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 1932Discovery of Sulphonamides 19401st identification of a  -lactamase % resistance to penicillin in Staphylococcus aureus 1950s50% resistance to sulphonamides in E. coli 1970s Resistance began to be taken seriously

Worldwide Streptococcus pneumoniae resistance (2003) CountryAzithromycin %Penicillin % Ireland18.9 France USA Hong Kong Australia

Effect of antibiotic concentration on growth rate Time () Time (hours) Bacterial Concentration (CFU/ml) Resistant variant Sensitive strain at sub-MIC Sensitive strain at >MIC Sensitive strain

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 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 Target replicon Target replicon res tnpAtnpR 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

Mechanisms of Plasmid-encoded resistance

How do bacteria resist the action of antibiotics? tetracyclines active efflux penicillins inactivation altered target sulphonamides 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

β-lactamases

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 RPβ-lactamase gene 1 2Induction: Interference with repressor protein 3RPβ-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 group B.Adenyl-transferase – adenyl group C.Phospho-transferase – Phosphate group Chloramphenicol (acetyl) and aminoglycosides (All 3)

Action of Chloramphenicol Acetylase OO Ac H N O 2 NO 2 CHCHCH HO HCC l O C H N O 2 NO 2 CHCHCH O HCC l O C N 2 NO 2 CHCHCH HCC l O C Acetyl CoA

Aminoglycoside Modifying Enzymes 2 O OH HO O CH NH 2 O O OH CH OH NH AdenylaseAcetylase Phosphorylase Acetylase Phosphorylase Adenylase

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 Chromosome Plasmid 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