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Medical Microbiology > 2. The Evolution of Antibiotic Resistance > Flashcards

2. The Evolution of Antibiotic Resistance Flashcards

1. To explain how minimum inhibitory concentration of an antibiotic is linked with the definition of antibiotic resistant. 2. To described, with appropriate examples, the various strategies that bacteria use for antibiotic resistance. 3. To explain the rise of ß-lactam resistance in Gram-negative and Gram-positive bacteria with reference to the mobile genetic elements and recombination mechanisms primarily involved.

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1
Q

What is a composite transposon?

A

A segment of DNA located between 2 copies of the same insertion sequence and the entire things moves as 1 unit of DNA.

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2
Q

What is a complex transposon?

A

They are a movable genetic element that contains the transposase machinery and other genes that are not needed for insertion.
They copy and paste themselves into genomes.

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3
Q

What is mostly responsible for the rise of opportunistic infections?

A

Antibiotics resistance

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4
Q

Why are we using ß-lactam resistance as an example?

A
  1. There are lots of different mechanisms of resistance that are mirrored in other antibiotic classes.
  2. It makes up about 60% of antibiotics, so arguably, it is the most important type of resistance.
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5
Q

What are the 4 main types of resistance?

A
  1. Target site modification
  2. Reduced permeability or efflux
  3. Replacement of antibiotic target by a non-susceptible target
  4. Enzymatic degradation or modification
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6
Q

Types of resistance: Target site modification

A
  1. Most antibiotics bind to a protein target.
  2. If you change the structure of the protein through mutation the antibiotic cannot bind.
  3. However these protein targets have essential functions so mutation could cause more harm than the antibiotic.
  4. Therefore, this is a rare form of resistance.
  5. Acquired by mutation/transformation/mobile gene
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7
Q

Types of resistance: Reduce permeability

A
  1. Prevent antibiotic entry
  2. efflux pump out antibiotics
  3. Intrinsic or acquired by mutation
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8
Q

Types of resistance: Replacement

A
  1. The gene of the target of the antibiotic is replaced by a resistant version acquired by horizontal gene transfer
  2. Pre-evolved for this purpose
  3. Acquired through mobile genes
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9
Q

Types of resistance: enzymatic degradation

A
  1. Main form of ß-lactam resistance
  2. Pre-evolved
  3. Mostly acquired on mobile genes
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10
Q

What is resistance?

A

A binary category. Something either is or isn’t resistant.

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11
Q

How is resistance measured?

A

Using minimal Inhibitory Concentration

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12
Q

What is Minimal Inhibitory Concentration?

A
  1. The concentration of antibiotic required to prevent the growth of a population of bacteria
  2. Not always about killing the population
  3. Numerical scale so that if it is high enough, you can define a bacteria as resistant.
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13
Q

How is the point of decision of resistance determined?

A
  1. Based on the dose of antibiotic used in a clinical setting.
  2. This prevents giving too much antibiotic
  3. And determines if the dose that will be given is enough to work.
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14
Q

What does the higher MIC mean?

A

More antibiotic is needed to inhibit growth of bacteria

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15
Q

Why is dosing of antibiotic important?

A
  1. The dose affects the concentration in the blood
  2. We want to maximise the time the antibiotic concentration in the blood is higher than the MIC while keeping the dose safe.
  3. This maximises the time for the antibiotic to inhibit growth and the immune system to take effect.
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16
Q

What really matters in antibiotic treatment?

A

If the bacteria in the infection killed

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17
Q

What are the issues with MIC?

A
  1. It assumes the dose curve is the same for everyone which is not the case.
  2. This could be due to poor circulation, poor kidney function, and obesity.
  3. The break point is only as good as the information available and is different for every patient.
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18
Q

How do we determine the MIC of an Antimicrobial?

A
  1. Dilution tests using different concentrations of antibiotic growth
  2. very dependent on medium, temperature, density, and salt levels.
  3. Every test must be done in exactly the same conditions using standardised methods
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19
Q

What is disc susceptibility testing?

A
  1. This is a proxy measure for MIC
  2. There is a concentration of antibiotic in the disc that spreads through the agar.
  3. There are higher concentrations of antibiotics closer to the disc.
  4. Therefore the closer the growth to the disc = higher MIC
  5. Measure distance
  6. Easy to automate.
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20
Q

How do streptococci gain resistance?

A
  1. They don’t very easily but when they do it’s target site modification.
  2. They are naturally competent so they take up naked linear DNA from the environment.
  3. This passes mutations from one cell to another within populations
  4. Once in the cell the DNA recombines with the host genome using non-reciprocal homologous recombination
  5. This transforms the cell
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21
Q

What makes bacteria naturally competent?

A

They have a series of proteins that pulls in the DNA and protects it.

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22
Q

How does non-reciprocal homologous recombination occur?

A
  1. The DNA has to be very similar to the sequence in the genome.
  2. RecA gets hold of the donor DNA and randomly shoves it into the genome DNA.
  3. Sometimes it hits a region of complementarity and hydrogen bonds form between the genome DNA and the donor DNA. This stabilises the interaction.
  4. The rest of the donor DNA is cut away and the genomic strand is replaced with the donor strand.
  5. If the donor sequence introduces a different sequence it can introduce a mutation.
  6. The DNA is replicated and 1 of the daughter cells carries the mutation.
  7. The 2 bacteria fight it out with natural selection to determine if the mutation is beneficial.
23
Q

What is the target of penicillin?

A

Penicillin binding protein or PBP

24
Q

How do Penicillin binding proteins gain mutations and resistance?

A
  1. As long as the dosing of antibiotic is correct mutations tend to be unsuccessful.
  2. Single mutations can slightly increase the MIC of penicillin.
  3. Multiple mutations can gradually increase the MIC but this is very slow.
  4. This process is sped up by the construction of mosaic PBP genes.
25
Q

What are Mosaic PBPs?

A

A bacteria in the population picks up all the single mutations from the other bacteria to create a mosaic PBP with up to 83 different mutations that massively increase the MIC.

26
Q

Where do mutations in penicillin binding proteins occur?

A
  1. Off the penicillin binding site
  2. This is so the protein can continue its normal function.
27
Q

What speeds up the formation of mosaic PBPs?

A
  1. Incorrect dosing of antibiotics creating a selection pressure so the mutation carrying bacteria survive.
  2. This speeds up resistance due to transformation and not relying on single mutations.
28
Q

How did the fear of resistance cause more resistance?

A
  1. The fear of underdosing causing mutation by target site modification was a massive concern.
  2. This lead to the overdosing of antibiotics, causing resistance through other methods like ß-lactamases.
29
Q

What is the ß-lactamase from S. aureus?

A

BlaZ,

30
Q

When was BlaZ S. aureus resistance discovered?

A

1941 - penicillin introduce
1944 - resistance observed in S. aureus
1948 - >50% of S. aureus in UK was resistant
This is due to BlaZ

31
Q

How did ß-lactamase enzymes evolve?

A
  1. They evolved a long time ago to help bacteria fight fungi that produce antibiotics.
  2. The genes that encode these enzymes are on plasmids so they can easily move between species and genus.
  3. This causes a sudden increase in the MIC.
32
Q

How does the dose of antibiotic affect ß-lactamase resistance?

A
  1. Underdosing has no effect
  2. Overdosing of antibiotics creates a selection pressure so bacteria with the resistance plasmid will survive.
33
Q

When was MecA S. aureus resistance discovered?

A

1961 - methicillin introduce to combat penicillin resistant S. aureus
1963 - Methicillin resistance seen in S. aureus
1984 - resistance was shown to be due to MecA

34
Q

What does MecA do?

A

It gives resistance to every ß-lactam

35
Q

What is MecA?

A
  1. It is encoded on a plasmid
  2. It is part of a complex transposon that often encodes many types of resistance and virulence factors (SCCmec II).
  3. Resistance is caused by the acquisition of a modified target and a sudden gain of resistance.
  4. It is inserted into the same place in the chromosome every time
36
Q

What is methicillin?

A

A larger ß-lactam that is not destroyed by most ß-lactamases

37
Q

What is ampicillin and why is it used?

A
  1. A chemically modified penicillin that can be used to treat gram negative pathogens
  2. Resistance developed quickly as gram negatives contained a ß-lactamase that hadn’t been encountered before.
38
Q

TEM ß-lactamase in Gram-negatives

A

1961 - ampicillin introduced
1965 - ampicillin resistance observed in E. coli
A Penicillinase called TEM causes the resistance.
1995 - TEM is responsible for 90% of ampicillin resistance in E. coli
Now there are many different TEMs

39
Q

Where is TEM encoded?

A

On a plasmid containing the Tn1 complex transposon

40
Q

What is the Tn1 complex transposon?

A
  1. A relatively simple complex transposon
  2. Contains the transposase genes and the TEM ß-lactamase gene surrounded by a pair of inverted repeats.
  3. It moves with a copy and paste mechanism.
  4. Gets everywhere especially if there is an ampicillin selection pressure.
41
Q

SHV ß-lactamase resistance mobilisation

A

1970 - SHV on the chromosome of and produced by K. pneumoniae but it is not mobile
1974 - SHV found in E. coli suggesting it has become mobile.
1979 - confirmed to be on a plasmid

42
Q

How did SHV ß-lactamase become resistant?

A
  1. A composite transposon was generated.
  2. The same insertion sequence is on either side of the SHV gene and others.
  3. The transposase enzymes can move all the genes between the insertion sequence.
  4. SHV has two different mobile forms.
43
Q

Why is mobilisation of chromosomal genes bad?

A

The composite transposon can contain strong promoters and drive the expression of resistance genes.

44
Q

Why were cephalosporins introduced ?

A

To treat resistant gram-negative infections like TEM and SHV producing E. coli and K. pneumoniae.

45
Q

How did cephalosporin resistance occur?

A
  1. AmpC is an E. coli protein that can destroy cephalosporins
  2. But it is not normally produced at high enough concentrations.
  3. Mutations occurred in the AmpC promoter and attenuator sequences to massively increase expression.
46
Q

What caused cephalosporin resistance?

A

Mutations that amplified existing resistance mechanisms

47
Q

Why could cephalosporins treat resistance infections?

A

They are bigger molecules so didn’t fit in the active site of most ß-lactamases.

48
Q

What are Extended-spectrum ß-lactamases (ESBLs)?

A

These are mutated ß-lactamases with larger active sites, so they can degrade cephalosporins.

49
Q

What are some examples of ESBLs?

A

SHV-2 and TEM-3 which both carry the gly238ser mutation

50
Q

What is now the most common ESBL?

A

CTX-M

51
Q

Where did CTX-M come from?

A

The chromosome of Kluyvera

52
Q

How did CTX-M spread?

A

Kluyvera lives in the gut near E. coli and passed it onto pathogenic E. coli strains

53
Q

how is CTX-M mobilised?

A
  1. Through a composite transposon with the ISEcp insertion sequence.
  2. This sequence only needs weak matches in the 2nd insertion sequence.
  3. This means it is much more likely to insert into a chromosome as only 1 insertion sequence needs to match to have the initiation mobilisation event.
54
Q

How can intrinsic resistance genes suddenly cause resistance?

A
  1. Mutation causing over expression
  2. Mobilisation leading to activation

Medical Microbiology (16 decks)

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