Antibiotic Resistance

Question 1

Give an overview of antibiotic resistance?

Answer 1

Definition - the ability of bacteria to survive and grow in the presence of antibiotic concentrations that can be safely achieved in patients at site of infection
= an antibiotic is no longer effective for treatment

With the increase of antibacterial chemotherapy we saw a dramatic increase in antibiotics resistance
Antibiotic resistance is inevitable

Question 2

What are types of resistance in general?

Answer 2

Acquired and Intrinsic
Acquired resistance is when a previously-antibiotic susceptible bacterial population becomes resistant

Intrinsic resistance results from inherent features of a particular bacterial population
Mycoplasmae are intrinsically resistant to β-lactams and glycopeptides, because they have no peptidoglycan cell wall
E. coli is intrinsically resistant to vancomycin and rifampicin because they can’t get through the outer membrane

Therefore acquired is problematic for us

Question 3

What are the negative consequences of antibiotic resistance?

Answer 3

Increased mortality
resistant infections often more fatal

Increased morbidity
prolonged illness
increased opportunities for spread of resistant organisms

Increased cost
length of stay in hospital
new, more expensive drugs

Question 4

Why do we need to prevent antibiotic resistance?

Answer 4

Modern medicine relies on antibiotics
Treatment of bacterial infection
Dentistry, surgery, transplants

Massive benefits to individual health and societies
Survival/ longevity
Quality of life
Productivity

Modern economies underpinned by antibiotics

Question 5

How has the evolution of antibiotic resistance come about?

Answer 5

Antibiotic resistance is not a new phenomenon - many natural pathogens therefore this has been happening before our discovery of them
In recent history we have now seen resistance in pathogenic bacteria
= evolution by natural selection

A suseptable pathogen can have a mutation or horizontal transfer of resistance genes resulting in a resistant pathogen -> transmission

Question 6

What is driving antibiotic resistance?

Answer 6

Antibiotic use
Selection pressure - 33K tonnes penicillin manufactured per year
Overuse, misuse and unregulated sales (e.g. Asia)
Antibiotic use in livestock (>10X that for humans)
Large fraction of most antibiotics leave the body in an active form

Question 7

What are the routes bacteria can take to confer antibiotic resistance?

Answer 7

Endogenous - spontaneous mutation to resistance
Point mutation (during replication) often in the gene encoding the drug target 

Exogenous - horizontal acquisition of antibiotic resistance
Gene transfer to another bacterial/pathogenic organism

Question 8

What are the mechanisms of antibiotic resistance?

Answer 8

Altered target site
Decreased uptake
Inactivation/modification
Bypass

Question 9

Describe altered target site as a mechanism of antibiotic resistance?

Answer 9

Mutation/modification of antibacterial drug target

Simplest mutation = point mutation

Question 10

Give some examples of altered target site mutations?

Answer 10

Alteration of RNA polymerase mediates rifampicin resistance in S. Aureus
It inhibits RNA polymerase by binding to the catalytic b subunit - blocking the exit of RNA
Substituting just one amino acid the loss in activity of rifampicin is drastic

Methylation of ribosomal RNA by Cfr methylase of a A2503 on 23S rRNA
This is horizontally-acquired resistance to linezolid in S.aureus
Also has resistance to chloramphenicol, lincosamides, streptogramins and pleuromutilins

Vancomycin resistance in enterococci
It normally binds to D-ala-D-ala (peptidoglycan precursor)
Substituting the terminal D-ala with D-lactate
Results in a single hydrogen bond being lost between the drug and the target which rapidly reduces the binding effect to the target

Question 11

Describe how the mutation effecting vancomycin works?

Answer 11

vanR and vanS are normally functioning in the operon
When vancomycin is detected the resistance machinery is turned on - vanH, vanA, vanX
These produce D-lactate, combine it with the D-ala and prevent any other D-ala binding to D-ala normally

Question 12

What is another altered target site mechanism?

Answer 12

Target overexpression
It normally interacts with the target in a set ratio of 1:1
When the bacterial cell increases the target you need to try to match the antibiotic level
Therefore the cell can out titrate the antibiotic
This is very energy costly for the cell - less fit

This is often experimented in the lab but not a very widely used method of resistance in the ‘wild’

Question 13

Give an example of target overexpression to induce antibiotic resistance?

Answer 13

Vancomycin-intermediate S. aureus

Numerous genetic/ biochemical changes described in VISA strain (Mu50)
Common denominator: significant thickening of cell-wall
Affinity-trapping of vancomycin

Reduced cross-linking increases the presence of D-ala-D-ala that can bind to vancomycin
They act as decoy sites to bind vancomycin before it can act on the deeper areas of the bacteria

Question 14

Describe decreased antibiotic uptake as a mechanism of antibiotic resistance?

Answer 14

Either:
Reduced permeability - can’t penetrate the cell membrane
Seen in gram negative - they can delete specific porins (but not common)
OR
Efflux - transport back out the cell

Question 15

Describe active efflux?

Answer 15

Toxic compounds pumped from the cell by a transporter protein or complex (‘efflux pump’)
There are several different families of efflux pumps
Commonly require proton motive force or ATP to function
They can recognise multiple antibiotic classes, dyes, heavy metals, solvents, detergents
Resistance may arise from up-regulation of an endogenous pump, or from horizontal acquisition of a new pump

Question 16

Give an example of decreased uptake of antibotics?

Answer 16

Multiple Drug Efflux Systems, such as AcrAB/TolC System in E. Coli

This is very important due to a broad substrate profile = large recognition of substrates
Therefore can often undergo upregulation
Efflux transporter connected to adaptor protein linked to OM channel protein
Drug crosses OM and/or CM
Transporter pumps drugs out, bypassing OM

Question 17

Describe enzymatic inactivation or modification as a mechanism of antibiotic resistance?

Answer 17

This is catalysing a chemical change within the antibiotics - which reduces the activity of these compounds
Destruction/modification

Question 18

Give an example of a enzyme that mediates resistance to an antibiotic?

Answer 18

B-lactamase
This mediates resistance to β-lactam antibiotics
>1500 described
Catalyse hydrolysis of the cyclic amide bonds of β-lactam ring
The open ring forms cannot bind to target sites

Serious problem in gram negative bacteria as B-lactams are fundamental in curing this type of bacterial infection

Question 19

Describe b-lactamases?

Answer 19

They are categorised into 4 classes
These categories are established due to their protein sequence

Class A, C and D have serine in the active site
They share an evolutionary history - similar proportions of alpha/beta sheets
Class A were most under concern - as they catalysed 1st, 2nd and 3rd cephalosporins
This drove towards the use of carbapenems (heavy weight antibiotics)

Class B have a metallo enzyme - contains 2 metal ions usually zinc
Responsible for catalysing the hydrolysis of b-lactams

Question 20

Give another example of an enzymatic modification of aminoglycosidases?

Answer 20

Inhibitors of protein synthesis
The groups added interfere between the interaction of the drug and the target

Aminoglycoside adenyltransferase  (ANT) - catalyses ATP-dependent adenylation of hydroxyl group 
Aminoglycoside acetyltransferases (AAC) - catalyses acetyl CoA-dependent acetylation of an amino group 
Aminoglycoside phosphotransferases (APH) - catalyses ATP-dependent phosphorylation of a hydroxyl group

Question 21

Describe target bypass as a mechanism of antibiotic resistance?

Answer 21

Acquisition of alternative target of an enzyme that can substitute for the original drug target - but it is not antibiotic sensitive
This is acquired via horizontal gene transfer from another bacterial/pathogenic organism
When the antibiotic binds, it will inactive the drug target - but the bacteria can still function

Question 22

Give an example of target bypass for antibiotic resistance?

Answer 22

Methicillin resistance in S.aureus
MRSA carries mecA which encodes PBP2a - mainly B-lactam insensitive
PBP2a catalyses sufficient cross-linked peptidoglycan to allow the organism to survive

Question 23

What is significant about these antibiotic resistant mechanisms?

Answer 23

They can work together to achieve the resistance

Question 24

What can we do in the future to improve antibiotic resistance?

Answer 24

Better training for prescribers
Co-ordination of surveillance of resistance in human and animal sectors

Better guidelines for therapy
Cycling of antibiotics to reduce selective pressure
Restriction on use of antibiotics as growth promoters

Improved infection control practices
Development of novel antibacterials to circumvent existing resistance mechanisms - but difficult, lengthy, costly