Application of cyanobacteria oligonucleotide probes designed for identifying bacterial cells in surface water

Cyanobacteria colonize different environments and blooms can occur both in contaminated and noncontaminated water bodies (freshwater, brackish and marine areas). In some cases, some bacterial species can produce toxins and this phenomenon can have a negative impact on ecosystem and human health. Inside 150 genera of cyanobacteria known, more than 40 species are able to produce toxins. The latter are natural compounds which differ from both chemical and toxicological point of view; they are responsible for both acute and chronic poisoning in animals and humans. Among the main classes of cyanotoxins, microcystins are frequently found in the environment. These toxins are mainly accumulated in liver, but also in intestine and kidneys and can be very dangerous for animal and human health [1]. Fast and sensitive methods to identify unequivocally species belonging to Microcystis and Planktothrix genera are necessary to discriminate these genera respect to the other non-toxic cyanobacteria. For this purpose, we designed, developed and validated some oligonucleotide probes for FISH (Fluorescence In Situ Hybridization) analysis in order to detect them in freshwater. The FISH probes were designed using the ARB software with the Silva database in the framework of the MicroCoKit project. We have focused on identification of Microcystis aeruginosa and Planktothrix agardii species because they are among the most common toxic cyanobacteria in freshwater. We tested different fixative methods to minimise the natural autofluorescence from chlorophyll-a and some accessories pigments (e.g. phycobilins and carotenoids) in order to visualize Microcystis aeruginosa and Planktothrix agardii under a laser confocal microscope. Firstly, the FISH probes designed (GNPlankS02, PkAgD03, MicAerD03) have been tested on pure cultures of M. aeruginosa and P. agardii species. Then the probes were successfully applied to several water samples collected from the River Tiber and Lake Albano (Italy).