Download From Bedside to Bench and Back again

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?
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
1
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
2
Gross autopsy: Necrotic lung
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
3
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
4
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
5
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
6
P.aeruginosa strains from blood
• 92 unique strains divided into 4 groups
Profiles of TTSS protein secretion on SDS-PAG
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
7
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
8
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
9
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
10
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
11
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?
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
12
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
13
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 dayand 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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
14
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
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
15
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
16
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
17
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
18
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
19
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
20
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
21
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
22
Acknowledgements
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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.**
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
23
Acknowledgements continued:
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Karine Faure, M.D.**
Kendra Rumbaugh, Ph.D.
Razzu Allmond, M.D.**
Matthew Haight, M.D.**
Temitayo Ajayi, M.D.
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
24
Acknowledgements continued:
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Karine Faure, M.D.**
Kendra Rumbaugh, Ph.D.
Razzu Allmond, M.D.**
Matthew Haight, M.D.**
Temitayo Ajayi, M.D.
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
25
And Students who helped:
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Jim Nemechek
Lauren Rattray
Denise Grimaldo
Thong Nguyen
Timur Karaca**
Dustin Mark
Vinh Nguyen**
Jennifer Lee
Elizabeth Thomas
Ticey Long
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
26
Students cont:
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Mehdi Meghood
James Kang
Robert Su
Lauren Rattray
Denise Grimaldo
Thong Nguyen
Dustin Mark
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
27
Final Students:
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David Doroquez
Krishna Surti
Mueen Ghani
Dai Pho**
Arlene Kavanagh
Department
University
of of
Anesthesia
California,San
and Perioperative
Francisco Care
28