A mechanistic basis for Bordetella biofilm formation: In vitro and in vivo.

摘要

It is becoming increasingly apparent that the majority of bacteria exist as a surface associated community in both the environment and within a host. Biofilms are major contributors to virulence for a number of bacterial species and have been defined as a sessile group of organisms embedded in a self produced polymeric matrix. The regulatory circuitry governing biofilm development can be complex and is controlled, in some species, by two component regulatory systems.; Bordetellae are Gram negative bacteria which colonize the respiratory tract of mammals. The majority of virulence factors expressed by Bordetella are regulated by the BvgAS two component system. Our data demonstrate that Bordetella biofilm development is controlled by the BvgAS system. Utilizing phase locked strains we demonstrate that the BvgAS effect is exerted at a postattachment stage. Additionally, we demonstrate that Bordetella biofilms are tolerant to a number of different antimicrobial compounds, including clinically relevant antibiotics.; The biofilm matrix plays an important role in biofilm structure and resistance to antimicrobial compounds. We have identified a putative polysaccharide locus in Bordetella, bpsABCD, and have shown that it contributes to the stability and maintenance of structures within mature biofilms. The bps locus is homologous to several bacterial loci that are required for the production of poly-beta-1,6-GlcNAc and have been implicated in bacterial biofilm formation. Utilizing immunoblot analysis and an enzyme which specifically cleaves poly-beta-1,6-GlcNAc, we demonstrated that Bordetella produce a similar polysaccharide which was both antigenically and biochemically similar to the poly-beta-1,6-GlcNAc. Our data also demonstrate that the production of this specific polysaccharide is dependent on the presence of the bps locus.; Despite evidence for Bordetella biofilm formation in vitro, direct evidence of the biofilm lifestyle during mammalian infections has been lacking. We hypothesize that the biofilm mode of existence in the respiratory tract, especially in the nasopharynx, allows Bordetella to escape immune defenses thereby leading to persistent infections. Our results demonstrate the ability of both the animal (B. bronchiseptica ) and human adapted species (B. pertussis) to form biofilms on the nasal septum epithelium.; Based on the similarity of the bps locus with bacterial loci that have been implicated in virulence, we hypothesized that Bps contributes to the survival and persistence of Bordetella in mammalian hosts. To address this, we utilized a murine model of Bordetella infection and inoculated animals intranasally with the wild type strain of B. bronchiseptica and the isogenic Deltabps strain containing an in frame deletion of the entire bps locus. Our results show that while the wild type is capable of forming robust biofilms, the Deltabps was impaired in biofilm formation at 38 days post-inoculation. In conclusion, the studies described here begin to define the biofilm mode of existence for Bordetella species both in vitro and in vivo.