Transcript Antimicrobials 2: - Trinity College, Dublin

Antimicrobials 3:
Resistance
Dr Fiona Walsh
Objectives of lecture
• Genetics of resistance
• Mechanisms of resistance
• Current and future problems
What’s new About Resistance?
Emergence of Resistance
• 1928
Discovery of Penicillin
• 1932
Discovery of Sulphonamides
• 1940
1st identification of a b-lactamase
• 1945
50% resistance to penicillin in Staphylococcus aureus
• 1950s 50% resistance to sulphonamides in E. coli
• 1970s Resistance began to be taken seriously
Worldwide Streptococcus
pneumoniae resistance (2003)
Country
Azithromycin % Penicillin %
Ireland
18.9
18.9
France
54.2
35.4
USA
35.4
28.7
Hong Kong
82.9
64.3
Australia
16.8
13.6
Effect of antibiotic concentration on
growth rate
Bacterial Concentration (CFU/ml)
10
10
Sensitive
strain
Resistant variant
8
10
Sensitive strain at sub-MIC
6
10
4
10
2
10
Sensitive strain at >MIC
0
2
4
6
8
Time (hours)
10
12
14
Genetics of resistance
• Intrinsic
– Proteins or impenetrable
• Acquired
– Chromosomal mutation and selection
– Plasmid-borne resistance
– Transposition (Transposons)
– Integrons
Chromosome mutation
Selection of a Resistant Variant
Spontaneous Mutation
- Usually Independent of
Antibiotic Usage
Selection of Mutation
- Often by the Killing of
Antibiotic Sensitive
Bacteria
• Single mutation or series of mutations required for resistance
• Clonal spread of resistance by dissemination of resistant clones
Plasmid-borne resistance
• Plasmid is a mobile replicating DNA circle
not attached to the chromosome
• May carry resistance genes
• Transfer by conjugation
• May move between strains and species
• Faster than chromosomal selection
• Selective pressure not vital
• Most clinically important mechanism
chromosome
R-plasmid
Bacterial cell resistant
to ampicillin
Plasmid Transfer
of Antibiotic
Resistance genes
sex pilus
Bacterial cell
sensitive to
ampicillin
Resistant to
ampicillin
Transposition
• Transposition is migration of a cluster of
genes
• Transposon is the cluster of genes, which
is unable to replicate independently. It
requires a plasmid or chromosome to
replicate
How do plasmids acquire new genes?
TRANSPOSITION - “jumping genes”
transposon
chromosome
plasmid
Conservative Transposition of Class I
Transposons from a Chromsomal Site
Chromosomal donor replicon
Tn
Host cell replication of chromosome
and hence transposon
Plasmid target replicon
Conservative transposition
mediated by transposase to
target replicon
Degradation of
donor replicon
Replicative Transposition of
Class II transposons
res
Donor
replicon
Target
replicon
tnpA tnpR
Fusion mediated by the action of transposase (tnpA gene product)
res
Cointegrate
Replication
by plasmid
res
Resolution of cointegrate by site-specific recombination between the two
res sites mediated by resolvase ( tnpR gene product)
res
Donor
replicon
res
Target
replicon
Transposition
Integrons
• Non-replicating cluster of genes found on
plasmids and transposons of gramnegative bacteria
How do transposons acquire new genes?
INTEGRONS - gene capture and expression systems
“natural” genetic engineering
plasmid
integron
chromosome
integrase
resistance gene cassette
transposon
Resistance
gene
expressed
Mechanisms of resistance
•
•
•
•
•
•
•
Impermeability
Efflux
Destruction/Inactivation
Modification
Alteration of target
Additional target
Hyperproduction of target
Mechanisms of Chromosomal Resistance
Impermeability
Tetracycline
Most antibiotics with Pseudomonas
Efflux
Tetracycline
Fluoroquinolones
Inactivation
b-lactamb-lactamases)
Aminoglycosides (modifying enzymes)
Hyperproduction
Trimethoprim
Altered Target
Trimethoprim
Sulphonamides
Fluoroquinolones
Aminoglycosides
Mechanisms of Plasmid-encoded
resistance
Inactivation
b-lactamases
Aminoglycoside modifying enzymes
Acetyl transferases
Adenyl transferases
Phosphotransferases
Chloramphenicol acetyl transferase
Efflux
Tetracycline
Chloramphenicol
Altered target
Trimethoprim
Sulphonamides
How do bacteria resist the
action of antibiotics?
penicillins
tetracyclines
Permeability
inactivation
sulphonamides
altered target
active
efflux
Permeability
• Cannot penetrate cell wall
• Permeability problems inherent resistance
• Example:
– Pseudomonas aeruginosa few porins
• Rare by mutation as energy cost
• If transport system required then stopping
transport energy is easy mechanism of
resistance
• Example:
– Tetracycline needs active transport
– Cell stops transport tetracycline cannot get into cell
Efflux
• Efflux is pumping antibiotic out of cell
• Active efflux requires energy
• Usually associated with porin as needs
way to pump out antibiotic through cell
wall
• Mainly low level resistance
• Examples:
– Streptococcus pneumoniae fluoroquinolone
resistance efflux
Destruction
•
•
•
•
Only example are β-lactamases
Very efficient and successful
Resistance to β-lactam antibiotics
Hydrolysis of β-lactam ring common to all
• Gram positives: Surrounding cell
• Gram negatives: Periplasmic space
between membranes
b-lactamase Action on Amoxycillin
HN
H
2
H
HO
H N
2
N
N
S
S
O
HO
N
O
O
COO-
b-lactamase
O
O
H
N
H
COO-
β-lactamases
b-lactamase
Class
Class &
Active Site
Substrate
Chromosomal
Plasmid
A
Serine-
B
Metallo-
C
Serine-
D
Serine-
Penicillins
Carbapenems
Cephalosporins
Penicillins
Gram + & Gram -
Gram + & Gram -
Gram -
Gram + & Gram -
Gram -
Gram -
Gram -
Class A β-lactamases
•
•
•
•
•
•
•
Chromosomal and plasmid
At least 75% of all β-lactamases TEM-1
Highly efficient against amoxycillin
β-lactamase inhibitors developed
TEM-1 type mutated
Cephalosporins developed
TEM-1 mutated
Class B and E β-lactamases
• Metallo β-lactamases
• Carbapenems
• Chromosomal induction required for
sufficient production
• Combined with reduced permeability
• Limited number plasmid mediated =
constitutively produced
Class C β-lactamases
• Chromosomally mediated
• Gram negative rods
• Induced
• De-repression: mutation in repressor gene
• Constitutive production of enzyme
Class C β-lactamases
• Repression/Derepression
1
R
2
P
β-lactamase gene
Induction: Interference with repressor protein
3
R
P
β-lactamase gene
Mutation
β-lactamase
Class D β-lactamases
• Oxa
• Initially oxacillin now wide range of β-lactams
• Mainly plasmid mediated
• Origins unknown
• Diversity of bacterial species
Modification
•
Plasmids encode a gene that adds a functional group
to antibiotic
•
An inactive drug no longer inhibits bacteria
A.
B.
C.
Acetyl-transferase – acetyl group
Adenyl-transferase – adenyl group
Phospho-transferase – Phosphate group
•
Chloramphenicol (acetyl) and aminoglycosides (All 3)
Action of Chloramphenicol Acetylase
O2N
CH
NH
CO
CH
CH
OH
CCl 2
OH
Acetyl CoA
O2N
CH
NH
CO
CH
CH
CCl 2
O Ac
OH
Acetyl CoA
O2N
CH
O Ac
NH
CO
CH
CH
O Ac
CCl 2
Aminoglycoside Modifying Enzymes
Adenylase Acetylase
CH2 NH2
HO
O
Acetylase
HO
OH
Phosphorylase
NH2
O
HO
NH2
O
CH2 OH
Phosphorylase
Adenylase
O
OH
HO
NH2
Modification
• Produced in cytoplasm act at entrance site
of antibiotic
• Only small portion of antibiotic is modified,
suggests resistance occurs by antibiotic
blocking path for more antibiotic to enter.
• Moderately high levels of resistance
Target Alteration
• Most common mechanism of chromosomal mutation
Aminoglycosides
• Target on 30S ribosomal subunit alters
• No antibiotic binding
Quinolones
• Target DNA topoisomerases mutate
• Prevents quinolones binding
Macrolides
• Plasmid mediated addition of methyl group to target in ribosome
• Chromosome mediated alteration of binding site in ribosome
• Prevent macrolide binding
Additional target
• Usually plasmid mediated
• Antibiotic binds to target
• Plasmid produces additional target – less
susceptible to antibiotic
• Only work if quantity of product required is low
By-Pass Mechanism of Plasmid-encoded
Trimethoprim Resistance
Production of an Additional Dihydrofolate Reductase
Chromosome
DHFR
Tp
Dihydrofolate
Tetrahydrofolate
Plasmid
DHFR
Tp
Hyperproduction of target
• Chromosomal dihydrofolate reductase hyperproduced by
100-fold
• Bind many trimethoprim molecules
• Still sufficient enzyme to function
• Highly expensive to cell
• Selective disadvantage when antibiotic not present
Current and future problems
• Multi-drug resistance current
– Vancomycin-resistant Staphylococcus aureus
– Vancomycin-resistant Enterococcus faecium &
Enterococcus faecalis
– Carbapenem-resistant Acinetobacter baumannii
• Multi-drug resistance future
–
–
–
–
Carbapenem-resistant Pseudomonas aeruginosa
Carbapenem-resistant Klebsiella spp
Multi-resistant Mycobacterium tuberculosis
Penicillin-resistant Streptococcus pneumoniae
Key points
• Genetic methods used by bacteria in resistance
spread/development
• Mechanisms used by bacteria to stop antibiotics
working (Resistance)
• Examples
• Think of what we need to do to curb resistance