Antimicrobial Resistance

Question 1

Where does antimicrobial resistance come from?

Answer 1

-Healthcare
-Community
-Food/Farms
-The world

Question 2

What are the problems of antimicrobial resistance

Answer 2

Some bacteria are resistant to nearly all current antibiotics

Unchecked, by 2050, the global death toll will rise to ~10 million

Question 3

What are the forms of antimicrobial resistance?

Answer 3

Intrinsic resistance- inherited or natural resistance (e.g. Chlamydia do not have peptidoglycan, so are not susceptible to penicillins).

Acquired resistance- developed through alteration of the microbial genome

Question 4

Compare & contrast intrinsic & acquired resistance.

Answer 4

Intrinsic resistance:
– Chromosomic genetic support
– Affect almost all species strains
– Existed before antibiotic use (Enterobacter sp.- amoxicillin)

Acquired resistance:
– Chromosomic, plasmidic or transposon genetic support
– Affects a fraction of strains
– Increased with antibiotic use
(extended spectrum beta-lactamase producing E. coli)

Question 5

How can you get alteration of the microbial genome in acquired resistance?

Answer 5

‘Vertical evolution’ (Darwinian, mutation and natural selection) ‘Horizontal evolution’ (transfer of genes between microbes).

Question 6

How does transfer of genes between microbes happen?

Answer 6

Usually will include either:
Transposons- small, mobile sequences of DNA that can move/be copied to other regions of the genome, either within the gene or to other genes.

Plasmids- circular, ‘mini chromosomes’ that replicate independently of chromosomal DNA.

Question 7

How does resistance evolve in a strain?

Answer 7

Mutations can arise through: spontaneous point mutations; mistakes in DNA repair; transposon insertion

Genes can be duplicated/amplified by: homologous recombination; other forms of recombination event

Genes can be transferred by: lysogenic bacteriophage infection (transduction); pili mediated sex (conjugation); transformation (‘leaky’ bacterial uptake of nuclear material)

Question 8

Is genetic exchange of antimicrobial resistance genes limited to their own species?

Answer 8

No, it can happen between members of different species as well.

Question 9

How do you test for antimicrobial susceptibility?

Answer 9

Measure the MIC- this can be assessed through 2 methods:

1. Liquid media (dilution)
2. Solid media (diffusion) e.g. disc diffusion (Kirby-Bauer) or E-tests

Question 10

What would the setup be if you were measuring MIC in a liquid medium?

Answer 10

• Tubes containing increasing antibiotic concentrations
• Incubation during 18 hr at 37°C
• Tedious

Question 11

Describe Kirby-Bauer disc testing.

Answer 11

Antibiotic-impregnated discs are placed on an agar plate at the interface between test organism and susceptible control organism

Resulting zones of inhibition are compared, use of controls Susceptibility is inferred (from standard tables)

Question 12

What are the 6 main mechanisms of antibiotic resistance?

Answer 12

-Antimicrobial exclusion
-Enzymatic degradation of the drug
-Modification of the drug target
-Target bypass
-Enhanced production of the target
-Efflux mechanisms

Question 13

What is antimicrobial exclusion?

Answer 13

Preventing the antimicrobial from entering the microbe- e.g. the outer membrane of Gram-negative bacteria.

The outer membrane acts as a barrier to extracellular compounds

Question 14

What is the consequence of the outer membrane of Gram-negative bacteria acting as a barrier?

Answer 14

Large (e.g. glycopeptides) and hydrophobic agents (e.g. macrolides, rifamycins) cannot readily penetrate and diffuse across the outer membrane.

Small, hydrophilic solutes can pass through the outer membrane through aqueous channels/pores formed by transmembrane proteins (porins).

Question 15

What is an example of enzymatic degradation being a mechanism of antibiotic resistance?

Answer 15

Resistance in penicillin

Penicillins work by irreversibly binding to the transpeptidase enzymes, stopping the process of peptidoglycan cross linking Binding occurs via the β-lactam ring.

Question 16

What is resistance to penicillin mediated by and how?

Answer 16

Mediated by β-lactamases

β-lactamases hydrolyse the β-lactam ring, preventing penicillin from binding the transpeptidases.

Gram-negative bacteria produce one type of β-lactamase, whilst Gram-positive bacteria produce a wide variety

Question 17

How do the β-lactamases differ in Gram-negative & Gram-positive bacteria?

Answer 17

Gram-negative bacteria:
β-lactamases are cell-bound:
-Found in the periplasmic space, between the outer and inner membrane
– Concentrated at a strategic
location

Gram Positive bacteria:
β-lactamases are inducible:
– Produced in response to the
drug
– Economical for the bacteria

β-lactamases are extracellular
– Long-range action
– Dilution of effectiveness

Question 18

How can you overcome β-lactamases?

Answer 18

Develop resistant β-lactams:
E.g. methicillin- resistance to β-lactamases is created through increased steric hindrance

Inhibit the β-lactamases with another drug:
E.g. clavulanic acid- isolated from Streptomyces spp. & there is no antimicrobial activity when it binds to transpeptidases BUT causes irreversible acylation of β-lactamases

Question 19

What are some other examples of enzymatic degradation?

Answer 19

Chloramphenicol- resistance is usually due to acetylation by chloramphenical acetyl transferases (CAT)

Aminoglycosides- modification by e.g. acetylation, phosphorylation or conjugation with a nucleotide

Question 20

How does modification of drug target induce resistance?

Answer 20

Glycopeptides inhibit peptidoglycan synthesis.

They bind to amino acids in the peptidoglycan, preventing extension (in particular, they bind acyl-D-alanyl-D-alanine) BUT you can have resistance to glycopeptides which will result in antibiotic resistance

Question 21

What are the 2 main methods of glycopeptide resistance?

Answer 21

Intrinsic resistance:
1. Gram-negative bacteria– Due to the outer membrane and drug exclusion

2. Some gram positive bacteria
3. Those bacteria with intrinisic
   resistance to glycopeptides
   have precursors similar to
   Acquired resistance

Acquired resistance:
* e.g. Enterococcus faecium
Resistance to Vancomycin and
teicoplanin is acquired from a
plasmid. Results in the production of 7 new polypeptides. 3 of these polypeptides confer resistance through the formation of a modified peptidoglycan precursor.

Question 22

What is the modified peptidoglycan precursor and how does it result in antibiotic resistance?

Answer 22

D-Ala - D-Ala is replaced by D-Ala – D-Lac (Lac= lactic acid, with the NH2 group replaced by an OH group)

This results in the loss of an H-bond critical for vancomycin & teicoplanin binding

Affinity for the antibiotics reduces by ~100x

Thus peptidoglycan extension is no longer inhibited

Lac residue is lost during cross-linking of peptidoglycan

Question 23

Give more examples of when modification of drug target has led to antibiotic resistance.

Answer 23

1. Trimethoprim resistance occurs when the target dihydrofolate reductase enyzme (DHFR) is modified
2. Aminoglycoside resistance can result from modifications to the structure of the bacterial ribosome
3. Quinolone resistance can be a result of mutations in topoisomerase IV (DNA gyrase) reducing binding affinity or can also be a result of production of novel proteins that bind to & mask the topoisomerase

Question 24

How does drug target bypass work in order to achieve antibiotic resistance?

Answer 24

Antibiotic targets are mostly key steps in biological processes. Some microbes have intrinsic resistance to an antibiotic by not utilising the key step or by having an alternative.

Microbes can acquire resistance by acquiring mechanisms to side-step these steps.

Question 25

Give some examples of drug target bypass.

Answer 25

Methicillin- production of an additional transpeptidase which is not susceptible to penicillins, meaning that you can then carry on cross-linking the peptide chains because you’re no longer dependent on the penicillin-sensitive transpeptidases

Sulfonamides- Resistance has developed as bacteria have developed an alternative route for folic acid biosynthesis that doesn’t use the dihydropteroate synthase enzyme (DHPS)

Question 26

How does enhanced target production lead to antibiotic resistance?

Answer 26

Some microbes develop resistance by increasing production of the drug target to overwhelm the antibiotic e.g. trimethoprim- resistance to trimethoprim can result from an overproduction of the target, dihydrofolate reductase (DHFR).

Question 27

Efflux mechanisms involve efflux pumps. What are they?

Answer 27

Efflux pumps are transporter proteins that pump things out of the cell & are located in the cytoplasmic membrane (they are “active” transporters so require a source of energy to function i.e. ATP or p.d.).

There are many different types of efflux pumps that fall into 5 different groups, based on protein sequence & energy source utilised.

Question 28

What has drug resistance via efflux mechanisms arisen to?

Answer 28

Tetracyclines, macrolides, quinolones, & chloramphenical.

Question 29

How do efflux mechanisms work?

Answer 29

It’s usually a stress-mediated response so first there will be antibiotic stress which will activate the efflux pumps & pump the antibiotic out.

Because there’s a high conc. of antibiotic causing the stress, that causes a stress response meaning you get over expression of the efflux pumps and so more of them are activated, so antibiotic is all boosted out of the cell.

The more efflux pumps there are, the more antibiotic can be removed.

Question 31

How do azoles work?

Answer 31

Inhibits the fungal cytochrome p450 enzyme, 14α- demethylase.

This prevents the conversion of lanosterol to ergosterol- a major constituent of fungal cell membranes.

Question 31

How can resistance to azoles develop?

Answer 31

Several mechanisms

1. Point mutations in the 14α
   demethylase enzyme gene,
   ERG11, this leads to reduced affinity for Azoles– or the overexpression of enzyme.
2. Alterations in other enzymes of
   the ergosterol biosynthetic pathway. Production of various sterols supporting growth. Cross-resistance to other azoles
3. Overexpression of the CDR and
   MDR efflux pump genes. this Reduced drug accumulation in cell

Question 32

What is a resistome?

Answer 32

Collection of antimicrobial resistance genes

Question 33

How can we combat antibiotic resistance?

Answer 33

-Use the correct antibiotic
-Use the correct dose of antibiotic
-Use multiple antibiotics
-Use a secondary, non-antibiotic drug to reduce resistance
-Use of bacteriophages to disrupt resistance genes

Question 34

What are bacteriophages?

Answer 34

Bacteriophages are natural viruses of bacteria which are specific to individual bacterial species and there is no chance of developing resistance

Question 35

By what mechanisms can we use bacteriophages as ‘antibiotics’?

Answer 35

1. direct antibiotic
   -phage attaches to the host cell and injects DNA
   -phage DNA circulates and enters the lytic cycle.
   -new phage DNA and proteins are synthesised and assembled into to virions
   -cell lyses, releasing phage virions
2. remove resistance gene
   -phage attaches to the host cell and injects DNA
   -phage DNA circulates and enters the lysogenic cycle
   - phage DNA integrated within the bacterial chromosome by recombination to become a prophage
   -lysogenic bacteria reproduced normally
   initiate lytic cycle.