An innovative approach to biofilm-associated infections: CRISPR/Cas9-Mediated Genetic Intervention

Methicillin Resistant Staphylococcus aureus is the major cause of biofilm-associated infections. S.aureus biofilms can be formed through surface proteins (bap) as well as the ica operon. We used pCasSA to target ica A, ica D and bap genes for the first time. Suppression of these genes expression was confirmed by qPCR. Crystal violet assay was performed to quantify the biofilm formation. Mutations in the related genes were shown by Sanger sequence analysis. Antibiotic susceptibility testing was used to assess the effect of suppression of biofilm-associated genes on methicillin susceptibility. Compared to the Wild-type strain, icaA, icaD, bap genes decreased by 70%, 60%, 40%, respectively. Biofilm formation was reduced 6-fold in Knock-out(KO)- ica A strain, 5.6-fold in KO- ica D and 3-fold in KO- bap . KO- ica A, KO- ica D and KO- bap strains exhibited a 64, 16, 4-fold decrease in oxacillin MIC, respectively. Cefoxitin zone increased approximately 2.5-fold in the KO strains. We conclude that the CRISPR/Cas system may be an alternative strategy to inhibit bacterial biofilm.