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Hunting intracellular bacteria with antibiotics

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. The medical campus of the Université catholique de Louvain.

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Hunting intracellular bacteria with antibiotics

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

  2. The medical campus of the Université catholique de Louvain Brussels The Cellular and Molecular Pharmacology Group But before that, where are you from ? Belgium slides are available on www.facm.ucl.ac.beLectures 1st International and 12th Iranian Congress of Microbiology

  3. Intracellular infection: bacterial strategies Carryn et al.Infect Dis Clin North Am. 2003;17:615-34. 1st International and 12th Iranian Congress of Microbiology

  4. Intracellular infection and recurrence/relapses: the case of S. aureus(as an example) 1st 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 1st International and 12th Iranian Congress of Microbiology

  6. Pharmacochemistry and Biology to your help ! A real need... • Staphyloxanthin and intracellular survival • Activity of availailable antibiotics • What for the future ? 1st International and 12th Iranian Congress of Microbiology

  7. Settting up the model … Seral et al. Antimicrob. Agents Chemother. 2003 47:2283-2292 1st International and 12th Iranian Congress of Microbiology

  8. Adhesion – Invasion(early steps of infection) S. aureus virulence factors fibrinogen IgG vWF ClfA ClfB SpA fibronectin elastin internalization EbpS S. aureus α5β1integrin FnBPA FnBPB collagen Cna Skin infections & necrotizing pneumonia staphyloxanthin Diseases-related toxins PVL SEs Food poisoning Protection vs. host defenses Coa Scald skin syndrome & impetigo KatA ETs TSST-1 Toxic shock syndrome H2O2 Hemolysins Degradatives enzymes prothrombin H2O Hla nucleases tissues necrosis and inflammation Hlb proteases Hlg Eukaryotic cell lipases Hld collagenase hyaluronidase Olivier et al., Role of virulence factors in S. aureus infections. Submitted Colonization - Degradation - Nutrition (late steps of infection) 1st 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 1st International and 12th Iranian Congress of Microbiology

  10. Staphyloxanthinsynthesis 1st 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 1st International and 12th Iranian Congress of Microbiology

  12. The results…. Olivier et al. J. Infect. Dis. (2009) 200:1367-1370 1st 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 1st 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. 1st International and 12th Iranian Congress of Microbiology

  15. -lactams: a pharmacokinetic paradox Accumulation of antibiotics in macrophages • 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 Tulkens PM, Intracellular distribution and activity of antibiotics. Eur J Clin Microbiol Infect Dis. 1991; 10:100-6 1st International and 12th Iranian Congress of Microbiology

  16. Cs Cs Emax Emax intracellular extracellular Lemaire et al., JAC 55:897-904, 2005 -lactams and S. aureus... (MSSA) 1st International and 12th Iranian Congress of Microbiology

  17. Hint: pH in vacuoles is acidic The intracellular pharmacodynamic paradox of -lactams… Lemaire et al., AAC (2007) 51:1627-1632 1st International and 12th Iranian Congress of Microbiology

  18. Back to the future… 1st International and 12th Iranian Congress of Microbiology

  19. Biochemistry comes to your help… 1st International and 12th Iranian Congress of Microbiology

  20. Acid pH favors penicillin binding to PBP2a Lemaire et al., JBC (2008) 283:12769-12776 1st 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 1st International and 12th Iranian Congress of Microbiology

  22. Screening available antibiotics Barcia-Macay et al. Antimicrob. Agents Chemother. 2006, in press 1st International and 12th Iranian Congress of Microbiology

  23. New glycopeptides (oritavancin,telavancin, dalbavancin)… Hemi-synthetic derivatives derived from vancomycin teicoplanin Phase III, Intermune Targenta Awaiting FDA approval, Vicuron  Pfizer Phase III, Theravance  Astellas Van Bambeke, Current Opinion in Pharmacology (2004) 4:471-478 1st 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 1st International and 12th Iranian Congress of Microbiology

  25. lipophilic side chain VAN TLV this causes increase in bacterial membrane permeability Telavancin … polar group Higgins et al., Antimicrob Agents Chemother. (2005) 49:1127-34 1st International and 12th Iranian Congress of Microbiology

  26. Telavancin intracellular accumulation and subcellular disposition Van Bambeke et al., unpublished 1st International and 12th Iranian Congress of Microbiology

  27. 24h CFU  at Cmax: • vanco:  0.5 log • TLV:  2 log Intracellular activity of telavancin vs.vancomycin: MSSA MRSA Barcia-Macay et al., JAC, in the press 1st International and 12th Iranian Congress of Microbiology

  28. 24h CFU  at Cmax: • vanco: static • TLV:  1.2 log Intracellular activity of telavancin vs.vancomycin: VISA VRSA Barcia-Macay et al., JAC, in the press 1st 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 1st International and 12th Iranian Congress of Microbiology

  30. SCVs of S. aureus as a cause of persistent foreign body infection 1st International and 12th Iranian Congress of Microbiology

  31. Intracellular SCVs… 1st International and 12th Iranian Congress of Microbiology

  32. it's hard to exceed 2 log drop… Intracellular SCVs and antibiotics … Nguyen et al., Antimicrobial Agents and Chemotherapy (2009) 53:1434–1442 • Perhaps due to membrane-detabilization effect … • Domenech et al. Biochim Biophys Acta. 2009 1788:1832-1840 • Baudoux et al. ICAAC 2009 [C1-1354] 1st International and 12th Iranian Congress of Microbiology

  33. Will this be successful ? 1st International and 12th Iranian Congress of Microbiology

  34. And now Listeria… Carryn et al.Infect Dis Clin North Am. 2003;17:615-34. 1st International and 12th Iranian Congress of Microbiology

  35. escape from vacuole in cytosol As if you were there … phagocytosis Ouadrhiri et al. Antimicrob Agents Chemother. 1999; 43:1242-51. 1st International and 12th Iranian Congress of Microbiology

  36. Emax ampicillin is MORE active intracellularly A surprise … Lemaire et al. J Antimicrob Chemother. 2005; 55:897-904. 1st 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. 1st International and 12th Iranian Congress of Microbiology

  38. What may change in intracellular Listeria… ABC transporters hexose transporters metabolism of sugars/aminosugars/glycerolipids synthesis of dTDP-rhamnose UDP-glucose glyceraldehyde-3-P pentose phosphate pathway teichoic acid lipoteichoic acid Synthesis of thiamine precursors nucleotide synthesis glutamate fatty acids citrate D-Ala-D-Ala synthesis of peptidoglycan D-alanine pyruvate Oxidative stress response 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. 1st International and 12th Iranian Congress of Microbiology

  39. Anyway, the intracellular infection kept us busy and happy … Cristina Seral Maritza Barcia-Macay Hoang Anh Nguyen Sandrine Lemaire Pierre Baudoux AurélieOlivier Françoise Van Bambeke Sébastien Van de Velde and many others 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… 1st International and 12th Iranian Congress of Microbiology

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