Characterization of Cr(VI)-hyper-resistant Pseudomonas alcaliphila 34 biofilm

Biofilm, the form commonly taken by microorganism in environment, consist of surface-attached cells embedded in a self-produced extracellular polymeric matrix. Pseudomonas alcaliphila 34, a biofilm producing bacterium characterized by a high level of resistance to Cr(VI), was used as a model to examine the influence of Cr(VI) on bacterial biofilm. Planktonic cultures of this strain underwent a previous phenotypic characterization through the high-throughput technology of Penotype Microarray (PM) - Biolog, allowing to test up to 2000 phenotypes simultaneously. The bacterium was grown into 96 wells polistirene microplates in mineral medium (TMM) supplemented with gluconate and Cr(VI) at different concentrations for 24 h. Then cultures were removed by the wells and biofilm was quantified by cristal violet assay. Metabolic activity of biofilm was also quantified in a separate experiment, using a tetrazolium dye. The results showed that bacterial biofilm production got enhanced by Cr(VI) concentrations ranging from 6.25 to 50 mM, compared to the control condition [0 mM Cr(VI)]. On the contrary biofilm metabolic activity decreased with increasing Cr(VI) concentrations. This suggested that Cr(VI) enhances formation of biofilm, mainly composed by metabolic inactive cells. In order to confirm this result, biofilms of P. alcaliphila 34 grown in TMM added with gluconate or TMM added with gluconate and 6.25 mM of Cr(VI) were stained with Syto19 (a freely diffusible, nucleic acid intercalator that labels all cells in the microbial population regardless of viability) and propidium iodide (a membrane impermeant DNA intercalator that only stains cell with compromised membrane integrity) and analysed by confocal laser scanning microscopy. Since extracellular DNA (eDNA) is one of the main polymers contributing to the biofilm structure, we assessed the role of eDNA in biofilm structure of P. alcaliphila 34: cultures untreated and treated with Cr(VI) at different concentrations were grown in presence or absence of DNase I and subsequently biofilm production was measured by cristal violet assay while biofilm metabolic activity was analyzed by a tetrazolium dye.Our results showed that, for all samples tested, biofilm formation and biofilm metabolic activity were significantly reduced in cultures treated with DNase, suggesting that eDNA plays a key role in the formation of P. alcaliphila biofilm both in presence and absence of Cr(VI). The research was supported by the MIUR (PRIN2008)