What

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Whats 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?

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Gross autopsy Necrotic lung
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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 1831767, 2001

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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
  20011472659

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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
  38of patients with non-type III isolates
• Isolates either had exoS gene or exoU gene
• Crit Care Med
  200230521

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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 34h no TTSS
• 4- 4 no cytotoxicity no TTSS
• J Infectious Disease 2003188512

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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 2003188512

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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 2003413526-3531

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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 200442 554-562

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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 200442
  554-562

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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?

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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

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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

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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

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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.

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Acknowledgements continued

• Karine Faure, M.D.
• Kendra Rumbaugh, Ph.D.
• Razzu Allmond, M.D.
• Matthew Haight, M.D.
• Temitayo Ajayi, M.D.

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Acknowledgements continued

• Karine Faure, M.D.
• Kendra Rumbaugh, Ph.D.
• Razzu Allmond, M.D.
• Matthew Haight, M.D.
• Temitayo Ajayi, M.D.

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And Students who helped

• Jim Nemechek
• Lauren Rattray
• Denise Grimaldo
• Thong Nguyen
• Timur Karaca
• Dustin Mark
• Vinh Nguyen
• Jennifer Lee
• Elizabeth Thomas
• Ticey Long

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Students cont

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

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Final Students

• David Doroquez
• Krishna Surti
• Mueen Ghani
• Dai Pho
• Arlene Kavanagh