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Rapid and sensitive method for evaluating Pseudomonas aeruginosa virulence factors during corneal infections in mice
M J Preston1, S M Fleiszig, T S Zaidi
1Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
A murine corneal scratch model has been used extensively to study various aspects of the pathogenesis of Pseudomonas aeruginosa, a common etiologic agent of corneal infections. This model uses mild inhalation anesthetics which keep the animals immobile for a relatively short time and promote the interaction between the infecting organisms and the corneal wound. Under these circumstances, only a small number of P. aeruginosa isolates delivered at inocula of > 10(7) CFU are infectious. We determined that this model is useful for studying other P. aeruginosa strains given at lower doses if injectable anesthetics are administered prior to infection to keep the animals immobile for 15 to 30 min. Under these conditions, eight clinical isolates of P. aeruginosa tested at doses of 10(8) CFU per eye induced corneal perforation and/or phthisis in C3H/HeN mice. The 50% infective doses of several strains were between 3 x 10(2) and 1 x 10(5) CFU per mouse eye. When this modified anesthetic procedure was used to evaluate the roles of different P. aeruginosa virulence factors in eye infections, pathology was not observed when eyes were inoculated with 10(8) CFU of strains deficient in production of a complete lipopolysaccharide or the RpoN sigma factor. A strain with a point mutation in the fur gene, involved in production of iron-regulated factors, showed decreased virulence, while a mutant deficient in both hemolytic and nonhemolytic phospholipase C was fully virulent. By modifying the anesthesia procedure, the corneal scratch model allows rapid evaluations of the roles of P. aeruginosa virulence factors in corneal infections.
Insights
Modifying anesthesia in a murine corneal scratch model enhances Pseudomonas aeruginosa infection studies. This improved model allows for lower inocula, revealing virulence factors crucial for corneal infections and perforation.
Area of Science:
- Ophthalmology
- Microbiology
- Infectious Diseases
Background:
- The murine corneal scratch model is widely used for studying Pseudomonas aeruginosa pathogenesis in corneal infections.
- Current anesthetic methods limit the model's sensitivity, requiring high bacterial inocula (>10^7 CFU) for infection.
- This limitation hinders the study of less virulent strains and specific virulence factors at lower doses.
Purpose of the Study:
- To modify the murine corneal scratch model for enhanced sensitivity to Pseudomonas aeruginosa infection.
- To investigate the roles of specific Pseudomonas aeruginosa virulence factors in corneal infections using a modified model.
- To establish a more efficient method for evaluating bacterial virulence factors in ocular infections.
Main Methods:
- Utilized injectable anesthetics for prolonged animal immobility (15-30 min) during infection.
- Inoculated mouse eyes with lower doses of Pseudomonas aeruginosa clinical isolates (10^8 CFU).
- Evaluated corneal pathology, including perforation and phthisis, and determined 50% infective doses (ID50).
- Assessed the virulence of mutant strains lacking specific factors like lipopolysaccharide, RpoN, fur gene, or phospholipase C.
Main Results:
- The modified anesthesia procedure enabled infection with lower inocula, with ID50 values ranging from 3 x 10^2 to 1 x 10^5 CFU.
- Eight clinical isolates induced significant pathology, including corneal perforation and phthisis, at 10^8 CFU.
- Strains deficient in lipopolysaccharide or RpoN sigma factor did not cause pathology.
- A fur gene mutant showed reduced virulence, while a phospholipase C-deficient mutant remained fully virulent.
Conclusions:
- The modified corneal scratch model with injectable anesthetics significantly enhances sensitivity to Pseudomonas aeruginosa.
- This improved model allows for rapid and accurate evaluation of Pseudomonas aeruginosa virulence factors in corneal infections.
- Key virulence factors, including lipopolysaccharide and RpoN, are essential for P. aeruginosa-induced corneal pathology.