(93d) The Pseudomonas aeruginosa Biofilm matrix

A critical element of Pseudomonas aeruginosa pathogenesis is its ability to form biofilms in the lungs of CF patients. Biofilm bacteria produce one or more extracellular polymeric substances (EPS) that act as a scaffold, holding biofilm cells together and to a surface. We discovered that P. aeruginosa has the capacity to encode at least two alternative exopolysaccharides, designated Psl and Pel, which play a critical role in biofilm formation. Psl has been reported to consist of a neutral polymer of a pentasaccharide subunit. Recent discoveries by our group have determined that Pel is a positively charged amino sugar polymer. Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the biofilm matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance, as previous studies suggest fairly rapid movement of antibiotics through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid P. aeruginosa biofilms. To our surprise, we showed that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions, but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.