Lecture 19: Antimicrobials and resistance II

Question 1

What are the causes of ineffective treatment? (where all the microorgansims are not cleared)

Answer 1

1. Antibiotic resistance
2. Bacterial tolerance:
   - The ability of a whole bacterial population to survive longer treatments with bactericidal anitibiotics.
   - An example of developing tolerance is the formation of biofilms in bacteria which reduces antibiotic access and leads to inability or slower killing.
3. Suboptimal treatment regimen.
   - When all patients are treated the same in terms of dosage and treatment time. Not all patients exhibit the same reaction to antibiotics in terms of excretion/degradation and absorption profiles.

Question 2

What is the difference between bacterial tolerance and bacterial persistance?

Answer 2

• Bacterial tolerance: bacterial populations which can outlive exposure to raised concentrations of an antibiotic, by slowing down essential bacterial processes.
• Bacterial persistance: a sub-population of bacteria that are able to survive antimicrobial treatments without acquiring resistance-conferring genetic changes. (Often associated with bacteria that are in a dormant, non-growing state)

Question 3

1. What drives antibiotic resistance?
2. What determines the ongoing survival/transmission of antibiotic resistant bacteria?

Answer 3

1. Antibiotic use and natural selection.
   - Resistant cells have a strong selective advantage and will rapidly dominate the population in response to antibotic treatment.
2. Survival and transmission is determined by whether the resistance brings some survival cost (defect).
   - For example if a mutation causes a bacteria to change its RNA polymerase so it is no longer susceptible to a specific antibiotic, this change in RNA polymerase is likely to cause a fitness defect in the bacteria and therefore these resistant bacteria are less likely to survive.

Question 4

1. What are natural antibiotics?
2. Does selection occur for these antibotics?
3. How do microorganisms that produce antimicrobials develop resistance to the antimicrobials that they produce? And can this resistance be spread to other species?

Answer 4

1. Natural antibotics are produced by microorganisms to defend themselves from other microorgansisms and to compete for limited resources, e.g. penicillin.
2. Yes, selection occurs naturally.
3. The microorganism that is producing an antimicrobial must be resistant to it; the organism either does not have the target for the antimicrobial or it already has genes for resistance to the antimicrobial in its genome.

Resistance determinants can be spread to other species by exchanging genetic material, e.g conjugation.

Question 5

1. What is resistance?
2. What are the different categories of resistance?

Answer 5

1. When the growth of an organism is not inhibited by an antibiotic at concentrations achievable in the body after approved dosage.
2. -Multi-drug resistant (MDR): acquired non-susceptibility to atleast one agent in three or more antimirobial categories.
   - Extremely-drug resistant (XDR): non-susceptibilty to atleast one agent in all but two or fewer antimicrobial categories.
   - Pan-drug resistant (PDR): non-susceptibilty to all agents in all antimicrobial categories.

Question 6

1. What is intrinsic resistance? Give examples.
2. What is acquired resistance?

Answer 6

1. Intrinsic resistance: when bacteria are naturally resistant to antibiotic. This usually occurs due to poor antibiotic uptake or lack of targets.
   e. g. Gram-positive bacteria are intrinsically resistant to polymyxins as the target, LPS, is not present in gram-positive bacteria.
2. Acquired resistance: can occur due to aquisition of new genetic elements that give resistance or the result of mutations.

These mutations can either alter gene/target expression, or, directly affect the target molecule.

Question 7

What are the four main mechanisms of resistance?

Answer 7

1. Blocked penetration
2. Efflux pump
3. Inactivation
4. Target modification

Question 8

How does blocked penetration lead to antibiotic resistnace? Explain.

Answer 8

• Many antibiotics enter the cell via membrane porins/ specific channels. This allows th entry of small hydrophillic molecules such as β-lactmas, tetracycline and chloramphenicol that are usually unable to cross the hydrophobic membrane.
• Bacteria can develop resistance via blocking the entry of these molecules through these porins/channels.
• It does this by acquiring mutations that affect the porins/channels and alter antibiotic access:
• Mutation in gene that encodes porin so that porin is no longer there- porin loss.
• Mutation causing an alteration in sequence of porin which affects its structure - narrow porin.
• Mutation leading to altered expression of porin- decrease porin expression.

Question 9

How are efflux pumps in bacteria involved in contributing to antibiotic resistance?

Answer 9

• Bacteria express various efflux systems to export toxic compounds, either from cytoplasm or completely out of the cell.
• Since efflux pumps are naturally expressed in bacteria, they are involved in intrinsic resistance.
• Mutations or transcriptional responses can lead to increased efflux expression, and therefore increased antibiotic efflux and therefore decreased access of antibotics to their targets.
• Many of the efflux systems are either non-specific or reasonable non-specific, therefore, increased expression of one of these efflux systems can export various antibiotcs and therefore leads to resistance to more than one type of antibiotic.

Question 10

How are antibiotics inactivated?

Answer 10

• Antibiotics are either modified or degraded.

Question 11

How does antibiotic modification occur?

Answer 11

• Enzymes such as aminoglycoside modifiying enxymes (AMEs), add phophate groups or acetyl groups to the aminoglyoside antibiotic (e.g. streptomycin).
• This addition reduces the effciiency by which aminoglycosides interact with ribosomes, this leads impacts protein translation (reduction or inhibition) and therefore inactivates the antibiotic.

Question 12

1. How do enzymes degrade antibiotics?
2. How can this degradation be overcome?

Answer 12

• Enzymes such as β-lactamase break down the β-lactam ring in β-lactam antibiotics.
• This structure is essential in forming suicide bond in order to kill bacteria. Therefore, breaking down the β-lactam ring and inactivates the antibiotic.
• Use of β-lactamase inhibitors - drugs that are administered with the β-lactam antimicrobial.
• E.g. clavulanic acid + amoxicillin
• β-lacatamase inhibitors mimic the structure of β-lactams, therefore can interact with β-lactamse to prevent the antibiotic from degradation.

Question 13

1. What are carbapenems?
2. What are carbapenemases? What allows them to become rapidly spread?
3. What is horizontal gene transfer and what are the main types of horizontal gene transfer.

Answer 13

• Carbapenems are a class of broad-spectrum β-lactam antibiotics that exhibit resistance against most types of β-lactamase.
• Carbapenemases are β-lactamases with versatile hydrolytic capacities.
• e.g. NDM1 β-lactamase
• Rapid dissemination is due to horizontal gene transfer; transformation, transduction and conjugation.
• Horizontal gene transfer is the movement of genetic material from a donor organism to a recipient organism that is not its offspring.
• Transformation: naked DNA uptake by bacteria
• Transduction: bacterial DNA transferred by viruses
• Conjugation: DNA transfer between bacterial cells

Question 14

How does the modification of antibiotic targets lead to resistance? Use rifampin and vancomycin resistance as examples.

Answer 14

• Resistance result of mutations that alter the antibiotic binding site.
• E.g. rifampin resistance is due to mutations that alter the antibiotic target binding site in the RNA polymerase, rifampin cannot bind to target and therefore cannot inhibit RNA polymerase.
• E.g. vancomycin resistance is due to the van A gene which alters peptidoglycan biosynthesis; D-Ala-D-Ala is changed to D-Ala-D-Lac, vancomycin cannot target D-Ala-D-Ala and therefore cannot inhibit cell wall biosynthesis.

Question 15

What can we do to change the futue and present of antibiotic resistance?

Answer 15

1. Prolong the lifespan of current antibiotics by best practice use; best practice infection contol, and best practice prescribtion.
2. Use current antibiotics in combinations that give improved activity and slow resistance development.
3. Develop new antibiotics.
4. Develop new strategies, e.g. bacteriophage therapy

Question 16

What is best practice use of antibiotics?

Answer 16

• Infection prevention and control practices
• Diagnostic tools
• Awareness
• Vaccines
• Waste management

Question 17

What are the possible outcomes of using multiple antibiotics together?

Answer 17

• Additive effect: level of activity is addition of the two seperate activities. Will reduce the level of resistance development as the organsism has to become resistant to two antibiotics at once.
• Synergistic effect: level of activity is higher than the addition of the two seperate activities.
• Antagonistic effect: level of activity is lower than the addition of the two seperate activities.

Question 18

What causes antibiotic synergy?

Answer 18

Increased activity can be due to:

• one antibiotic increasing access of the second antibiotic
• action of one antibiotic can be saved from resistance mechanisms by the second.
  e. g. Treatment with polymyxin permeabilizs cell membranes, this increases access for antibiotics that have intracellular targets.