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 -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 Effect of antibiotic concentration on growth rategrowth rate

0 2 4 6 8 10 12 14

Time (Time (hours))

Bac

teria

l Con

cent

ratio

n (C

FU

/ml)

Resistant variant

Sensitive strain at sub-MIC

Sensitive strain at >MIC

Sensitive strain

10

10

10

10

10

1010

88

66

44

22

Genetics of resistance

• Intrinsic– Proteins or impenetrable

• Acquired– Chromosomal mutation and selection– Plasmid-borne resistance– Transposition (Transposons)– Integrons

Chromosome mutationSelection of a Resistant Variant

• Single mutation or series of mutations required for resistance• Clonal spread of resistance by dissemination of resistant clones

Spontaneous Mutation - Usually Independent of Antibiotic Usage

Selection of Mutation- Often by the Killing of Antibiotic Sensitive Bacteria

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

Bacterial cell sensitive to ampicillin

Plasmid Transfer of Antibiotic

Resistance genes

Bacterial cell resistant to ampicillin

chromosome

R-plasmid

sex pilus

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”

chromosome plasmid

transposon

Conservative Transposition of Class I Transposons from a Chromsomal Site

Chromosomal donor replicon

Plasmid target replicon

Host cell replication of chromosome and hence transposon

Conservative transposition mediated by transposase to target replicon

Degradation of donor replicon

Tn

Donor replicon

Fusion mediated by the action of transposase (tnpA gene product)

Cointegrate

Resolution of cointegrate by site-specific recombination between the two res sites mediated by resolvase ( tnpR gene product)

Donor replicon

res res

res

res

Target replicon

Target replicon

res

tnpA tnpR

Replicative Transposition of Class II transposons

Replication by plasmid

Transposition

Integrons

• Non-replicating cluster of genes found on plasmids and transposons of gram-negative bacteria

How do transposons acquire new genes? INTEGRONS - gene capture and expression systems

“natural” genetic engineering

chromosome

plasmid

integron

resistance gene cassette

integrase Resistance gene expressed

transposon

Mechanisms of resistance

• Impermeability

• Efflux

• Destruction/Inactivation

• Modification

• Alteration of target

• Additional target

• Hyperproduction of target

Mechanisms of Chromosomal Resistance

Impermeability TetracyclineMost antibiotics with Pseudomonas

Efflux TetracyclineFluoroquinolones

Inactivation -lactam-lactamases)Aminoglycosides (modifying enzymes)

Hyperproduction Trimethoprim

Altered Target TrimethoprimSulphonamidesFluoroquinolonesAminoglycosides

Mechanisms of Plasmid-encoded resistance

Inactivation -lactamasesAminoglycoside modifying enzymes

Acetyl transferasesAdenyl transferasesPhosphotransferases

Chloramphenicol acetyl transferase

Efflux TetracyclineChloramphenicol

Altered target TrimethoprimSulphonamides

How do bacteria resist the action of antibiotics?

tetracyclines

active efflux

penicillins

inactivation

altered target

sulphonamides

Permeability

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

-lactamase Action on Amoxycillin

HO

H NH

N

SN

OO

COO -

2

-lactamase

N

S

COO -H

β-lactamases

-lactamase Class

ASerine-

Penicillins

Gram + & Gram -

BMetallo-

Carbapenems

Gram + & Gram -

CSerine-

Cephalosporins

Gram -

DSerine-

Penicillins

Gram + & Gram - Gram - Gram - Gram -

Class & Active Site

Substrate

Chromosomal

Plasmid

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

R P β-lactamase gene1

2 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. Acetyl-transferase – acetyl groupB. Adenyl-transferase – adenyl groupC. Phospho-transferase – Phosphate group

• Chloramphenicol (acetyl) and aminoglycosides (All 3)

Action of Chloramphenicol Acetylase

O OAcAc

H

N

O

2NO

2

CH CH CH

HO

H CClOC

H

N

O

2NO

2

CH CH CH

AcO

H CClOC

N

2NO

2

CH CH CH

H CClOC

Acetyl CoA

Acetyl CoA

Aminoglycoside Modifying Enzymes

2

OOH

HO

O

HO

HO

HO CH NH 2 O

O OH

CH OH

NH2

NH2

2

NH2

Adenylase Acetylase

Phosphorylase

Acetylase

PhosphorylaseAdenylase

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

Dihydrofolate

Tetrahydrofolate

DHFR DHFR

ChromosomePlasmid

Tp Tp

By-Pass Mechanism of Plasmid-encoded Trimethoprim Resistance

Production of an Additional Dihydrofolate Reductase

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