1. University of California,San Francisco 1 Department of Anesthesia and Perioperative Care What ’ s all this got to do with patients? What evidence is there that the type III secretion system and toxins are involved in disease? What evidence is there regarding the difference between colonizing strains and strains that cause infections?

2. University of California,San Francisco 2 Department of Anesthesia and Perioperative Care

3. University of California,San Francisco 3 Department of Anesthesia and Perioperative Care Gross autopsy: Necrotic lung

4. University of California,San Francisco 4 Department of Anesthesia and Perioperative Care Analysis of P.aeruginosa strains from critically ill patients P.aeruginosa strains from patients produced either ExoU or ExoS, but not both All of the deaths in ICU patients infected with P.aeruginosa were associated with a strain secreting type III toxins; strains secreting type III toxins were also associated with pneumonia, bacteremia and sepsis J Infect Disease 183:1767, 2001

5. University of California,San Francisco 5 Department of Anesthesia and Perioperative Care Prevalence of type III genes in P.aeruginosa strains Evaluated 100 strains from urine, blood, wound and lungs All isolates have genes for type III apparatus but toxin genes vary among isolates 72% isolates have exoS and 28% have exoU genes--no strains had both CF strains did not have exoU gene Microbiology 2001;147:2659

6. University of California,San Francisco 6 Department of Anesthesia and Perioperative Care Virulence Factors in VAP 35 VAP isolates; performed pulse-field gel electrophoresis (non-clonal) 19/35 died (54%) 9/35 recovered from VAP; 6/35 relapsed 27/35 (77%) + for type III secretion 10/35 (29%) + for ExoU--90% severe disease vs 38%of patients with non-type III isolates Isolates either had exoS gene or exoU gene Crit Care Med 2002;30:521

7. University of California,San Francisco 7 Department of Anesthesia and Perioperative Care P.aeruginosa strains from blood 92 unique strains divided into 4 groups Profiles of TTSS protein secretion on SDS- PAGE and kinetics of cytotoxicity #1-28% isolates--rapidly (1h) cytotoxic (ExoU and ExoT) #2-52% slower cytotoxic (ExoS & ExoT) #3-15% some cell death 3~4h ; no TTSS #4- 4% no cytotoxicity & no TTSS J Infectious Disease 2003;188:512

8. University of California,San Francisco 8 Department of Anesthesia and Perioperative Care O serotypes-Utilized to characterize P. aeruginosa LPS O antigen used to classify strains; 20 different serotypes based on B-band of LPS The presence or absence of exoU correlates with O serotype 1, 10, 11 The presence of O serotypes 3,4,6,12,16 had exoS gene J Infectious Disease 2003;188:512

9. University of California,San Francisco 9 Department of Anesthesia and Perioperative Care Single-nucleotide-Polymorphism Analysis of Type III toxins in Strains Causing Disease Not all strains have genes for the type III toxins; PAO1 missing exoU and PA103 missing exoS Evaluated 23 clinical strains: exoU had smallest number of SNPs (14)-- highly conserved exoY had 34 SNPs Targeting of toxins or other bacterial proteins will require analysis of clinical strains --could utilize PCR for diagnostic purposes J Clin Microb 2003:41:3526-3531

10. University of California,San Francisco 10 Department of Anesthesia and Perioperative Care Quorum Sensing in VAP 442 P.aeruginosa isolates colonizing respiratory tract of 13 patients during first 3 days of colonization-9 genetically independent strains Evaluated the ability of the strains to produce QS dependent virulence factors--6/9 strains produced autoinducer associated virulence products J Clin Microb 2004;42: 554-562

11. University of California,San Francisco 11 Department of Anesthesia and Perioperative Care Conclusions re: QS and biofilms in intubated patients Results suggest P.aeruginosa strains colonizing intubated patients had less capacity for biofilm production compared to strains found in CF patients About 20% of isolates were deficient in autoinducer production and 2/3 strains involved in invasive infections were deficient in autoinducer production (and had been proficient prior to invasive disease) J Clin Microb 2004;42: 554-562

12. University of California,San Francisco 12 Department of Anesthesia and Perioperative Care New Research Goals Can we use genetic tools to diagnose and quantify P. aeruginosa in ICU patients? Can we detect which Pseudomonas toxins are being secreted in ICU patients? Can we distinguish P.aeruginosa strains that just colonize vs the strains that cause disease genetically? Can we design new therapeutics to block Pseudomonas-induced lung disease?

13. University of California,San Francisco 13 Department of Anesthesia and Perioperative Care New SCCOR Translational Grant Collect daily endotracheal aspirates from all intubated patients in the ICUs--find P.aeruginosa in patients who are not sick Screen for P.aeruginosa Characterize P.aeruginosa in all patients-- distinguish strains that colonize vs infect patients by genetics and phenotypes Lavage patients to obtain P.aeruginosa in lungs and to evaluate for bacterial genetic and protein expression

14. University of California,San Francisco 14 Department of Anesthesia and Perioperative Care SCCOR interim results So far have screened endotracheal aspirates from 600 intubated patients every day and will add a childrens hospital and neonatal unit 75 patients grew P.aeruginosa 19/75 patients had bronchoalveolar lavages 11/75 patients grew P.aeruginosa for 1 day- and either were extubated or the bacteria did not grow again **Have now obtained permission to routinely screen patients and lavages are now routine care to diagnose VAP

15. University of California,San Francisco 15 Department of Anesthesia and Perioperative Care CONCLUSIONS Pseudomonas aeruginosa has multiple virulence systems and products-- however, it appears that the type III secretion system and the type III toxins are very important in the pathogenesis of acute lung injury and invasive disease Blockade of the type III system is a reasonable therapeutic target

16. University of California,San Francisco 16 Department of Anesthesia and Perioperative Care

17. University of California,San Francisco 17 Department of Anesthesia and Perioperative Care

18. University of California,San Francisco 18 Department of Anesthesia and Perioperative Care

19. University of California,San Francisco 19 Department of Anesthesia and Perioperative Care

20. University of California,San Francisco 20 Department of Anesthesia and Perioperative Care

21. University of California,San Francisco 21 Department of Anesthesia and Perioperative Care

22. University of California,San Francisco 22 Department of Anesthesia and Perioperative Care

23. University of California,San Francisco 23 Department of Anesthesia and Perioperative Care Acknowledgements Ichidai Kudoh, M.D., Ph.D.** Satoru Hashimoto, M.D., Ph.D.** Hiroshi Miyazaki, M.D.** Jean Francois Pittet, M.D.** Christian Jayr, M.D.** Kiryoyasu Kurahashi, M.D.** Junichi Fujimoto, M.D.** Arup Roy-Burman, M.D. Britta Swanson, Ph.D. Noburou Shime, M.D., Ph.D.**

24. University of California,San Francisco 24 Department of Anesthesia and Perioperative Care Acknowledgements continued: Karine Faure, M.D.** Kendra Rumbaugh, Ph.D. Razzu Allmond, M.D.** Matthew Haight, M.D.** Temitayo Ajayi, M.D.

25. University of California,San Francisco 25 Department of Anesthesia and Perioperative Care Acknowledgements continued: Karine Faure, M.D.** Kendra Rumbaugh, Ph.D. Razzu Allmond, M.D.** Matthew Haight, M.D.** Temitayo Ajayi, M.D.

26. University of California,San Francisco 26 Department of Anesthesia and Perioperative Care And Students who helped: Jim Nemechek Lauren Rattray Denise Grimaldo Thong Nguyen Timur Karaca** Dustin Mark Vinh Nguyen** Jennifer Lee Elizabeth Thomas Ticey Long

27. University of California,San Francisco 27 Department of Anesthesia and Perioperative Care Students cont: Mehdi Meghood James Kang Robert Su Lauren Rattray Denise Grimaldo Thong Nguyen Dustin Mark

28. University of California,San Francisco 28 Department of Anesthesia and Perioperative Care Final Students: David Doroquez Krishna Surti Mueen Ghani Dai Pho** Arlene Kavanagh