Geographical area studied and sediment sampling
The Tiber River belongs to one of the largest river systems in Italy, with a catchment area of 17,375 km2 . The river is 405 km long, and runs from the Tuscan-Emilian Apennines to the Tyrrhenian Sea, through four administrative regions. The water volume ranges from 60 m3s-1 to 3200 m3s-1 with a yearly average of 230 m3s-1 .
Thirteen sampling sites were selected, four along the main stem of the Tiber, four along the Farfa, four along the Treja and one along the Aniene (Table 4). The aim was to allow a comparison of the microbial community composition in sediments upstream and downstream of the city of Rome Sediment samples were collected seasonally from February 2002 to June 2003 for all sites located along Tiber River and Aniene tributarie while sites along Treja and Farfa were sampled only during the hot season. Sampling was performed using a bucket attached to a winch, that was immersed at a depth of between 1 and 3 meters below the water surface. Samples were transported and stored in sterile 50 ml tubes, at 4 °C . Granulometric characterization was performed on each sediment sample, based on the different sedimentation speeds of silt, clay and sand particles [58, 59].
Isolation of bacteria
Five grams of sediment from each sample were suspended in 45 ml of sterile phosphate buffered saline (PBS) [K2HPO4 3 g/ml, KH 2PO4 1 g/ml, NaCl 8,5 g/ml; (pH 7,2 ± 0,2)] + 0.1% Tween 80. Three replicates were used for each sample. Suspension samples were then homogenized on a magnetic stirrer (Heidolph, MR 3001) for 30 minutes in order to break up clumps of bacteria. The samples were heat-shocked at 80–85°C for 10 minutes before cultivation, so as to inactivate vegetative bacteria and enhance sporulation. Five ml of the spore-containing suspension was diluted (1:10) with sterile PBS, in 15 ml Falcon vials (BD, USA). For optimal growth, we adopted the following plating technique: 8 ml of SPS (Sulphite Polymixine Sulphadiazine Agar, Oxoid) medium culture was placed in Petri dishes with a 1 ml aliquot of diluted suspension, and incubated in an anaerobic jar equipped with a manometer and a CO2 generator, at 36 ± 1°C for up to 24 hours. Black colonies characteristic of anaerobic bacteria appeared.
Colonies were counted directly, and the results expressed as colony-forming units per gram of dry weight of sediment (CFU/gss). In order to calculate dry weight, about five grams of sediment were weighted and dried, in triplicate, at 120 ± 1°C for 12 hours. The procedure was repeated until a stable weight was reached.
After counting, colonies were transferred into new plates with TSA (Tryptone Soya Agar, Oxoid) medium and incubated in anaerobic conditions as described above. Single colonies were isolated from each of the three replicates and resuspended in sterile water for DNA extraction. Biochemical identification of strains was then performed using API 20A Strips (Biomerieux, France) according to the manufacturer's instructions.
Positive and negative controls were used to validate the microbiological method. A negative control was generated as follows: after sterilization at 121°C for 15 min, five grams of sediment were suspended into 45 ml of sterile PBS, then plated in medium culture as described above. The same procedure, followed by in vitro infection with C. perfringens (ATCC 12918), was used to generate a positive control.
Microbiological analyses and genome extractions were performed within 24 hours from sampling; extracted DNA were redissolved in sterile water, aliquoted in small volumes and stored at -80°C until use. All molecular experiments were completed within six months from extraction.
DNA extraction and 16S ribosomal RNA amplification
One and a half ml of microbial suspension was pelleted by spinning at 6000 rpm for 5 min. Extraction of nucleic acids was carried out following the method described in Ausubel et al. . The pellets were resuspended in 570 μ1 TE buffer [10 mM Tris-HCl, 1 mM EDTA, (pH 8)] and the suspension mixed with 80 μl CTAB/NaCl solution (10% CTAB in 0.7 M NaCl), 30 μl of 10% SDS and 3 μl of Proteinase K (20 mg μ/ml), and incubated at 55°C for 10 min.
An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added, and the mix was centrifuged at 17000 rpm for 5 min. Supernatants were transferred to new tubes, washed with 1 volume of chloroform/isoamyl alcohol (24:1) and spinned at 17000 rpm for 5 min. Supernatants were again transferred to new tubes and DNA precipitated at -20°C with two equivalent volumes of 95% ethanol for 30 min. After precipitation pellets were washed with 70% ethanol, dried in a thermoblock at 40°C, and resuspended in 100 μl of 10 mM TE buffer. A 701 bp fragment of the 16S rRNA coding region was selectively amplified with two primers: 728 forward (5'-GGGGAATATTGCACAATGG-3') and 729 reverse (5'-GGGACTTAACCCAACATCTCA-3'). Primers were designed (using Primer3 software) , so as to anneal to conserved positions of different Clostridium species published on the NCBI database (accession numbers: NC_003366, AF371836, X68174, AF479585, AF458779). In particular the forward primer anneal at position 351–371 and the reverse primer at position 1047–1060 of accession number NC_003366 (Clostridium perfringens str. 13, complete genome, GeneID: 988236). Annealing temperature was calculated by the Primer 3 software. The final concentrations of PCR reaction components were: 10 mM PCR Buffer [Tris-HCl (pH 8.8)], 2 mM MgCl2, 0.2 mM of each dNTP, 0,2 mM of the two primers, 1 U Taq polymerase (Roche, Mannheim, Germany).
The PCR reaction was performed in a Personal cycler (Biometra T4, Göttingen, Germany), under the following conditions: an initial denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, annealing at 57°C for 30 s and elongation at 72°C for 60 s. The reaction was terminated with a 10-min final elongation step at 72°C. A negative control (sterile deionized water) and a positive control (C. perfringens genomic DNA) were included in the experiment to confirm the validity of results.
PCR products (5 μl of each reaction mixture) were visualized by electrophoresis on a 1% agarose gel, in TBE (Tris Borate EDTA) buffer (100 mM Tris-HCl, 90 mM boric acid, 1 mM Na2EDTA), with ethidium bromide staining (10 mg/ml), at 90 V for 1 h.
Partial sequencing of the 16S ribosomal RNA gene
Before sequencing, unincorporated nucleotides and primers were removed from PCR products using the Promega DNA Purification System (Madison, WI, USA), following the manufacturer's protocol. The sequencing reaction was prepared using the CEQ 2000 Dye Terminator Cycle Sequencing with the Quick Start Kit (Beckman Coulter, Fullerton, USA), and was performed in a Personal cycler (Biometra T4, Göttingen, Germany) according to the manufacturer's instructions. Sequences were generated with a Beckman 3000 sequencer (Beckman Coulter, Fullerton, USA) and analyzed using Chromas 2.23 software (Technelysium Pty Ltd, Tewantin, Australia). BLAST searches were performed for each of the sequences obtained, to find its closest phylogentic neighbors.
Sequences alignment and analysis
The partial sequences of 16S rRNA genes obtained in this study were aligned to each other using ClustalX software, version 1.81 [62, 63]. Phylogenetic relationships were inferred by the neighbor-joining method  using the phylogenetic inference software Paup*, version 4.0 . The evaluation of the most appropriate model of sequence evolution was computed using ModelTest version 3.0 . For the 16S rRNA gene, this resulted in the selection of the HKY85 + G model. Genetic relationships were also plotted by non-metric multidimensional scaling (MDS) using SYSTAT 9.0 software (SPSS-SCIENCE), which provides an ordering of n variables on a similarity/dissimilarity matrix by means of n points in a three dimensional space [Figure 2].
Accession numbers of 16S rRNA sequences
The 83 nucleotide sequences reported in this paper have been deposited in the GSDB, DDBJ; EMBL and NCBI nucleotide sequence databases under accession nos. EF153846 to EF153928. Eighty three environmental isolates of Clostridium: C. bifermentans [GenBank: EF153846 to 153861], C. ghoni [GenBank: 153862 to 153868], C. glycolicum [GenBank: 153869 to 153870], C. perfringens [GenBank: 153871 to 153918], C. barati [GenBank: 153919 to 153921], C. thiosulfatireducens [GenBank: 153922 to 153925] and C. butyricum [GenBank: 153926], and two environmental isolates of Bacillus cereus [GenBank: EF153927, EF153928].