Penetration of clinical isolates of Pseudomonas aeruginosa through MDCK epithelial cell monolayers
Y Hirakata1, B B Finlay, D A Simpson
1Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia, Vancouver, Canada V5Z 4H4. hirakata@net.nagasaki-u.ac.jp
Abstract:
Pseudomonas aeruginosa causes both invasive (bacteremic) and chronic noninvasive infections. A simple in vitro system to screen P. aeruginosa clinical isolates for their capacity to penetrate MDCK cell monolayers has been developed. By means of this system, P. aeruginosa clinical isolates, including 32 blood and 45 respiratory isolates, were examined. When monolayers were infected with 3.5x107 cfu of bacteria, significantly more blood (93.7%) than respiratory (54.4%) isolates (P<.001) were detected in the basolateral medium after 3 h. Penetration ability was usually independent of cytotoxicity. Only 8 (4 blood and 4 respiratory) isolates were cytotoxic, possessed exoU, and passed through the monolayer after epithelial cell death, associated with a marked drop in transepithelial electrical resistance. Conversely, noncytotoxic isolates with high penetration ability but without severe epithelial damage were invasive. This system is well suited for screening clinical isolates and their mutants for specific genes conferring the invasiveness phenotype.
Insights
Pseudomonas aeruginosa from blood samples more frequently invaded cell layers than those from respiratory samples. A new in vitro system can screen bacterial invasiveness and identify genes responsible for this Pseudomonas aeruginosa phenotype.
Area of Science:
- Microbiology
- Cell Biology
Background:
- Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for diverse infections, ranging from bloodstream infections to chronic respiratory conditions.
- Understanding the mechanisms of bacterial invasion is crucial for developing effective therapeutic strategies against P. aeruginosa.
Purpose of the Study:
- To develop and validate a novel in vitro system for assessing the invasive potential of Pseudomonas aeruginosa clinical isolates.
- To differentiate between invasive and non-invasive P. aeruginosa strains based on their ability to penetrate epithelial cell monolayers.
Main Methods:
- A simple in vitro model using Madin-Darby Canine Kidney (MDCK) cell monolayers was established to evaluate bacterial penetration.
- Clinical isolates of P. aeruginosa (32 blood, 45 respiratory) were infected onto MDCK monolayers, and bacterial presence in the basolateral medium was quantified after 3 hours.
- Cytotoxicity assays and detection of the exoU gene were performed on select isolates.
Main Results:
- A significantly higher percentage of blood isolates (93.7%) demonstrated penetration compared to respiratory isolates (54.4%) (P<.001).
- Bacterial penetration was generally independent of cytotoxicity, with only 8 isolates exhibiting both cytotoxicity and the exoU gene, leading to epithelial cell death.
- Non-cytotoxic isolates with high penetration ability but minimal epithelial damage were characterized as invasive.
Conclusions:
- The developed in vitro system effectively screens P. aeruginosa clinical isolates for invasiveness.
- This model can be utilized to identify specific genes and mechanisms underlying the invasive phenotype of P. aeruginosa.
- Distinguishing between invasive and non-invasive P. aeruginosa strains has implications for understanding disease pathogenesis and guiding treatment.
