University of California,San Francisco

1Department of Anesthesia and Perioperative Care 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?

University of California,San Francisco

2Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

3Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

Gross autopsy: Necrotic lung

University of California,San Francisco

4Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

5Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

6Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

7Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

8Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

9Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

10Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

11Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

12Department of Anesthesia and Perioperative Care 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?

University of California,San Francisco

13Department of Anesthesia and Perioperative Care 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--distin

guish 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

University of California,San Francisco

14Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

15Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

16Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

17Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

18Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

19Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

20Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

21Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

22Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

University of California,San Francisco

23Department of Anesthesia and Perioperative Care 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.**

University of California,San Francisco

24Department of Anesthesia and Perioperative Care 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.

University of California,San Francisco

25Department of Anesthesia and Perioperative Care 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.

University of California,San Francisco

26Department of Anesthesia and Perioperative Care 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

University of California,San Francisco

27Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

Students cont:

• Mehdi Meghood• James Kang• Robert Su• Lauren Rattray• Denise Grimaldo• Thong Nguyen• Dustin Mark

University of California,San Francisco

28Department of Anesthesia and Perioperative Care Department of Anesthesia and Perioperative Care

Final Students:

• David Doroquez

• Krishna Surti

• Mueen Ghani

• Dai Pho**

• Arlene Kavanagh