Microbial whole genome sequencing analysis: from de novo single strains reconstruction to metagenomics to characterize bacterial communities

Microbial genomes analysis is the field that takes the greatest advantages from the breakthroughs in genomics and computational biology. Whole-genome sequencing strategies allowed the fulfilment of first complete genome sequence of a bacteria in 1995, since then technological improvements reduced the time required for sequencing and the cost per base pair while at the same time dramatically increased the quality of final products. These enhancement leads to the possibility to extend microbial genomes analysis ranging from the study of the single strain to whole microbial communities. In this thesis two application of whole-genome sequencing are presented: I) the genome’s reconstruction and characterization of Acinetobacter johnsonii ICE_NC and II) the analysis of the microbial community characterizing the oral plaque of human patients in different clinical conditions of periodontitis and type 2 diabetes. Chapter 1. Acinetobacter johnsonii ICE_NC was isolated from industrial soil and showed a role in the biotransformation of bile acids (BAs). Here are described the use of hybrid de novo assemblies, that combine long-read Oxford Nanopore and short-read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α-hydroxysteroid dehydrogenase (12α-HSDH), involved in bile acids metabolism. The hybrid de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α-HSDH was detected on the plasmid. Our findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These also allowed the identification of the A. johnsonii ICE_NC gene for the 12α-HSDH enzyme, whose activity was confirmed at the biochemical level. Chapter 2. Periodontitis and type 2 diabetes have a long established bi-directional relationship. Twelve subjects, falling into one of the four study groups based on the presence/absence of poorly controlled type 2 diabetes and presence/absence of moderate-severe periodontitis, were selected. The high-resolution whole metagenomic shotgun sequencing was used to characterize the subgingival microbiome of patients with different health status regarding periodontitis and type 2 diabetes. Taxonomic profile was analysed with a BLAST analysis combined with MEGAN6 or MetaPhlAn 3.0. Both outputs were statistically tested with STAMP and LEfSe. The investigation of functional activities was performed by DIAMON combined with MEGAN6 -KEGG and by HUMAnN 3.0; statistical analysis was carried out with LEfSe. Taxonomic analysis results unlighted: I) the significant presence of Anaerolineaceae bacterium oral taxon 439 in periodontitis patients, II) that the presence of bacteria historically associated with onset and progression of periodontitis seems more linked to a general inflammatory status, rather than specifically with periodontal disease and III) the presence a core microbiome, that is resilient to changes in the oral health status. Main results in functional investigation were that most of the significantly enriched pathways involved in amino acid biosynthesis and carbohydrate or energy metabolism belonged to patients without periodontitis. Pathways related to cell structure biosynthesis, fatty acid and lipid biosynthesis, nucleoside and nucleotide metabolism and ferroptotic death and/or iron homeostasis were significantly abundant in patients affected by periodontitis, diabetes or presenting comorbidity.