Mechanisms of Antibiotic Resistance (Pilch) - 4/26/16

Question 1

Where is anti-microbial resistance most prevalent?

Answer 1

In strains that cause infections in hospitals

During outbreaks of infection, patients w/ resistant strains more likely to have received prior anti-microbial therapy (not good, didn’t comply, didn’t take it long enough –> resistance arises)

Areas in hospitals w/ highest antibiotic use have highest incidence of drug-resistant bacteria.

Likelihood of patient being colonized by resistant organisms increases w/ increased duration of exposure to antibiotics in hospitals

Question 2

What are the four ways that bacterial transfer resistance-related DNA to each other? “DNA sex”

Answer 2

1. Transformation - small pieces of DNA containing genes for drug resistance taken up from environment –> incorporated into genome of drug-sensitive bacterium –> expressed
2. Transduction - resistance genes transferred from one bacterium to another by bacteriophage (carrier… shuttle)
3. Conjugation - “sex” - two bacterium become linked via pilus (“penis”)… NO CARRIER. DNA (plasmid) gets ejected right through pilus.
4. Transposition - allows resistance genes to jump between plasmid and chromosomal (genomic) DNA

Question 3

What is the biochemical impact of DECREASED INTRACELLULAR DRUG LEVEL?

Example?

Answer 3

Alteration in drug entry and exit from host bacteria

Anything that allows less and less of drug to be inside bacteria –> resistance (either can’t get in… or bacteria actively pump it out)

Example:
Gm- bacteria w/ mutations that alter porin proteins –> decrease passive drug diffusion
- Resistance to tetracyclines through acquisition of genes that encode an active efflux system for the drug

Question 4

What is the biochemical impact of INCREASED INACTIVATION OF DRUG?

Example?

Answer 4

Bacteria can produce enzymes that render antibiotics biologically inert.

Examples:

• Enzymatic cleavage of penicillins and cephalosporins by beta-lactamases
• Enzymatic modification of aminoglycosides

Question 5

What is the biochemical impact of DECREASED CONVERSION OF A DRUG TO A MORE ACTIVE COMPOUND?

Example?

Answer 5

• Occurs most often w/ drugs that act as antimetabolites
• Arises via mutation and selection

Example: Resistance to the antifungal agent 5-flucytosine due to decreased activity of UMP pyrophosphorylase.

Question 6

What is the biochemical impact of INCREASED CONCENTRATION OF A METABOLITE THAT ANTAGONIZES DRUG ACTION?

Answer 6

If drug acts as an antimetabolite, then increased production of the normal metabolite will compete for the drug effect and lead to resistance.

aka:

Pathway that your drug is competing with has a change …. result: your drug doesn’t compete effectively anymore

Example: resistance to sulfonamides by increase in p-aminobenzoic acid (PABA) levels

Question 7

What is the biochemical impact of ALTERED AMOUNT OF TARGET ENZYME OR RECEPTOR?

Answer 7

1. Resistance plasmids containing multiple copies of the gene encoding the target or that confer inducible expression of the gene (aka making 5 times as much of resistant gene)
2. Mutation in regulatory gene (e.g. a repressor) that controls expression of the gene encoding the target

Antibiotic will still work but the amount required will need to be so much more…. but could lead to toxicity! (window: therapeutic window/index)

Question 8

What is the biochemical impact of DECREASED AFFINITY OF THE RECEPTOR FOR THE DRUG?

Examples?

Answer 8

Gene for receptor undergoes a mutation that changes conformation of drug binding site –> reducing affinity of drug for target

Example:
1 . Trimethoprim resistance - produces DHFR w/ reduced affinity for drug
2. Streptomycin resistance - due to mutation in S12 protein of 30S ribosomal subunit

Question 9

What is the biochemical impact of DECREASED ACTIVITY OF AN ENZYME REQUIRED TO EXPRESS THE DRUG EFFECT?

Answer 9

Mutants deficient in autolytic enzymes (make new cell wall), which breakdown sections of the proteoglycan (PG) matrix during growth.

In absence of autolytic enzymes, drugs that inhibit cell wall synthesis are bacteriostatic rather than bactericidal (tolerance).

aka:

If you can screw up the ability of bacteria to form new wall, it can’t live. But what happens is that instead of these drugs being toxic and causing bacteria to die, causes it to become static.

Question 10

What do gram (+) have that gram (-) don’t have?

The 7 bio. mechs above are mainly for gram (-) bacterium.

Describe vancomycin resistance.

Answer 10

Gram (+) have huge PG outer wall…

Cell wall drugs that target gram (+) tend to target big PG outer wall.

Vancomycin = classic gram (+) drug - specific for that! Don’t effect gram (-)

Vancomycin binds D-Ala-D-Ala moiety –> interferes with formation of PG matrix.

VanHAX operon:

• Produces enzymes that generate D-Ala-D-lactate termini –> form PG matrix but do not bind vancomycin
• Enterococci that express VanHAX operon = vancomycin resistant

Question 11

What are the principals of antimicrobial chemotherapy to combat drug resistance?

Answer 11

1. Give FULL dose
2. Administer enough to obtain clear killing or growth-inhibitory levels
3. Make sure you finish the WHOLE duration of antibiotics
4. Administer combinations of two drugs with different mechanisms of action and different biochemical pathways of resistance (if resistance mutation frequency is 10^-6 for each drug alone, then it will be 10^-12 when both drugs are administered in combination)