Rapid and sensitive method for evaluating Pseudomonas aeruginosa virulence factors during corneal infections in mice

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.