survive will become dominant in a culture. Some resistant mutations are reversible though! They can flare up depending on the environment but tone down when nothing’s attacking the cell. This is a reversible mutation. These are usually the high-cost mutations, since the cell can’t afford to have them on at all times.

Question 44

What should we consider when choosing antibiotics for medication?

Answer 44

You want to attack cells through a mechanism that works! But you also want to consider encouraging the bacterial population that does survive to do so through the most costly mechanisms. So you would want to pick an antibiotic that can only be nullified by an extremely costly genetic mutation (something like mutating PBPs or totally decreasing intake of nutrients/external molecules from the environment)

Question 45

How can bacteria alter the intake/output (efflux) of a drug?

Answer 45

They can decrease the activity of porins (decrease intake). This can be bad for the cell’s health, because it will also lose key nutrients. They can decrease membrane permeability to hydrophobic molecules (same issue with cell health).

Cells can increase efflux of drug by either mutating or acquiring genetic data for production of pump proteins that grab drug and push it out of the cell. Or there could be a mutation that increases the binding affinity of a current pump or changes an unrelated pump’s structure so it optimally binds and pushes the drug out. Remember that mutations don’t happen within a cell’s exposure, but would have happened before. Then the cells with this mutation (just randomly there) survive and pass it on.

Question 46

What are ways that penicillin transport have been modified by resistant bacteria?

Answer 46

Membrane permeability has been decreased (this only matters in gram -, since there are the two membranes).

They’ve acquired pumps that push penicillin out of the cell.

Question 47

How would plasmid borne b-lactamase impact antiobiotic effectiveness? What impact would it have on overall cell fitness?

Answer 47

It would stop b-lactams from inhibiting cell wall formation. It would not negatively impact the cell’s overall fitness much because the enzyme is easy to make and doesn’t cost much in terms of energy or function.

Question 48

How would PBP mutations impact antiobiotic resistance? What impact would this have on a bacteria’s fitness?

Answer 48

PBP mutations would be super effective in stopping b-lactams from doing anything useful. But they would also cost the cell a lot (and because of this we don’t see PBP mutations much). You use PBPs so often that to have a suboptimal mutation costs the cell a lot of energy.

Question 49

How would increased efflux pump activity impact antibiotic impact on a cell? How would a mutation for increased efflux pumping impact genetic fitness of a bacteria cell?

Answer 49

Efflux pump would push drug out, decreasing the cell’s exposure to the drug. It would be reasonably costly, as the pump would not be something normally present in the cell, and it could also potentially pump out stuff you wanted in the cell.

Question 50

How would decreased uptake of drug through porins impact antibiotic effect and cell fitness?

Answer 50

If you decrease uptake of extracellular materials, you will eliminate some but not all drug exposure. But you’ll also lose access to key nutrients and signal molecules. So this would be pretty costly.

Question 51

What do glycopeptides do?

Answer 51

Glycopeptides inhibit glycoslylation (elongation) of peptidoglycan chains (addition of n-acetylmuramic acid and n-acetylglucosamine)

Question 52

How is resistance for vancomycin developed?

Answer 52

d-ala-d-ala target is not coded by genes but is added in post-translation modification. So a mutation in d-ala-d-ala cannot come about through random mutation. There aren’t any enzymes that are known to deactivate glycopeptides. So the mutation has to be a couple steps “back” the protein which adds modifications to n-acetylmuramic acid and n-acteylglucosamine would need to be changed

Question 53

What peptide modification defeats the effectiveness of vancomycin? Why is this surprising?

Answer 53

the d-ala-d-ala peptide branch is changed to be d-ala-d-lac. it’s still recognized by the transglycosylase enzymes and by the PBPs, but vancomycin and other glycopeptides can’t bind and inhibit anymore. This is surprising because the peptide branch (either on n-acetylmuramic acid or n-acetylglucosamine, can change) isn’t actually modified by the transglycosylase enzymes but is just used for recognition

Question 54

What is characteristic of mycobacteria?

Answer 54

They have mycolic acid, arabinogalactan, instead of having any peptidoglycan.

Question 55

What drugs can work on mycobacteria? What are their mechanisms of action?

Answer 55

They need to be targeted by drugs entirely differently than gram + or gram - drugs. Isoniazid inhibits mycolic acid synthesis, and ethambutol inhibits arabinogalactan synthesis by acting on arabinotransferases

Question 56

What are lipopeptides? Why don’t they hurt our cells?

Answer 56

Lipopeptides target specific regions of the cell membrane, puncturing the membrane, increasing porosity, and ultimately causing cell death. They work on G+ bacteria, but are too chunky to get through the strict outer membrane of gram - bacteria. They bind to phosphatidyl-glycerol, which is prevalent in bacteria but extremely rare in most of our cells. After binding they form large pores in the cell membrane.

Question 57

What cells in the body would lipopeptides impact?

Answer 57

The lungs! Alveolar cells produce all that surfactant have a ton of phosphatidyl-glycerol in their membranes. So lipopeptides cannot be used to treat pneumonia or any infections in the lungs.

Question 58

What drug classes target the 30s subunit for bacterial ribosomes?

Answer 58

aminoglycosides and tetracyclines

Question 59

What drug classes target the 50s subunit for bacterial ribosomes?

Answer 59

chloramphenicols, macrolides, lincosamides, streptogramins, oxazolidinones

Question 60

What is an aminoglycoside? What cell types does it work/not work against?

Answer 60

antibiotics that target the 30s subunit. Bactericidal (kill bacteria) that work on gram - by penetrating the lipopolysaccharide layer (lps) where it connects to the membrane. Streptomycin and gentamicin are common aminoglycosides. Aminoglycosides only work on aerobes. Any ANAEROBIC bacteria will be immune to aminoglycosides.

Question 61

What are some ways that bacteria can develop resistance to aminoglycoside?

Answer 61

Enzymatic deactivation of drug (phosphorylation, adenylation, acetylation)
Methylation of 30s ribosomal RNA target (it can still function but aminoglycosides can’t bind anymore)
Decrease drug exposure (decreased intake, increased efflux)

Question 62

What is combinatorial therapy? What would a synergistic interaction look like on a graph?

Answer 62

When two drugs have mechanisms that complement each other and produce a result higher than either would produce independently (or the combination of both independent impacts). Synergy on a graph of necessary concentration of drug A vs. Drug B to get a set impact would be concave. Additive would be linear. Antagonistic would be convex.

Question 63

How can antibiotics be paired together for a more effective attack?

Answer 63

If one antibiotic penetrates the cell wall, and another goes in to attack the ribosome, you’d probably get a synergistic effect. But you would never want to pair a bactericidal drug that required the cell to attempt proliferating with a bacteriostatic drug that halts the bacteria from doing anything.
B-lactam and aminoglycoside would be a good combo for gram -. you could do tetracylcine and oxazolidinones for gram +

Question 64

How could antibiotics be paired together poorly?

Answer 64

you wouldn’t want to pair a bactericidal drug with a bacteriostatic drug.

Question 65

What are tetracyclines?

Answer 65

Bacteriostatic drugs that target the 30s ribosomal subunit. Work against G-, G+, mycobacteria, and even intracellular bacteria (bacteria that live inside our cells). One drug in the tetracycline family is called tetracycline, another is called doxycycline. Divalent cations (cations with 2+ charge, so mg 2+ ca2+) inhibit uptake, so you can’t drink milk with medicine.

Question 66

What are some bacterial resistance mechanisms against tetracyclines?

Answer 66

there are a bunch of tetracycline efflux pumps that can be added to the genome of bacteria, but most only work on tetracylcine, not doxycycline. Ribosome protection proteins can be developed to stop tetracyclines from binding to the ribosome. They do this by competitively inhibiting the binding sites.

Question 67

What does chloramphenicol do?

Answer 67

Bacteriostatic drug used against 50s subunit. Can be used in all bacteria. Acetyl transferases can modify chloramphenicol to inactivate it.

Question 68

What do macrolides do?

Answer 68

Bacteriostatic drugs that work against G+ (erythromicin and azithromycin). They’re used to target the 50s subunit of ribosomes in bacteria.

Question 69

What do lincosamides do?

Answer 69

Bacteriostatic, works against G+ and G- anaerobes. Selective to 50s ribosomes. THEY ONLY WORK AGAINST ANAEROBES!!!! So this is the opposite of aminoglycosides
clindamycin is a common one

Question 70

What are the different classes of cell wall synthesis inhibitor antibiotics?

Answer 70

b-lactams and glycopeptides

Question 71

What is included in the cell wall of a bacteria?

Answer 71

Everything past the inner cell membrane. In gram + this is just the peptidoglycan layer. In Gram - this is the lipopolysaccharide layer, the outer lipid bilayer, the thin peptidoglycan layer, and anything else in the periplasm)

Question 72

How do bacteria develop resistance to lincosamides/streptogramins?

Answer 72

Drug target alteration: ribosome can be methylated and no longer accessible to drug but still functional. This can come from up to 40 different mutations in the erm gene. Usually resistance to lincosamide equates to resistance to streptogramins and vice versa.

Question 73

What are oxazoilidinones?

Answer 73

Bacteriostatic drugs that bind to the 50s subunit of ribosomes. They work against a lot of gram + drugs that are resistant to b-lactams. They bind to the 23s RNA portion of the 50s subunit in bacterial ribosomes. The only known resistance to oxazoidones is to mutate the shape of the 23s region of the ribosome.

Question 74

What is included in the cell wall of a bacteria?

Answer 74

Everything past the inner cell membrane. In gram + this is just the peptidoglycan layer. In Gram - this is the lipopolysaccharide layer, the outer lipid bilayer, the thin peptidoglycan layer, and anything else in the periplasm)

Question 75

What does vancomycin do?

Answer 75

a glycopeptide, blocks the glycosylase reaction that extends the chain of a peptidoglycan. It binds to the d-ala-d-ala branch and stops the transglycosylase enzymes from binding. While they don’t actively cause reactions on the peptide branch, the transglycosylase enzymes recognize and bind to the branch in preparation of bringing n-acetylmuramic acid and n-acetylglucosamine together.

Question 76

What would induced resistance look like on a disk diffusion test?

Answer 76

You’d have a full circular zone of clearance on one side and a blunted circle on the other side (indicating that bacteria within exposure range for the other drug or environmental factor were now resistant)