FT-IR SPECTROSCOPY AND OTHER CORROBORATIVE TECHNIQUES APPLIED TO THE STUDY OF BORDETELLA PERTUSSIS BIOFILM AND ITS EXTRACELLULAR POLYMERIC SUBSTANCES (EPS)

DIEGO SERRA; ALEJANDRA BOSCH; OSVALDO YANTORNO

Heidelberg, Alemania

Congreso; Spec 2006. Shedding Light on Disease: Optical Diagnosis for the New Millennium; 2006

Robert Koch Institute, Berlin

Recent studies provided evidence supporting the hypothesis that B. pertussis, a persistent human pathogen, may choose a community-based existence (biofilm) as a strategy to colonize the host [1,2]. Biofilm is a microbial lifestyle where organisms grow on surfaces organized in communities [3]. Novel approaches combining molecular, microscopic and phenotypic techniques were successful in providing primary information about structural and physiological features of the biofilm development process in many organisms [4]. However, little is known about the development process, the architecture, and the changes that occur throughout biofilm development in B. pertussis. In the current study we use a continuous flow culture system to characterize the biofilm development and the extracellular polymeric substance (EPS) produced by B. pertussis wild type strain. Additionally, we examined the relevance of the Bordetella virulence gene activator/sensor (BvgAS) system and the role of virulence factors such as Adenylate cyclase (AC) and the adhesin Filamentous hemagglutinin (FHA) in biofilm growth. B. pertussis wild type Tohama I strain (WT), an avirulent strain Bvg- phase-locked (BP537 strain), mutants lacking FHA expression (BPGR4 strain), and AC expression (BPM3183 strain) were cultured in continuous flow chamber systems on ZnSe windows or borosilicate coverslips and monitored over time by FT-IR spectroscopy, Fluorescence microscopy and Scanning Laser Confocal Microscopy (SLCM). Microscopy images were analysed by LSM 5 Image Browser software, and in order to obtain quantitative information of biofilm structures, SLCM images were evaluated by COMSTAT software. FT-IR spectra of biofilms formed by B. pertussis wild type strain, revealed an increase of biomass (Amide II) over the time with enhanced intensities of bands assigned to carbohydrates groups (1200-900 cm-1) relative to proteins and modifications in bands referred to as biofilm growth markers, as 1629, 1372 and near 1400 cm-1 (due to asymmetric and symmetric carboxylate ion stretching), a shoulder in Amide II band at 1516 cm-1, bands at 1733 and 1260 cm-1 (assigned to O-acetyl groups), and 860 and 800 cm-1 bands due to carbohydrates anomeric configuration. The later bands were also found in a further FT-IR characterization of the extracellular substance recovered and purified from biofilms. Biomass increase over the course of biofilm development was also observed by fluorescence microscopy examination. Wild type bacteria formed strong biofilm that result in large, irregularly shaped microcolonies scattered on the surface. Interestingly, the development of these structures correlated with the higher production of EPS observed by FT-IR spectroscopy, suggesting that the progress toward a mature biofilm in B. pertussis is associated with the production of a polysaccharide-enriched matrix which may contribute to the biofilm architecture. Quantitative analysis of SLCM images provided additional information about the structure of the biofilm formed by the wild type strain and showed the inability of the avirulent strain and FHA deficient mutant to form thick and strong biofilms. Our results remark that the biofilm development in B. pertussis occurs in stages, which are accompanied by changes at the protein and carbohydrate levels. Furthermore, biofilm growth correlates with the production of an extracellular matrix, and that this process is mainly dependent on the expression of a complex array of virulence factors regulated by the BvgAS system, with specially contribution of FHA. Current investigations are directed to determine weather the virulence control system, BvgAS, is involved in the production of the extracellular polysaccharide-enriched matrix. Likewise, to determine weather this component directly contributes, like FHA adhesin, to the development of the mature biofilm formed by B. pertussis.