Abstract
Abstract: :
Purpose: Bacterial growth in a biofilm has been associated with pathogenic infections and increased resistance to antibiotics, but this has not been extensively studied in ocular models of infection. Analysis of a biofilm mutants of Pseudomonas aeruginosa (PA) has revealed that biofilm initiation, development and maintenance are under tight genetic regulation. We now describe the in vitro characterization of a new transposon mutant of PA as well as its phenotype in a murine model of corneal infection. Methods: The PA14 strain of PA was used to construct a Tn5 mutant library which was screened for defects in biofilm formation. These mutants were designated as surface attachment defective or sad mutants. The sad–210 mutant was analysed for its ability to form biofilms in a flow cell and under static conditions. Twitching motility was assessed using a thin Luria–Bertani broth (LB) plate. Swarming motility was studied using a minimal medium plate with 0.5% agar. Swimming motility was measured using an LB plate with 0.3% agar. The site of the transposon insertion was determined by arbitrary PCR, followed by a sequencing reaction. Virulence was assayed in a corneal scratch model of bacterial keratitis using C57/B6 mice. Results: Under static and flowing conditions the sad–210 mutant is defective for biofilm formation. Despite being able to attach to the surface of the flow cell, no microcolonies were observed, indicating a defect in the early stages of biofilm development. The sad–210 mutant demonstrated normal twitching motility. Swimming motility was slightly diminished compared to wild type PA14 and swarming motility was completely absent. The transposon insertion mapped to the PA4953 locus, an open reading frame encoding for a putative second flagellar motor. The polar flagellum was visible by electron microscopy. Wild type PA14 was highly virulent in the mouse bacterial keratitis model. The sad–210 mutant was not virulent in this model. However, other sad mutants blocked in later stages of biofilm formation did produce disease. Conclusions: We have presented data that a mutant strain of PA defective in the early stages of biofilm formation lacks virulence in a murine model of corneal infection. This suggests that in vitro models of biofilm formation may be useful in identifying novel genes involved in the ocular virulence of PA and supports the hypothesis that there is a relationship between biofilm formation and corneal infection.
Keywords: keratitis • bacterial disease • Pseudomonas