1. Antimicrobials 3: Resistance Dr Fiona Walsh

2. Objectives of lecture Genetics of resistance Mechanisms of resistance Current and future problems

3. What’s new About Resistance? Emergence of Resistance 1928 Discovery of Penicillin 1932Discovery of Sulphonamides 19401st identification of a  -lactamase 194550% resistance to penicillin in Staphylococcus aureus 1950s50% resistance to sulphonamides in E. coli 1970s Resistance began to be taken seriously

4. Worldwide Streptococcus pneumoniae resistance (2003) CountryAzithromycin %Penicillin % Ireland18.9 France54.235.4 USA35.428.7 Hong Kong82.964.3 Australia16.813.6

5. 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 10 10 8 6 4 2

6. Genetics of resistance Intrinsic –Proteins or impenetrable Acquired –Chromosomal mutation and selection –Plasmid-borne resistance –Transposition (Transposons) –Integrons

7. 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

8. 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

9. Bacterial cell sensitive to ampicillin Plasmid Transfer of Antibiotic Resistance genes Bacterial cell resistant to ampicillin chromosome R-plasmid sex pilus Resistant to ampicillin

10. 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

11. How do plasmids acquire new genes? TRANSPOSITION - “jumping genes” chromosome plasmid transposon

12. 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

13. 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

14. Transposition

15. Integrons Non-replicating cluster of genes found on plasmids and transposons of gram- negative bacteria

16. 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

17. Mechanisms of resistance Impermeability Efflux Destruction/Inactivation Modification Alteration of target Additional target Hyperproduction of target

18. Mechanisms of Chromosomal Resistance

19. Mechanisms of Plasmid-encoded resistance

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

21. 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

22. 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

23. 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

24.  -lactamase Action on Amoxycillin

25. β-lactamases

26. 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

27. 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

28. Class C β-lactamases Chromosomally mediated Gram negative rods Induced De-repression: mutation in repressor gene Constitutive production of enzyme

29. Class C β-lactamases Repression/Derepression RPβ-lactamase gene 1 2Induction: Interference with repressor protein 3RPβ-lactamase gene Mutation β-lactamase

30. Class D β-lactamases Oxa Initially oxacillin now wide range of β-lactams Mainly plasmid mediated Origins unknown Diversity of bacterial species

31. 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)

32. 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

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

34. 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

35. 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

36. 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

37. Dihydrofolate Tetrahydrofolate DHFR Chromosome Plasmid Tp By-Pass Mechanism of Plasmid-encoded Trimethoprim Resistance Production of an Additional Dihydrofolate Reductase

38. 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

39. 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

40. 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