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Medical Microbiology > 5. New developments in (fluoro)quinolone Resistance > Flashcards

5. New developments in (fluoro)quinolone Resistance Flashcards

1. Describe the mechanism of action of quinolone antibiotics. 2. Describe mechanisms of quinolone resistance. 3. Discuss how different mechanisms can work together to confer resistance.

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USMLE® Step 1 flashcards Biology 101 flashcards
1
Q

What are quinolones?

A
  1. They are synthetic antibiotics.
  2. They were relatively recently discovered in the 60s
  3. They are topoisomerase inhibitors.
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2
Q

How were quinolones discovered?

A
  1. Scientists were trying to quinine into a better anti-malaria treatment.
  2. Quinolones like Nalidixic acid were a product of this program.
  3. They failed to find antimalarials, but these had strong antibiotic activity when tested
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3
Q

What is Nalidixic Acid?

A
  1. The 1st quinolone.
  2. It is used to treat gram negative bacteria.
  3. They are used in humans and was moderately effective in humans.
  4. It has some issues with resistance and adverse pharmacological properties.
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4
Q

Why is Nalidixic acid unusual?

A

It mainly treats gram-negative bacteria. This is strange as gram-positives are easier to treat due to its membrane.

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5
Q

Why were quinolones developed into fluoroquinolones?

A
  1. There was a need for better treatment for GI infections like salmonella and shigella.
  2. There was a need for more and better gram-negative treatment.
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6
Q

How are 2nd generation (Fluoro)quinolones different from 1st generation?

A
  1. They have the addition of fluorine on carbon six on one of the rings.
  2. This expands their spectrum of activity to make it very potent against gram-negative bacteria.
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7
Q

What is the main 2nd generation (fluoro)quinolone and how is it different from 1st gen quinolones?

A
  1. Ciprofloxacin
  2. Contains the fluorine at carbon 6 as well as the addition of a 6 membered ring that contains a nitrogen and a piperazinyl groups.
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8
Q

What is a piperazinyl group?

A

A carbon ring with a nitrogen and a free NH group.

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9
Q

What increases the spectrum of activity of quinolones?

A

The addition of extra chemical groups

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10
Q

What additions were made to make 3rd generation quinolones?

A

Different chemical groups are added to the free N on the piperazinyl group. eg a methyl group

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11
Q

What can 3rd gen quinolones treat?

A
  1. The gram-negative GI etc infection of the previous generation
  2. Expanded to treat gram-positive infections like respiratory tract infections.
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12
Q

What is an example of 3rd gen quinolones?

A

Levofloxacin

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13
Q

What additional chemical groups were added to make 4th generation quinolones?

A

In the piperazinyl group, a carbon is changed to a nitrogen

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14
Q

What can 4th gen quinolone treat?

A

Everything the previous generations can with the addition of anaerobic infections.
They are very broad spectrum and very potent.

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15
Q

What is the base structure of quinolones?

A

a bicyclic ring

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16
Q

Additions to the quinolone base structure: carbon 1

A

These increases the overall potency.

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17
Q

Additions to the quinolone base structure: carbon 5

A

These increase the gram-positive activity

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18
Q

Additions to the quinolone base structure: carbon 6

A

Fluorine addition increases the gram-negative activity

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19
Q

Additions to the quinolone base structure: carbon 7

A

Additions of groups like piperazinyl increase the gram-negative and gram-positive activity

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20
Q

Additions to the quinolone base structure: Position 8

A

These increase activity to cover anaerobes and expand the gram-positive activity.

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21
Q

What is the clinical importance of (Fluoro)Quinolones?

A
  1. They are broad spectrum.
  2. Treat UTIs, Bacterial diarrhoea and pneumonias.
  3. They can treat some anaerobic infections
  4. They are synthetic agents
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22
Q

Why are quinolones being synthetic agents attractive?

A
  1. Naturally produced antibiotics have intrinsic resistance mechanisms in the organisms that make them and organisms that live in the same niche.
  2. This prevents them from being killed by their metabolites but they can mobilise to make resistant infections.
  3. Synthetic compounds would be expected not to have intrinsic resistance mechanisms which make them desirable.
  4. However this is not the case
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23
Q

What is the problem with quinolones?

A
  1. As they are used more, it has become apparent they can have very serious side effects.
  2. Joint damage, muscle damage, neurological complications, and adverse mental health effects.
  3. This has caused very tight regulation on their use to limit them to serious and complex infections.
  4. Due to this, it is expected quinolones will decline in use even more.
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24
Q

How do quinolones work?

A

They are bacteria type 2 DNA topoisomerase inhibitors.

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25
Q

How do bacteria package their DNA and what problems does this create?

A
  1. They have circular chromosomes and plasmids and they need to package it efficiently using supercoils.
  2. Fundamental processes like DNA replication and transcription puts the DNA under torsional stress and effects it topology.
  3. If this is not countered, the stress can break the DNA.
  4. During replication, the chromosomes are linked and need to be separated.
  5. This is done by topoisomerases
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26
Q

What do type 2 topoisomerases do?

A
  1. They control the shape and topology of the DNA.
  2. DNA gyrase controls super coiling in the negative direction.
  3. Topo 4 controls supercoiling in the positive direction.
  4. Topo 4 can also separate the chromosomes from replication.
  5. They are essential for cell function, so they make a good drug target.
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27
Q

What do quinolones target?

A

The type 2 topoisomerases DNA gyrase and Topo 4.

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28
Q

What does DNA gyrase do during DNA replication?

A

It coats the DNA and changes supercoiling in order to counter the topological stress caused by replication.

29
Q

How does quinolone target activity change depending on the bacterial target?

A
  1. In gram-negative bacteria quinolones mainly target DNA gyrase.
  2. In gram-positive bacteria, quinolones mainly target Topo 4.
30
Q

Does quinolones having 2 targets prevent resistance?

A
  1. Not really
  2. Due to the difference in activity depending on the bacteria a mutation in 1 along with another resistance mechanism like efflux is enough to provide resistance.
31
Q

What makes up the topoisomerase subunits?

A

2 subunits:
Gyrase A that binds the DNA
Gyrase B that is an ATPase

32
Q

What encodes the Gyrase A subunit?

A

ParC

33
Q

What encodes the Gyrase B subunit?

A

ParE

34
Q

What is the mechanisms of action of topoisomerases?

A
  1. The topoisomerase assembles around the gate (G) segment of the DNA
  2. Gyrase B grabs a 2nd strand of DNA called the transport (T) segment.
  3. A serious of conformational changes, driven by the hydrolysis of ATP, occurs to introduce a dsDNA break in the G segment.
  4. The T segment passes through the gap in the G segment.
  5. The break is resealed. This twists or untwists the DNA.
35
Q

Why is topoisomerase activity risky for bacteria?

A
  1. dsDNA breaks are toxic to cells.
  2. They are also hard to repair.
  3. They are essential for function but make bacteria vulnerable to drug attack.
  4. If something stops the gyrase from repairing the transient dsDNA breaks they accumulate.
  5. This kills the cell.
36
Q

What makes up the inhibitory complex in quinolone treatment?

A
  1. The quinolone
  2. The gyrase
  3. The DNA
37
Q

What is the structure of the gyrase A active site?

A
  1. It is a dimer protein so a quinolone binds to each active site.
  2. A tyrosine residue sits in the active site that is responsible for the dsDNA breaks.
38
Q

How do quinolones inhibit topoisomerases?

A
  1. The quinolone binds next to the tyrosine residue.
  2. Both gyrase A and B bind the quinolone and the DNA.
  3. The Bicyclic ring of the quinolone has a similar structure to the DNA.
  4. It intercalates with the DNA and sits across the bases.
  5. The quinolone interacts with the gyrase subunits via hydrogen bonds.
39
Q

How does the quinolone hydrogen bond with the gyrase?

A

It hydrogen bonds through Mg2+ and water with aspartate87 and serine83 in the active site of the gyrase.

40
Q

What are the major resistance mechanisms to quinolones?

A
  1. Target modification through mutation or transformation.
  2. Inactivation of antibiotic
  3. Removal of antibiotic
41
Q

What does QRDR stand for?

A

Quinolone resistance determining regions

42
Q

What is the QRDR?

A

A few key amino acids that are important for quinolone binding. Mutations in these residues cause resistance due to the quinolone not being able to bind.

43
Q

What are some common mutations in the QRDR?

A
  1. Mutations in ser83 to a residue that cannot hydrogen bond
  2. Asp87 to an alanine.
44
Q

How do mutations in the QRDR contribute to resistance?

A
  1. Individual mutations alone don’t cause massive resistance.
  2. If both ser83 and asp87 mutate, this starts to affect the MIC and create a resistant bacteria.
  3. A build-up of point mutations, as well as other mechanisms, are needed for resistance.
  4. You can also get mutations in gyrase B.
45
Q

What is the function of chromosomal efflux pumps?

A

To remove toxin and toxic metabolites from the bacterial cell.

46
Q

What is the NorA efflux pump?

A

It is associated with resistance in gram positives.

47
Q

What are the 2 types of gram-negative efflux pumps?

A
  1. One that sites in the inner membrane and pumps out molecules into the periplasm.
  2. One that sits in the inner and outer membrane to take molecules from the cytoplasm to outside the cell.
48
Q

What are RND efflux pumps?

A
  1. Resistance nodulation division pumps
  2. Sits across both membranes
  3. very complex proteins
49
Q

What are the different subunits of an RND efflux pump? (example from E. coli)

A
  1. AcrB inner membrane subunit that pumps out the molecules.
  2. TolC outer membrane subunit that creates a pore.
  3. AcrA that links the 2 together.
50
Q

What is the source of energy of RND efflux pumps?

A
  1. They are proton antiporters.
  2. The energy source is the proton motive force.
  3. AcrB take protons from the periplasm, pumps them into the cytoplasm, and uses the energy to drive cargo out of the cell.
51
Q

What type of resistance are RND efflux pumps?

A

Intrinsic resistance that is not exclusive to quinolones but heavily contribute to it.

52
Q

How do efflux pumps work with other resistance?

A

To get an MIC increase you need to upregulate efflux as well as target site modification or inactivation.

53
Q

When was plasmid mediate quinolone resistance discovered?

A
  1. In the 90s due to taking time to develop
  2. on an MDR plasmid in klebsiella.
54
Q

What is responsible for plasmid mediated quinolone resistance?

A
  1. When the plasmid was sequenced resistance was associated with the gene QNR.
  2. QNR was shown to prevent the action of quinolone by restoring the supercoiling function of the topoisomerases.
55
Q

What is QNR?

A

A pentapeptide repeat protein with repeating residues in position 2 and 3.

56
Q

What are the repeating residues in QNR?

A

Position 2 = acidic residue like aspartic acid
Position 3 = A hydrophobic residue.

57
Q

What is unusual about the QNR protein structure?

A
  1. Due to the pentapeptide repeats the protein forms a square rode like structure.
  2. It is very uniform.
  3. It has the same dimensions and charge distribution as B form DNA.
  4. The acidic amino acids mimic the phosphate backbone.
58
Q

What is the purpose of the QNR structure?

A
  1. It mimics the T segment of the DNA
  2. QNR interacts with gyrase B
  3. When QNR is present with ciprofloxacin the cleavage rate of DNA decreases.
59
Q

What is the function of QNR?

A

The exact mechanism is unknown but the evidence suggests that QNR helps religate the G segment of the DNA.

60
Q

Where is Qnr encoded?

A

Many QNR isoforms are mainly found on multi-drug resistance plasmids with other things like ß-lactamases.

61
Q

Where is Qnr mediated resistance found?

A

Globally with many isoforms discovered

62
Q

How does Qnr have synergy with chromosomal resistance mechanisms?

A

In order to make a high enough MIC a combination of resistance is needed. This includes Target site modification, efflux and QNR.

63
Q

What are Aminoglycosides?

A

An antibiotic class made up of 6 membered sugars.

64
Q

What is aac?

A

An Aminoglycoside N-acetyltransferase. This modifies aminoglycoside antibiotics by acetylation of a free N.

65
Q

What is aac(6’)-1b cr?

A
  1. A version of aac that causes resistance to quinolones, especially ciprofloxacin.
66
Q

What are the mutation in aac to make aac(6’) 1b cr?

A
  1. tryptophan 102 Arginine (W102R)
  2. Aspartic acid 179 tyrosine (D179Y)
67
Q

How does aac(6’)-1b cr provide quinolone resistance?

A
  1. The point mutations mean aac(6’)-1b cr can acetylate the free nitrogen on the piperazinyl group.
  2. The mutations at 102 and 179 opens up the active site enough that ciprofloxacin can enter.
  3. The free nitrogen can then be modified.
  4. This inhibits the function of the quinolones.
68
Q

Are Qnr and aac true resistance mechanisms?

A
  1. They alone are not enough to cause resistance.
  2. They are more mechanisms of tolerance.
  3. Not every bacteria is killed by initial treatment and 1 mutation is enough to give a few cells a survival advantage.
  4. These surviving cells can replicate and gain more resistance mechanisms,
  5. This creates an environment for further resistance to develop.

Medical Microbiology (17 decks)

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