Isolation and molecular characterization of Acinetobacter communities isolated from Epilithons

Objectives: Epilithons are microbial communities consisting of prokaryotes, fungi, and protists embedded in a polysaccharide matrix; these communities form on mineral surfaces and organic substrates (e.g. leaf litter) temporally or permanently submerged in water of rivers or benthic ecosystems. In streams, these sites are hot spots of carbon turnover. The microbial activity of epilithons is favoured by the dissolved carbon in the surrounding water during the initial development. Epilithic biofilms typically are physiologically heterogeneous structures with high biodiversity of bacteria that express distinct metabolic pathways. Therefore epilithons are interesting systems to study the horizontal gene transfer (HGT) as they provide an environment where sessile bacterial populations and high levels of activity can promote bacterial interactions. While microbiological studies mostly focused on the planktonic component, the bacterial communities of epilithic biofilms have remained poorly explored. Here we present the isolation and molecular characterisation of bacteria from epilithons forming on the rocks surface of the Acquarossa river (Viterbo, Italy) a peculiar riparian ecological niche in the volcanic area of central Italy, characterized by high level of iron deposition (Acquarossa = "red water") and geothermal activity. Methods: Two epilithons, differing in the prominent colour, red or black, were analysed; 16 rDNA sequencing of 200 randomly selected isolated was performed followed by sequence analyses in databases and phylogenetic trees construction. Results: 16S sequences analyses pointed out that more that 56% and 87% of isolated bacteria, respectively from the red and black epilithon, belongs to the genus Acinetobacter with Pseudomonas as the second most abundant genus (20 and 5%, respectively). Phylogenetic tree construction showed that the Acinetobacter strains isolated from the 2 epilithons form separated clusters, with a low number of shared haplotypes, showing, i) a high diversity of the two epilithons at strain level, but a low diversity of genus level, ii) a diversity in the composition between the two epilithons. Conclusions: Epilithons can represent interesting ecosystems to study bacterial interactions and HGT; the molecular characterisation of the two communities is going ahead two deepen the evaluation at strain level; furthermore plasmid extraction and sequencing is planned to study HGT and natural transformation inside the two epilithons.