A pilot study on the effect of the exoU gene on biofilm formation by a strain of Pseudomonas aeruginosa isolated from microbial keratitis.

PURPOSE: To investigate the role of the exoU gene in biofilm formation by comparing wild-type and an exoU knockout mutant of Pseudomonas aeruginosa isolated from microbial keratitis (MK).

METHODS

Biofilm formation by nine exoU-positive P. aeruginosa strains from MK was assessed using the crystal violet assay. The strain exhibiting the highest biofilm production was selected for exoU deletion via homologous recombination using the recombineering plasmid pCasPA. Successful deletion was confirmed using exoU-up-F and exoU-down-R primers and exoU gene specific primers. Cytotoxicity of the wild-type and mutant strains was compared in human corneal epithelial cells using MTT assay. Changes in biofilm between wild-type and mutant strains were assessed using crystal violet assays and confocal laser scanning microscopy (CLSM). Biofilm-associated antibiotic tolerance was assessed by treating pre-formed biofilms of the wild-type and exoU mutant strains with ciprofloxacin and levofloxacin, followed by quantification of surviving bacteria.

RESULTS

PA169 P. aeruginosa produced the highest biofilm amount (OD570/OD660 nm = 2.2 ± 0.15) among the tested strains, and the exoU gene was subsequently deleted from this strain. Biofilm formation was significantly reduced in the mutant (OD570/OD660 nm = 1.4 ± 0.08) compared to its wild-type counterpart (OD570/OD660 nm = 2.2 ± 0.15, p < 0.01). CLSM confirmed a decrease in biofilm thickness in the mutant strain relative to the parent strain (8.33 ± 0.58 μm vs 11.67 ± 0.58 μm, respectively, p < 0.01). Biofilm-associated antibiotic tolerance was also reduced in the exoU mutant, which showed significantly lower survival than the wild-type strain after exposure to ciprofloxacin at 1× MIC (66.87 ± 4.46% vs 88.38 ± 10.09%) and 10× MIC (49.47 ± 6.36% vs 73.74 ± 7.63%), and levofloxacin at 4× MIC (55.29 ± 14.14% vs 79.80 ± 12.25%) and 20× MIC (12.63 ± 1.59% vs 38.13 ± 3.06%) (all p ≤ 0.03). Additionally, the mutant produced lower cytotoxicity than the wild-type (OD570 8.2 ± 0.23 vs 2.1 ± 0.25, p < 0.01).

CONCLUSION

This pilot study suggests that the exoU gene may be associated with biofilm formation and biofilm-associated antibiotic tolerance in the PA169 strain. However, these findings are based on a single clinical strain, and no complementation assay was performed to rule out polar effects of the gene knockout. Further experimental work, including knockout of additional strains, complementation, and strand-specific transcriptomic analysis, is needed to determine whether the observed phenotypic changes are directly caused by deletion of the exoU gene.