Antibiotics Part 1

1. Antibiotics Part 1 Dr P Gayo Munthali Consultant Microbiologist UHCW Honorary Associate Clinical Professor University of Warwick

2. Objectives • By the end of this lecture you should be able to: • Explain the mode of action of beta lactams, aminoglycosides, fluoroquinolones, macrolides, tetracyclines and glycopeptides • Mention the major side effects of the antibiotic groups in (1) • Appreciate different types of resistance and in simple terms, explain the mechanisms of resistance to beta lactams • Explain some limitations in the use of antibiotics in (1) • Understand the general spectrum of activity of antibiotics in (1)

3. Antibiotics, Point of Action Folic acid Metabolism Trimethoprim, Sulphonamides Cell membrane Polymixin, bacitracin,colistin Cell wall Synthesis Beta-lactams, Glycopeptides Daptomycin Fosfomycin 50S 30S DNA Replication Quinolones DNA Dependent RNA Pol. Rifampicin Protein Synthesis 30S Tetracyclines,Aminoglycosides 50SChloramphenicol, Clindamycin, Erythromycin, Linezolid,Streptogramin

4. Important mechanisms of resistance to antibiotics

5. ß-Lactams Β-Lactam Ring Thiazolidine Ring

6. Penicillins and Cephalosporins s R-CONH Penicillins 1940- N o COOH R-CONH s R N Cephalosporins 1948- o COOH

7. Carbapenems and Others CHз Carbapenems 1976- R HO S HO N o o COOH N o COOH Clavulanic acid 1976

8. Mobactams R Monobactam 1981- R-CONH N o R

9. Mechanisms of Action • Inhibit bacterial enzymes involved in cell wall synthesis • Penicillin binding proteins (PBPs) essential for peptidoglycan synthesis • Trigger membrane associated autolytic enzymes that destroy cell wall • Inhibit bacterial endopeptidase and glycosidase enzymes which are involved in cell wall growth • Time dependent activity

10. Beta Lactams Against Bacterial Cell Wall Cell wall Osmotic Pressure Cell Membrane Antibiotic against cell wall Osmotic Pressure Cell membrane Rapture

11. Spectrum of Activity • Very wide • Gram positive and negative bacteria • Anaerobes • Spectrum of activity depends on the agent and/or its group • Aztreonam only active against gram negatives

12. Pharmacokinetics • Absorption • PO forms have variable absorption • Food can delay rate and extent of absorption • Distribution • Widely to tissues & fluids • CSF penetration: IV – limited unless inflamed meninges IV 3rd & 4th generation cephalosporins, meropenem, & Aztreonam – penetrate well • Metabolism & Excretion • Primarily renal elimination • Some have a proportion of drug eliminated via the liver • ALL -lactams have short elimination half-lives

13. Adverse Effects Penicillin hypersensitivity – 0.4% to 10 % • Mild: rash • Severe: anaphylaxis & death • There is cross-reactivity among all Penicillins • Penicillins and cephalosporins ~5-15% • Penicillins and carbapenems~1% (may be higher) • Desensitization is possible for mild hypersensitivity • Aztreonam does not display cross-reactivity with Penicillins and can be used in penicillin-allergic patients

14. Resistance to ß-Lactams • Penicillin-Binding Protein (PBP) mediated Resistance • ß-Lactamase • Efflux pumps/loss of porins

15. Penicillin-Binding Protein (PBP) mediated Resistance • PBP over expression • Acquisition of Foreign PBPs genes • Mutation by recombination with foreign DNA • Point mutation

16. PBP over expression • Rare • The more PBPs are expressed, the more an organism becomes resistant • S.aureus increased resistance to methicillin by over expression of PBP4 • E.faecium strains thatover express PBP5 have increased resistance to penicillin. AAC 39:2415-2422, AAC 38:1980-1983, AAC 45: 1480-1486

17. Acquisition of Foreign PBPs • Represented best by Methicillin Resistant S.aureus (MRSA) • S.aureus acquires foreign PBP2a encoded by mecA gene • PBP2a has low affinity for all ß-lactams • PBP2a can perform all the combined functions of all the S. aureus PBPs • Almost all MRSAs express ß-Lactamase Clin. Microbiol. Rev.10:781-791, J.Infect.Dis.162:705-710

18. Result • All PBPs in S.aureus become redundant • MRSA is resistant to all ß-lactams

19. Mutation by Recombination with Foreign DNA • Streptococcus pneumoniae and Neisseria are capable of picking up foreign DNA and integrating it with their own DNA • Form mosaic gene • Pneumococcus picks up resistant genes from alpha haemolytic streps • Reduced affinity to beta lactams • Seen as penicillin resistant Pneumococci

20. MICs Isolate MIC for meningitis BSAC JAC 1992,30(3);279-288

21. Efflux pumps/Loss of Porins • Important type of resistance in Pseudomonas • A combination of ß-Lactamase production and porin loss can lead to complex resistance pattern • Can lead to carbapenem resistance without carbapenemase production

22. Porins and Pumps Porins Overexpressed Efflux pumps Adapted from Journal of Bacteriology, April 2006, p. 2297-2299, Vol. 188, No. 7

23. Resistance due to ß-Lactamases • Mode of action • Classification

24. ß-Lactamase ß -pleated sheet-5 ά-helices AAC 39:2593-2601

25. Bound ß-lactam by ß-Lactamase

26. ß-Lactamases-action R-CONH s CH3 C N CH3 o COOH Enzyme-Ser-OH s R-CONH CH3 o N C CH3 HOH O H COOH Enzyme Ser Annu.Rev.Microbiol.45:37-67

27. Beta Lactam Classification • You do not need to know the classification or detailed information on ß-Lactamases • However you need to appreciate the following concepts; • Simple betalactamases • Extended spectrum betalactamases (ESBL) • Betalactamases against the Carbapenems

28. Simpleß-Lactamases • Many Based on genes called TEM-1 and SHV-1 found on mobile DNA elements • TEM-1 and SHV-1 are simple penicillinases in Enterobacteriaceae • Inactive against cephalosporins • Confer resistance to Penicillins such as Benzylpenicillin and amoxicillin • On mobile elements and therefore transmissible • Staphylococci also produce simple beta lactamases not based on TEM-1 and SHV-1 • Flucloxacillin designed to resist betalactamases in Staphylococcus aureus AAC 33:1131-1136

29. Extended Spectrum ß-lactamases • Based on TEM-1 and SHV-1 • Amino acid mutations in active site progressively increase their activity against cephalosporins • When they hydrolyze extended-spectrum cephalosporins • They are then called ESBLs • Also attack a monobactam Aztreonam -On mobile elements thus transmissible • Carry other resistance genes, Gentamicin, Ciprofloxacin

30. ESBLs • Hydrolyze extended-spectrum cephalosporins with an oxyimino side chain • These include; • Cefotaxime • Ceftazidime • Ceftriaxone • Loose term • Among the beta-lactam, only the Carbapenems are stable against ESBLs • Imipenem, Meropenem, Ertapenem and Doripenem are in clinical use

31. Characteristics of ESBLs • May appear sensitive to some cephalosporins and combinations of piperacillin and tazobactam as well as amoxicillin and clavulanic acid • However, use of these ß-lactam agents will lead to microbiological and clinical failure • Only carbapenems among the ß-lactams can be used successfully

32. AmpC ß-Lactamases • Produced by almost all gram-negative bacteria • Chrosomally encoded versions important inCitrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Morganella morganii, Pseudomonas aeruginosa and Serratia marcescens (notfound in Salmonella and Klebsiella) • AmpCß-Lactamase genes have been found on transferable plasmids

33. Class C ß-Lactamases • All ß- lactams induce AmpC ß-lactamase production • Only carbapenems are resistant to AmpC ß-lactamases • If there is loss of porins as well, this will lead to carbapenem resistance • Other ß- lactams will be hydrolysed

34. Metallo-ß-Lactamases • Require Zinc or other heavy metal for activity • Hydrolyse all ß-Lactams including carbapenems • Most will be associated with resistance to many antibiotic classes • Currently New Delhi Metallo-ß-Lactamase (NDM-1) is a new flavour in the UK • Associated with India • Resistant to almost all antibiotics in use in the UK

35. Aminoglycosides • Highly positively charged compounds, concentration dependent activity • Inhibit bacterial protein synthesis by irreversibly binding to 30S ribosomal unit • Naturally occurring: • Streptomycin • Neomycin • Kanamycin • Tobramycin • Gentamicin • Semisynthetic derivatives: • Amikacin (from Kanamycin) • Netilmicin (from Sisomicin)

36. 30S Ribosomal Unit Blockage by Aminoglycosides • Causes mRNA decoding errors • Block mRNA and transfer RNA translocation • Inhibit ribosome recycling • Ribosome recycling follows the termination of protein synthesis

37. Spectrum of Activity • Gram-Negative Aerobes • Enterobacteriaceae; E. coli, K. pneumoniae, Proteussp. Citrobacter, Enterobactersp. Morganella, Providencia, Serratia • Pseudomonas aeruginosa • Acinetobacter • Gram-Positive Aerobes(Usually in combination with ß-lactams) S. aureus and coagulase-negative staphylococci Viridans streptococci Enterococcussp. (gentamicin)

38. Mechanisms of Resistance • Ribosome changes • Prevents binding • Loss of cell permeability • Expulsion by efflux pumps • Enzyme inactivation by Aminoglycoside modifying enzymes • This is the most important mechanism

39. Pharmacokinetics • All have similar pharmacologic properties • Gastrointestinal absorption: unpredictable but always negligible • Distribution • Hydrophilic: widely distributes into body fluids but very poorly into; • CSF • Vitreous fluid of the eye • Biliary tract • Prostate • Tracheobronchial secretions • Adipose tissue • Elimination • 85-95% eliminated unchanged via kidney • t1/2 dependent on renal function • In normal renal function t1/2 is 2-3 hours

40. Adverse Effects • Nephrotoxicity • Direct proximal tubular damage - reversible if caught early • Risk factors: High troughs, prolonged duration of therapy, underlying renal dysfunction, concomitant nephrotoxins • Ototoxicity • 8th cranial nerve damage – irreversible vestibular and auditory toxicity • Vestibular: dizziness, vertigo, ataxia • Auditory: tinnitus, decreased hearing • Risk factors: as for nephrotoxicity • Neuromuscular paralysis • Can occur after rapid IV infusion especially with; • Myasthenia gravis • Concurrent use of succinylcholine during anaesthesia

41. Macrolides • Erythromycin is the prototype antibiotic for this group • Bacteriostatic- usually • Inhibit bacterial RNA-dependent protein synthesis • Bind reversibly to the 23S ribosomal RNA of the 50S ribosomal subunits • Block translocation reaction of the polypeptide chain elongation

42. Macrolides Lactone Ring 14 14 Erythromycin Telithromycin 14 15 Clarithromycin Azithromycin

43. Mechanisms of Resistance • Altered target sites • Methylation of ribosomes preventing antibiotic binding • Resistance to macrolides , lincosamides (Clindamycin) and streptogramin B • Can be induced by macrolides • Efflux pumps • Resistance to macrolides only • Cross-resistance occurs between all macrolides

44. Spectrum of Activity • Gram-Positive Aerobes: • Activity: Clarithromycin>Erythromycin>Azithromycin • MSSA • S. pneumoniae • Beta haemolytic streptococci and viridans streptococci • Gram-Negative Aerobes: • Activity: Azithromycin>Clarithromycin>Erythromycin • H. influenzae, M. catarrhalis, Neisseria sp. • NO activity against any Enterobacteriaceae • Anaerobes: upper airway anaerobes • Atypical Bacteria • Other Bacteria: Mycobacterium avium complex

45. Pharmacokinetics 1 • Erythromycin ( Oral: absorption 15% - 45%) • Short t1/2 (1.4 hr) • Acid labile • Absorption (Oral) • Erythromycin: variable absorption of 15% - 45% • Clarithromycin: 55% • Azithromycin: 38% • Half Life (T1/2) • Erythromycin 1.4 Hours • Clarithromycin (250mg and 500mg 12hrly) 3-4 & 5-7 hours respectively • Azithromycin 68hours • Improved tolerability • Excellent tissue and intracellular concentrations • Tissue levels can be 10-100 times higher than those in serum • Poor penetration into brain and CSF • Cross the placenta and excreted in breast milk

46. Pharmacokinetics 2 • Metabolism & Elimination • Clarithromycin partially eliminated by the kidney • ALL hepatic elimination

47. Adverse Effects • Gastrointestinal (up to 33 %) (especially Erythromycin) • Nausea • Vomiting • Diarrhoea • Dyspepsia • Thrombophlebitis: IV Erythromycin & Azithromycin • QTc prolongation, ventricular arrhythmias • Other: ototoxicity with high dose erythromycin in renal impairment

48. Fluoroquinolones Quinolone pharmacore

49. Fluoroquinolones • Synthetic antibiotics • Concentration-dependent bactericidal activity • Broad spectrum of activity • Excellent pharmacokinetics • bioavailability, tissue penetration, prolonged half-lives • In common use • Ciprofloxacin • Levofloxacin • Moxifloxacin

50. Mechanism of Action • Inhibit bacterial topoisomerases which is used by bacteria to; • Relax supercoiled DNA before replication • DNA recombination • DNA repair • DNA gyrase – Primary target for gram-negatives • Topoisomerase IV – Primary target for gram-positives