The Evolution of Antibiotic Resistance AND Beta-Lactams and Resistance To Them

Question 1

What are the 4 main methods of antibiotic resistance

Answer 1

1) Target site modification –> not a common mechanism of resistance as mutations often compromise bacterial fiteness
2) Decreased permeability, increased efflux –> can convey resistance to many classes of Abx, mainly a method used in gram negatives
3) Enzymatic degradation –> the most important and most common method of antibiotic resistance
4) Replacement of antibiotic target by a non-susceptible target

Question 2

What is the most common cause of antibiotic resistance

Answer 2

Enzymatic degradation –> the most important and most common mechanism of resistance

Question 3

What are the 3 methods used to determine the MIC of an antimicrobial

Answer 3

1) Agar dilution test

2) Disc susceptibility - Zone of Inhibition

Question 4

Describe the agar dilution test

Answer 4

Put different concentration of antibiotic in the agar and see if the bacteria can grow

Question 5

Why is breakpoint setting political

Answer 5

if you set a higer breakpoint than it looks like you have less resistance

Question 6

Name 2 bacteria that are naturally competent

Answer 6

H. Influenzae and Strep Pneumoniae

Question 7

What does it mean that bacteria are ‘naturally competent’

Answer 7

The bacteria can take up naked linear Dna from the environment and then combine it with the host genome via non-reciprocal homologus recombination
This trasnforms the phenotype of the recipient cell

Question 8

Describe the process of non-reciprocal homologous recombination

Answer 8

The naked DNA is taken up from other dead bacteria
DNA enters as a linear strang
RecA proteins takes one strand of DNA and you get association of homolous segments
Strand seperation adn apiring –> endonucleases nick the donor and host strands –> the gaps in the strand are filled and ligated.

Can allow many mutations to be passed on

Question 9

Give an example of target site mutation conveying resistance in S. Pneumoniae

Answer 9

can accumulate 83 mutations in the PBP2 alpha protein nthat can retain the function of the PBP protein but prevents penicillin binding. Mainly gets this by creating ‘mosaic’ genes as non-reciprocal homologous recombination allows the individual mutations to accumulate

UNDERDOSING OF ABX FRIVES THIS –> however bacteria that aren’t naturally competent hence won’t really evolve resistance this

Question 10

describe how penicillin resistance arose

Answer 10

BlaZ penicillinase enzyme was produced

Penicillin was first used to treat gram +ves in 1941, just 3 years later 50% of S. aureus were resistant

Question 11

rWhat mobile element is BlaZ carried on

Answer 11

Tn552

Question 12

What is a transposon

Answer 12

A chromosomal segment that can undergo transposition –> they can move its location around the genome

Question 13

How do transposons actually work

Answer 13

Inverted repeat sequences ovvur at each end of the transposon
The transposon always encode a tnp enzyme that is responsible for moving the transposon!
Some transposons just cut themselves out and move, whereas other transposons cut themeselves out, duplicate themselves and then move

Question 14

Give an example of acquired resistance in S. Aureus

Answer 14

MecA gene in S. Aureus
This arose when methicillin was used to combat penicillin resistance in 1961.
However just 2 years later this arose –> MecA encoded an altered PBP2’

This is resistance through acquisition of an altered target

Question 15

What transposons is MecA being carried on

Answer 15

Generally carried on complex transposons. The transposons often carry resistance to other Abx.

Tn554 –> encodes erythromycin.spectinomycin
pUB110 –> encodes karamycin/neomycin

Question 16

Describe how the TEM: Beta-lactamase arose

Answer 16

Ampicillin was first used to treat E. Coli in 1961 but resistance was observed just 4 years later
This was due to TEM Beta-lactamase on a plasmid !!
TEM is responsible for 95% of ampicillin-resistance in E. Coli in 1995

Question 17

What antibitoic is E. Coli fundamentally resistant to

Answer 17

Pencillins as penicillin can not enter.

Question 18

What transposon is TEM generally carried on

Answer 18

Tn1

Question 19

Name an alternative beta-lactamase discovered other than TEM

Answer 19

SHV –> was first observed in K. Pneumoniae but wasn’t mobile or at high enoguh levels to convey resistance

SHV then seen in E. Coli, had been mobilised and seems to have activated it to incrased its expression

Question 20

Describe the transposons involved in SHV

Answer 20

SHV is part of a composite transposon
The Taiwanese transposon is common –> in this transposon the insertion sequence is right next to the SHV gene and drives expression of the gene making resistance more likely

Question 21

When were cephalosporins introduced into the UK

Answer 21

In the 1980s –> and killed E. Coli and K. Pneumoniae even TEM and SHV

Question 22

What did the use of cephalosporins fuel the rise of

Answer 22

Caused the explsion of previously rare pathogens that had intrinsic, inducible ESBLs and AmpC e.g. Beta lactamases

After just a few years EmpC hyperproductive was seen in E. COli

Question 23

Describe the mutations that arose in TEM and SHV to convey

Answer 23

Mutations at 238+240 i.e. Gly238–>Ser in SHV-2 and TEM –> this mutation gives the beta-lactamase a bigger mouth so that it can break down larger cephalosporins and are known as ESBLs

Question 24

What are the the 2 steps in PBP crosslinking

Answer 24

1) Transglycosylase reaction –> forms glycosylic bond between GlcNac and MurNAc and adjacent monomers to form the peptidoglycan chain
2) Transpeptidation reaction –> catalyses the crosslink between the amino group and the D-Ala residue on adjacent stem peptides

Question 25

What are penicillins

Answer 25

Structural analgoes of the d-ala-d-ala residue that is the end portion fo the stem peptides

Question 26

Describe the NORMAL action of the Transpeptidase action of PBPs

Answer 26

1) The TPase has an invariant serine residue in the active site with a terminal OH –> the terminal Serine OH attacks the carbonyl-carbon of the peptide bond between the 2 terminal d-ala-d-ala residues. This removed the terminal d-ala and forms an acyl-O-TPase intermediate.

2) A second stem peptide then comes in and attacks the carbonyl-carbon bond bewteen the peptide and the acyl bond
This regernates the TPase and the crosslinked peptidoglycan strand is fomred

Question 27

How do penicillins work

Answer 27

Structural analogues of the terminal d-ala-d-ala
The serine-OH of the PBP attacks the penicillin instead
This forms a penicillinoyl-O-TPase enzyme –> this eeffectively traps PBP. Only broken down by slow hydrolysis

This prevents crosslinking –> hence cells can die due to osmotic lysis

Question 28

what are the four classes of beta lactams

Answer 28

Penicillins
Cephalosporins
Carbapenems
Monobactams

Question 29

Describe the PBPs in S. Aureus

Answer 29

Has 3 HMW and 1 LMW

Question 30

Describe the mchanosm of S. Aureus resistance in MRSA

Answer 30

Acquires a MecA gene –> this encodes a single PBP2’ which is ab;e to take the role of the 3 HMW maitining the viability of the organism

Question 31

Where is PBP2’ thought to originate from

Answer 31

S. Sciuvi and it is carried on the SCCmec elements

Question 32

Describe the normal formatio of the PBP with penicillin

Answer 32

PBP+penicillin –> Michaelis Complex (non-covalent interactions –> Acyl PBP due to covalent bonds

The rate of this reaction is k2/kd

Question 33

What is the rate of reaction of forming acyl-PBP i n PBP2;

Answer 33

It is 1000x less than the normal value of PBPs –> this is because in PBP2’ a substantion confomrational change in its active site (particualrly moving the Beta3 strand) is needed in order for the beta lactam to bind

Question 34

what type of enzymes of PBPs

Answer 34

D-d-transpeptidases

Question 35

What do beta-lactamses catalyse

Answer 35

They catalyse the hydrolysis of beta-lactams forms pencillinoate with no antimicrobial activity

Question 36

what is the common motif of PBP and serine beta-lactamses

Answer 36

Also share a S-X-X-K conserved motif. In beta-lactamses 2 additional conserved sequences known as elements 2 + 3 then follow and these distinguish different classes of serine beta-lactamases

Question 37

Describe the hydrolysis of penicillins by beta lactamases

Answer 37

1) A general base makes the Ser-OH group a stronger nucleophile –> this attacks the Carbonyl-carbon bond of the Beta-lactam ring –> forms a short lived tetrahedral transition state before forming an acylenzyme intermediate
2) A general base also makes water a stronger nucleophile. This then attacks the carbon attached to the serine residue –> again a tetrahedral transition state is formed during this deacylation reaction and the hydrolysed penicillinoate is produced with no antimicrobial activity

Question 38

What is the general base for beta lactamases class A

Answer 38

Glu-166 –> activates water molecules

Question 39

What is the general base for beta lactamases class C

Answer 39

Tyr 150 for acylation. Beta lactams bind and enhance the ability of Tyr150 to deprotonate the serine

Question 40

What is the general base for beta lactamases class D

Answer 40

Carboxylated group on lysine 70 is the general base for both acetylation and deaceytlation

Question 41

How do Class B Beta lactamases work

Answer 41

Have 2 zn ions in the active site
Zn1 - interacts non-covalently with the beta lactam ring carboxy carbon
Zn2 –> interacts with the carbozylate ring attached to the beta lactam ing

This orientates the molecule so that a metal bound water molecule is positioned to attack the amide bound in the beta lactam ring

No covalent intermediate is formed

Question 42

What are the 3 ways beta-lactamases have been named

Answer 42

1) Patient names e.g. TEM
2) Biochemical properies e.g. SHV
Location of discovery: NDM