- Volume 8, Issue 2, 2022

The evolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) within passerines has resulted in pathoadaptation of this serovar to the avian host in Europe. Recently, we identified an S. Typhimurium lineage from passerines in North America. The emergence of passerine-adapted S. Typhimurium in Europe and North America raises questions regarding its evolutionary origin. Here, we demonstrated that the UK and US passerine-adapted S. Typhimurium shared a common ancestor from ca. 1838, and larids played a key role in the clonal expansion by disseminating the common ancestor between North America and Europe. Further, we identified virulence gene signatures common in the passerine- and larid-adapted S. Typhimurium, including conserved pseudogenes in fimbrial gene lpfD and Type 3 Secretion System (T3SS) effector gene steC. However, the UK and US passerine-adapted S. Typhimurium also possessed unique virulence gene signatures (i.e. pseudogenes in fimbrial gene fimC and T3SS effector genes sspH2, gogB, sseJ and sseK2), and the majority of them (38/47) lost a virulence plasmid pSLT that was present in the larid-adapted S. Typhimurium. These results provide evidence that passerine-adapted S. Typhimurium share a common ancestor with those from larids, and the divergence of passerine- and larid-adapted S. Typhimurium might be due to pseudogenization or loss of specific virulence genes.

Streptococcus mitis is a Gram-positive bacterium, member of the oral commensal microbiota, which can occasionally be the etiologic agent of diseases such as infective endocarditis, bacteraemia and septicaemia. The highly recombinogenic and repetitive nature of the S. mitis genome impairs the assembly of a complete genome relying only on short sequencing reads. Oxford Nanopore sequencing can overcome this limitation by generating long reads, enabling the resolution of genomic repeated regions and the assembly of a complete genome sequence. Since the output of a Nanopore sequencing run is strongly influenced by genomic DNA quality and molecular weight, the DNA isolation is the crucial step for an optimal sequencing run. In the present work, we have set up and compared three DNA isolation methods on two S. mitis strains, evaluating their capability of preserving genomic DNA integrity and purity. Sequencing of DNA isolated with a mechanical lysis-based method, despite being cheaper and quicker, did not generate ultra-long reads (maximum read length of 59516 bases) and did not allow the assembly of a circular complete genome. Two methods based on enzymatic lysis of the bacterial cell wall, followed by either (i) a modified CTAB DNA isolation procedure, or (ii) a DNA purification after osmotic lysis of the protoplasts allowed the sequencing of ultra-long reads up to 107294 and 181199 bases in length, respectively. The reconstruction of a circular complete genome was possible sequencing DNAs isolated using the enzymatic lysis-based methods.