An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates.
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.
Li
Li
L
Department of Internal Medicine (Infectious Diseases), Yale University School of Medicine, New Haven, CT, USA. lily1688@hotmail.com
Ledizet
Michel
M
Kar
Kalipada
K
Koski
Raymond A
RA
Kazmierczak
Barbara I
BI
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
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