University of California,San Francisco 1 Department of Anesthesia and Perioperative Care What ’ s...
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Transcript of University of California,San Francisco 1 Department of Anesthesia and Perioperative Care What ’ s...
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