1. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 1 Hunting intracellular bacteria with antibiotics Paul M. Tulkens, MD, PhD Cellular and Molecular Pharmacology Unit & Centre for Clinical Pharmacy Louvain Drug Research Institute Catholic University of Louvain, Brussels, Belgium

2. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 2 But before that, where are you from ? slides are available on www.facm.ucl.ac.be  Lectures Belgium Brussels The medical campus of the Université catholique de Louvain The Cellular and Molecular Pharmacology Group

3. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 3 Intracellular infection: bacterial strategies Carryn et al. Infect Dis Clin North Am. 2003;17:615-34.

4. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 4 Intracellular infection and recurrence/relapses: the case of S. aureus (as an example)

5. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 5 Intracellular infection and recurrence/relapses: the case of S. aureus (as an example) Phagocytic and non phagocytic cells in mastitis Brouillette et al., Microb. Pathog. (2003) 35:159-68

6. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 6 A real need... Pharmacochemistry and Biology to your help ! Staphyloxanthin and intracellular survival Activity of availailable antibiotics What for the future ?

7. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 7 Settting up the model … Seral et al. Antimicrob. Agents Chemother. 2003 47:2283-2292

8. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 8 S. aureus virulence factors EbpS ClfA ClfB FnBPA FnBPB SpA IgG vWF elastin fibrinogen fibronectin Cna collagen staphyloxanthin Diseases-related toxins Hemolysins Hla Hlb Hlg tissues necrosis and inflammation Hld Coa Degradatives enzymes S. aureus Eukaryotic cell internalization α 5 β 1 integrin nucleases proteases lipases collagenase hyaluronidase prothrombin KatA H2O2H2O2 H2OH2O Adhesion – Invasion (early steps of infection) Protection vs. host defenses Colonization - Degradation - Nutrition (late steps of infection) PVL SEs ETs TSST-1 Toxic shock syndrome Food poisoning Skin infections & necrotizing pneumonia Scald skin syndrome & impetigo Olivier et al., Role of virulence factors in S. aureus infections. Submitted

9. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 9 S. aureus virulence factors A.C. Olivier. Intracellular fate of Staphylococcus aureus in human phagocytic and non phagocytic cells and role of virulence factors. PhD Thesis, 2008

10. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 10 Staphyloxanthin synthesis

11. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 11 Our material…. 2 isogenic strains rsbu - and rsbu + inhibitors of staphyloxanthin synthesis * Olivier et al. J. Infect. Dis. (2009) 200:1367-1370 * made by E. Oldfield, Champaign, IL

12. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 12 The results…. Olivier et al. J. Infect. Dis. (2009) 200:1367-1370

13. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 13 And with other strains…. the more staphyloxanthin is produced, the more successful is the strain for intracellular growth … blocking staphyloxanthin synthesis makes all strain equally ill-successful… Olivier et al. J. Infect. Dis. (2009) 200:1367-1370

14. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 14 Antibiotics and intracellular infection… Carryn et al. Infect Dis Clin North Am. 2003 Sep;17(3):615-34.

15. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 15  -lactams: a pharmacokinetic paradox beta-lactams:  1x aminoglycosides: <1 to 2 x ansamycins: 2-3 x tetracyclines: 2-4 x fluoroquinolones: 10-20 x macrolides: 4 to > 100 x Accumulation of antibiotics in macrophages Tulkens PM, Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis. 1991; 10:100-6

16. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 16  -lactams and S. aureus... (MSSA) Lemaire et al., JAC 55:897-904, 2005 extracellular intracellular E max CsCs CsCs

17. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 17 The intracellular pharmacodynamic paradox of  -lactams… Hint: pH in vacuoles is acidic Lemaire et al., AAC (2007) 51:1627-1632

18. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 18 Back to the future…

19. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 19 Biochemistry comes to your help…

20. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 20 Acid pH favors penicillin binding to PBP2a Lemaire et al., JBC (2008) 283:12769-12776

21. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 21 Acid pH favors oxacillin-induced conformational change in PBP2a Lemaire et al., JBC (2008) 283:12769-12776

22. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 22 Screening available antibiotics Barcia-Macay et al. Antimicrob. Agents Chemother. 2006, in press

23. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 23 New glycopeptides (oritavancin,telavancin, dalbavancin)… Phase III, Theravance  Astellas Awaiting FDA approval, Vicuron  Pfizer Van Bambeke, Current Opinion in Pharmacology (2004) 4:471-478 Hemi-synthetic derivatives derived from vancomycin teicoplanin Phase III, Intermune  Targenta

24. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 24 Telavancin (and oritavancin) new modes of action … Possibility of dimerization  potential increase of intrinsic activity against D-Ala-D-Ala displaying organisms (MSSA, MRSA, VISA) Membrane destabilization effects…  strong concentration-dependent bactericidal effect (all strains...) * Beauregard et al., AAC 1995; 39:781-85

25. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 25 Telavancin … lipophilic side chain polar group VAN TLV this causes increase in bacterial membrane permeability Higgins et al., Antimicrob Agents Chemother. (2005) 49:1127-34

26. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 26 Telavancin intracellular accumulation and subcellular disposition Van Bambeke et al., unpublished

27. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 27 Intracellular activity of telavancin vs. vancomycin:  MSSA  MRSA Barcia-Macay et al., JAC, in the press 24h CFU  at C max : vanco:  0.5 log TLV:  2 log

28. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 28 Barcia-Macay et al., JAC, in the press 24h CFU  at C max : vanco: static TLV:  1.2 log Intracellular activity of telavancin vs. vancomycin:  VISA  VRSA

29. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 29 Telavancin intracellular activity in comparison with other drugs… Lemaire et al., ISSSI 2006 – Sept. 3-6, 2006

30. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 30 SCVs of S. aureus as a cause of persistent foreign body infection

31. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 31 Intracellular SCVs…

32. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 32 Intracellular SCVs and antibiotics … it's hard to exceed 2 log drop… Perhaps due to membrane-detabilization effect … Domenech et al. Biochim Biophys Acta. 2009 1788:1832-1840 Baudoux et al. ICAAC 2009 [C1-1354] Nguyen et al., Antimicrobial Agents and Chemotherapy (2009) 53:1434–1442

33. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 33 Will this be successful ?

34. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 34 And now Listeria… Carryn et al. Infect Dis Clin North Am. 2003;17:615-34.

35. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 35 As if you were there … phagocytosis escape from vacuole in cytosol Ouadrhiri et al. Antimicrob Agents Chemother. 1999; 43:1242-51.

36. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 36 A surprise … ampicillin is MORE active intracellularly Lemaire et al. J Antimicrob Chemother. 2005; 55:897-904. E max

37. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 37 A potential explanation … through proteomics Van de Velde et al. Isolation and 2D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9:5484-5496.

38. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 38 What may change in intracellular Listeria… pyruvate citratefatty acids glutamate Synthesis of thiamine precursors synthesis ofdTDP-rhamnose UDP-glucose hexose transportersABC transporters teichoic acid lipoteichoic acid Oxidative stress response nucleotide synthesis synthesis of peptidoglycan metabolism of sugars/aminosugars/glycerolipids pentose phosphate pathway glyceraldehyde-3-P D-alanine D-Ala-D-Ala Van de Velde et al. Isolation and 2D-DIGE Proteomic Analysis of Intracellular Listeria monocytogenes in comparison with its extracellular form. Proteomics (2009) 9:5484-5496.

39. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 39 In a nutshell intracellular bacteria is a reality… antivirulence therapy is a nice project … most available antibiotics are poorly active against intracellular bacteria, … and if they are, they most often are less potent than against extracellular bacteria (be prepared for the worse) we may have good (but rare) surprises…

40. Karmanshah, Iran, 24 May 20111st International and 12th Iranian Congress of Microbiology 40 Anyway, the intracellular infection kept us busy and happy … CristinaSeral Hoang Anh Nguyen Maritza Barcia- Macay Pierre Baudoux SandrineLemaire Aurélie Olivier Sébastien Van de Velde Françoise Van Bambeke In collaboration also with :  Y. Glupczynski, Cliniques universitaires de l'UCL à Mont-Godinne, Yvoir, Belgium  A. Vergison, O. Denis, M. Struelens, Hôpital Erasme, ULB, Brussels, Belgium  P. Appelbaum, Hershey Medical Center, Hershey, PA, USA  and many others… and many others