title: An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates.
abstract: BACKGROUND
  The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium.
  
  
  METHODS
  We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells.
  
  
  RESULTS
  Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates.
  
  
  CONCLUSIONS
  The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.
authors.affiliation: Department of Internal Medicine (Infectious Diseases), Yale University School of Medicine, New Haven, CT, USA. lily1688@hotmail.com
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Michel
authors.initials: M
authors.lastname: Ledizet
authors.forename: Kalipada
authors.initials: K
authors.lastname: Kar
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Koski
authors.forename: Barbara I
authors.initials: BI
authors.lastname: Kazmierczak
mesh_terms: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000276:immunology / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D010641:Phenotype; D052899:Pore Forming Cytotoxic Proteins; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D014774:Virulence
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D052899:Pore Forming Cytotoxic Proteins; C118546:antigen V, Pseudomonas; C111749:pseudomonas exoprotein A protein, Pseudomonas aeruginosa