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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-lactamb-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 HN 2 N N S S O HO N O O COO - b-lactamase O O H N H COO - β-lactamases b-lactam as e Clas s 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
