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Medical Microbiology > 4. Carbapenemases and ß-lactamase Inhibitors > Flashcards

4. Carbapenemases and ß-lactamase Inhibitors Flashcards

1. Discuss how class A ß-lactamases evolve to become ESBLs. 2. Identify the most clinically important carbapenemases. 3. Describe the basis for carbapenemase activity in Class A and class B enzymes. 4. Describe the usefulness and limitations of currently available ß-lactamase inhibitors.

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1
Q

What 2 class A ß-lactamases have evolved into ESBLs?

A
  1. TEM
  2. SHV
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2
Q

What is KPC?

A

A carbapenemase

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3
Q

What is OXA-48?

A

A carbapenemase

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4
Q

What ß-lactamases can break down penicillin?

A

All ß-lactamases.

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5
Q

What antibiotics can narrow spectrum ß-lactamases break down?

A

Penicillins and some cephalosporins

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6
Q

What antibiotics can ESBLs break down?

A

All penicillins and cephalosporins

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7
Q

What are ESBLs?

A

Extended spectrum ß-lactamases

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8
Q

What ß-lactamases can breakdown carbapenems?

A

None

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9
Q

What antibiotics can carbapenemases break down?

A

All ß-lactams including monobactams

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10
Q

What antibiotics do metallo-ß-lactamases break down?

A

The bicyclic ß-lactams.
So not monobactams.

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11
Q

Why is the development and improvement of ß-lactams important?

A

To improve potency

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12
Q

How has penicillin been developed and improved?

A
  1. An addition of a single amino group to make ampicillin.
  2. This means it can travel through the gram-negative double membrane.
  3. The further addition of an OH group to make amoxicillin improves solubility to pass through the membrane.
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13
Q

How have cephalosporins been improved?

A
  1. Developed from 1st generation in 1964 to 5th generation in 2010.
  2. Cephalosporins are classified mostly by their chemical structure but also the spectrum of activity.
  3. A bulky oxyimino group was added in the 3rd generation to prevent it from fitting in the active site of the narrow spectrum ß-lactamases.
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14
Q

What cephalosporins can be broken down by narrow spectrum ß-lactams?

A

1st and 2nd gen

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15
Q

What cephalosporins can be broken down by ESBLs?

A

All cephalosporins including the bulkiest ones.

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16
Q

How have TEM and SHV evolved?

A
  1. They have evolved via the same mutations to become ESBLs.
  2. They have very similar structures to begin with.
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17
Q

What forced the mutation of TEM and SHV into ESBLs?

A

The introduction of 3rd gen cephalosporins into clinics that are too bulky for narrow spectrum ß-lactams.

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18
Q

What are the mutations that TEM and SHV gain to become ESBLs?

A
  1. Mutation in position 238 that allows the 3rd gen cephalosporins to fit into the active site of the ß-lactamases.
  2. Mutation in position 240 only occurs with the 238 mutation and extends the spectrum of activity even further so it can break down the bulkiest cephalosporins.
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19
Q

Why do single amino acid changes in TEM and SHV cause massive alterations in their substrate profile?

A
  1. Ser70 is in the active site
  2. Positions 238 and 240 are very close to the active site, so they can directly affect the active site binding and catalysis.
  3. Small amino acid changes can change the structure of the enzyme.
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20
Q

What is the SHV-2 mutation into an ESBL?

A
  1. Glycine 238 to serine.
  2. This glycine is very close to the active site serine.
  3. The change to serine (which is a very bulky amino acid) alters a loop near the active site, which opens up the active site and effectively increases the size of the active site.
  4. This creates space for the oxyimino group on 3rd gen cephalosporins and allows the binding of bulky cephalosporins into the active site.
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21
Q

What has overtaken TEM and SHV as the most predominant ESBLs in clinic?

A

CTX-M

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22
Q

How many variants of CTX-M are there?

A

over 200

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23
Q

What is the most clinically important ESBL?

A

CTX-M

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24
Q

What can’t ESBLs break down?

A

Carbapenems

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25
Q

What are carbapenems?

A
  1. A class of ß-lactam antibiotics
  2. This most potent ß-lactams due to being unaffected by ESBLs
  3. They are used to treat resistant infections that produce other ß-lactamases.
  4. They are a last resort antibiotic that is mostly used on very ill patients.
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26
Q

What are carbapenemases?

A

ß-lactamases that can break down carbapenems.

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27
Q

Why is carbapenem resistance increasing?

A
  1. There is an increase in CTX-M producing infections that need treatment with carbapenems.
  2. The more we use carbapenems the more we select for carbapenem-resistant strains of bacteria.
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28
Q

What are the carbapenem resistance mechanisms?

A
  1. Efflux pumps
  2. Permeability modification working with weak affinity enzymes
  3. Acquired serine carbapenemases (KPC, OXA)
  4. Metallo-ß-lactamses
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29
Q

How does permeability modification and weak ESBL activity produce resistance to carbepenems?

A
  1. Some ESBLs can weakly break down carbapenems
  2. If you reduce the concentration of carbapenem in the periplasm even weak binding enzymes can have a large effect.
  3. Less antibiotic = more effective enzyme activity.
  4. You can reduce antibiotic concentration by reduced permeability or efflux pumps
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30
Q

What can break down all carbapenems?

A

Metallo-ß-lactamases

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31
Q

What is the structure and mechanism of the AcrAB-TolC efflux pump?

A
  1. It’s a tripartite pump
  2. TolC is the exit duct that creates a pore in the outer membrane.
  3. AcrB is the inner membrane efflux pump which captures the antibiotic in the periplasm that pumps out the antibiotic using energy from the proton motive force to change conformation.
  4. This change in conformation causes the TolC pore to open.
  5. AcrA is the periplasmic adaptor protein
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32
Q

What is the function of an efflux pump?

A
  1. To reduce the concentration of toxic molecules in the periplasm.
  2. In normal function this includes things like bile acids.
  3. They can also pump out antibiotics.
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33
Q

What are the big 5 carbapenemases?

A
  1. KPC
  2. OXA-48
  3. NDM
  4. VIM
  5. IMP
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34
Q

What are CPEs?

A

Carbapenemase-producing Enterobacteriaceae

35
Q

How has the profile of common carbapenemases changed over time?

A
  1. In the early 00s there was little evidence for carbapenemases.
  2. Around 2012 there was a huge rise in carbapenemases driving by KPC.
  3. KPC has overtaken OXA-48 as the most clinically relevant carbapenemase.
  4. NDM1 was discovered on 2009 and has seen a rapid increase in reported cases of CPE due to this.
  5. Often carbapenemases are produced together in a bacteria.
36
Q

What are the most clinically relevant carbapenemases?

A
  1. KPC
  2. NDM1
37
Q

What is KPC?

A
  1. A class A serine ß-lactamase
  2. That can turn lots of antibiotics, including ampicillin, 3rd gen cephalosporins, monobactams and carbapenems.
  3. Carbapenems bind very well into the active site
38
Q

What is Kcat?

A
  1. The catalytic rate constant
  2. Tells you how many molecules it can turnover every second.
39
Q

What is Km?

A
  1. It tells you how well the antibiotic binds
40
Q

How well does KPC bind carbapenems?

A

It has a moderate Kcat and a high Km, so KPC can bind carbapenems very well.

41
Q

Where is KPC encoded?

A

On a composite transposon that means it has spread all over the world.
It is particularly a problem in the USA.

42
Q

What makes KPC producers so worrying?

A
  1. They are often multidrug-resistant
  2. This is due to them often producing several ESBLs and carbapenemases.
  3. They can also produce efflux pumps.
43
Q

What is OXA-48?

A
  1. A class D carbapenemase
  2. Accounts for around 1/3 of all UK carbapenem-resistant infections.
  3. found everywhere particularly in India.
44
Q

What are most class D ß-lactamases?

A

ESBLs

45
Q

What is the OXA-48 spectrum of activity?

A
  1. Narrow spectrum then KPC2 due to their poor turnover of cephalosporins.
  2. Carbapenems are hydrolysed efficiently and they bind well into the active site.
  3. OXA-48 doesn’t need to have super high Kcat to Ertapenem as it works in conjunction with efflux pumps to lower the antibiotic concentration and make the enzymes more efficient.
46
Q

Why doesn’t OXA-48 effectively hydrolyse cephalosporins?

A

They don’t really fit in the active site of OXA-48 and have a high Km (bad binding).

47
Q

What other methods of resistance do OXA-48 producing bacteria often produce?

A
  1. TEM1
  2. CTX-M15
  3. Some efflux pumps
48
Q

What are the important metallo-ß-lactamases?

A
  1. IMP
  2. VIM
  3. NDM1
49
Q

What was the global spread of NDM1?

A

It was discovered in 2019 in India and in a short period it has now spread throughout the world.

50
Q

Why is NDM1 known as an epidemic gene?

A
  1. It is found on multiple different plasmid types and in lots of different bacteria.
  2. They are often found co-produced alongside other ß-lactamases like KPC2.
  3. It has been identified in multiple gram-negative bacteria predominately Enterobacteriaceae like E.coli and Klebsiella pneumoniae.
51
Q

what is the NDM1 spectrum of activity?

A
  1. It is not as efficient as other carbapenemases but can turn over all ß-lactams well.
  2. Due to it being a metallo-ß-lactamase it has a very open active site that allows for the binding of the bulkier ß-lactams.
52
Q

Does NDM1 break down Aztreonam?

A

No because it is a monobactam so it doesn’t bind into the active site of the metallo-ß-lactamases.

53
Q

What is a TOP10 lab strain of E.coli?

A

A lab strain of E.coli with no other resistance mechanisms.

53
Q

What happens when NDM1 is introduced to TOP10 E.coli?

A

NDM1 gives high levels of resistance to basically everything and all activity is due to the NDM1 which is worrying.

54
Q

What is Clavulanic acid?

A
  1. A ß-lactam but it is not an antimicrobial.
  2. It cannot inhibit PBPs, but it can inhibit some ß-lactamases.
55
Q

What ß-lactamases can clavulanic acid inhibit?

A

Some class A ß-lactamases

56
Q

How was clavulanic acid developed to be a ß-lactamase inhibitor?

A
  1. It was developed in the 70s and was derived from streptomyces clavuligerus.
  2. It was developed alongside Tazobactam and sulbactam.
  3. These are all ß-lactams that cannot bind PBPs but can bind serine ß-lactamases
57
Q

What is the reaction mechanism of Clavulanic acid and other serine ß-lactamase inhibitors?

A
  1. The inhibitor enters the active site and the serine nucleophile attacks the carbonyl bond in the ß-lactam ring.
  2. The ß-lactam ring breaks and the unstable tetrahedral intermediate forms.
  3. It then breaks down into the Acyl-enzymes intermediate.
  4. The clavulanic acid is covalently linked to the serine in the active site and the 4 membered ß-lactam ring is broken.
  5. The 2nd 5 membered ring also breaks open and produces lots of different produces.
58
Q

How do the different Clavulanic acid reaction products inhibit ß-lactamases?

A
  1. These don’t leave the active site easily so are an inhibitor for the ß-lactamases.
  2. Some products further break down and permanently alter the active site of the ß-lactamase preventing their function.
59
Q

What type of inhibitor is Clavulanic acid?

A

A suicide inhibitor as it cannot reform the starting compound.

60
Q

Why can’t clavulanic acid inhibit carbapenems?

A

The hydrolysis of the acyl-enzymes intermediate happens before it can break down into the inhibitory product.

61
Q

Why can’t clavulanic acid inhibit Metallo-ß-lactams?

A

Because it works by making a covalent bond with a serine in the active site.

62
Q

How can clavulanate be used in therapies?

A

It can be used with amoxicillin to cure otherwise amoxicillin resistant infections

63
Q

How have TEM and SHV developed resistance to inhibitors like clavulanic acid?

A
  1. Mutations at position 165 directly changes the clavulanic binding site to prevent binding.
  2. Mutations at position 244 prevents the opening of the 2nd ring and prevents the inhibitory products forming.
  3. Mutation at 69 and 276 arise in combination with ESBL mutations
64
Q

What is Avibactam?

A
  1. A diazabicyclooctane
  2. This is a 5 membered ring fused to a 6 membered ring.
  3. It looks like a ß-lactam but is not one.
  4. It contains the carbonyl bond needed to interact with the active site serine.
65
Q

How does Avibactam work?

A
  1. It enters the active site and undergoes nucleophilic attack on the carbonyl from the active site serine.
  2. This opens up the 5 membered ring and makes a covalent link between the avibactam and the active site serine.
  3. An acyl-enzyme intermediate forms which is very stable so there is little hydrolysis.
  4. Avibactam stays bound to the serine ß-lactamase and is an extremely strong inhibitor.
66
Q

What type of inhibitor is Avibactam?

A

A reversible inhibitor

67
Q

What is the recyclisation of Avibactam?

A
  1. Return to the original form.
  2. the NH group attack the carbonyl bond between the serine and avibactam and reforms the 5 membered ring.
68
Q

When did Avibactam become available?

A

2015

69
Q

What does Avibactam inhibit?

A
  1. Strong inhibitor of class A and C ß-lactamases including carbapenems
  2. Weak inhibitor of class D
  3. Cannot inhibit class B
70
Q

What did the success of Avibactam lead to?

A

The development of more DBO ß-lactamase inhibitors.

71
Q

What is Relebactam?

A
  1. A DBO with a similar structure to Avibactam.
  2. Slightly different side chain.
  3. It can be used in combination with imipenem and cilastatin.
72
Q

Can DBOs inhibit PBPs?

A
  1. Yes, they can be weakly.
  2. This is due to the carbonyl bond that can interact with the serine in the PBP active site.
  3. They currently cannot be used as antimicrobials on their own, but it is under development and would reduce the need for ß-lactams.
73
Q

What is ETX0462?

A
  1. A DBO that is a potent antimicrobial and is under development.
  2. Contains a pyrazol group that makes binding to PBPs better
74
Q

What is Vaborbactam?

A
  1. A cyclic Boronate inhibitor of Class A and C ß-lactamases.
  2. The active part is the boron ion that interacts with the serine in the active site.
75
Q

What is the mechanism of inhibition of Vaborbactam?

A
  1. The boron reacts with the active site serine
  2. A covalent link forms between the active site serine and the boron which changes the geometry of the boron from trigonal to tetrahedral.
  3. This covalent bond is very stable and hard to break.
  4. This is a mimic of the transition state of ß-lactam hydrolyses.
76
Q

What can Vaborbactam inhibit?

A
  1. It is a very potent class A (e.g. KPC2) and class C inhibitor. It is clinically approved to treat these.
  2. It is 5 times less potent against class D enzymes
  3. They can inhibit class B enzymes but 100 times less effective.
77
Q

Why can vaborbactam inhibit both serine and metallo ß-lactamase?

A

due to the common tetrahedral intermediate in the reaction mechanism.

78
Q

What are cross-class inhibitors?

A

ß-lactamase inhibitors that can inhibit all 4 classes of ß-lactamases by mimicking the tetrahedral transition state.

79
Q

what is Taniborbactam?

A
  1. A 6 membered boronate ring fused with another 6 membered ring to mimic bicyclic ß-lactams.
  2. It is a very effective inhibitor of serine and metallo ß-lactamases.
80
Q

How does Taniborbactam work?

A
  1. When bound to serine, the boron ion changes from trigonal to tetrahedral to mimic binding to PBPs. This is very stable.
  2. With Metallo-ß-lactamases, the boron interacts with the OH that sits between Zn ions and attacks the boronate to form the tetrahedral shape. The taniborbactam then sits in the active site. This is very stable
81
Q

What type of inhibitors are boron inhibitors?

A

Mechanism based inhibitors

82
Q

Where is the ESBL threat from?

A

It was SHV/TEM, but it is now CTX-M

Medical Microbiology (16 decks)

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