Physiological basis for biofilm resistance to antimicrobial agents

The physiological basis of Pseudomonas aeruginosa biofilm resistance to monochloramine (MCA) and hydrogen peroxide (H2O 2) was investigated. An experimental system consisting of bacteria attached to the surface of suspended alginate gel beads was developed to definitively eliminate the possibility of ineffective antimicrobial delivery to biofilm cells. After 20 hours or more of biofilm growth, disinfection efficacy of MCA was significantly reduced compared to planktonic cells (p ≤ 0.0018). Biofilms that were 24 hours and 48 hours old were also resistant to H 2O2 compared to planktonic cells (p ≤ 0.019). The ratio of disinfection rate coefficients measured for planktonic cells to that measured for biofilm cell was 1.6 -- 2.6 for MCA and 3.3 -- 3.9 for H 2O2. Susceptibility to the biocides did not significantly correlate with initial biofilm areal cell density (p = 0.84). These results indicate that physiological changes occurred in attached P. aeruginosa that protect them from oxidative stresses.; To address the issue of physiological changes in biofilm cells, the role of two sigma factors, AlgT and RpoS, in mediating resistance to H2O 2 and MCA was investigated. Two knock out mutant strains, SS24 ( rpos-) and PA06852 (algT- ) were compared to the wild type strain, PAO1, in their susceptibility to the biocides. When biofilms were grown on alginate gel beads, all strains were equally resistant to MCA disinfection and biofilm coefficients were significantly lower (p ≤ 0.015) than coefficients obtained from the same cells grown planktonically. However, 24-hour old rpoS- and algT- biofilms were more susceptible to H2O2 disinfection than were biofilms formed by PAO1; biofilm disinfection rate coefficients were statistically the same (p ≤ 0.15) as planktonic disinfection rate coefficients. While 48-hour old algT- biofilm cells became resistant to H2O 2, 48-hour old rpoS- biofilm cells remained highly susceptible.; To confirm the role of RpoS, rpoS was restored to SS24 by constructing a complement strain, SS240. This strain exhibited disinfection profiles similar to PAO1 (p = 0.12). The expression of RpoS in SS240 was confirmed by immunoblot analysis. It was concluded that algT and rpoS are two genes that are significantly involved in the protection of thin biofilm from H2O2.