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case_images: (A-B) Dental pulp sample from two immature individuals dated to the 16th century. (C-D) Isolation bacteraemia of Clostridium tetani. (A) The 16th century teeth of two prepubescent individuals are cleaned with pure ethanol and bleach. (B) The dental pulp is kept in a closed environment (the pulp cavity). (C) Appearance of a bacterial carpet of C. tetani after one week of incubation on COS Agar enriched with 5 % sheep blood. (D) Microscopic observation of C. tetani in rod form after staining with magnification 100X.
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case_images: Microscopic observation of colonies showed sporulated, gram-positive bacteria (Fig. 2) identified as C. tetani by MALDI-TOF-MS in the presence of negative control, with an identification score > 2.12.
case_images: Circular representation of the genomic comparisons using coding DNA sequences (CDS), display on Proksee online (https://proksee.ca/)(19, displaying Blast comparison of the ten C. tetani strain genomes, features an in the in red ring in middle depicting the CDS of C. tetani reference strain E88, surrounded by two rings depict GC content and the GC skew and a gray ring representing the backbone. Following the backbone, the CDS present at the level of the reference genome and absent within the two ancient genomes Q7451 and Q7452 marked in red, tracking of the genes of the nine genomes including the two genomes reported in this study with different colors detailed in the legend.
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case_images: Ancient Q7451 and Q7452 genomes specifically lacked 283 genes (Fig. 3); and specifically incorporated three genetic blocks for a total of 4780 bp (Supplementary data Fig. 3; Supplementary Table 1).
case_images: Pan-genome genetic relatedness analysis (utilizing the Roary pan-genome pipeline), of the ten C. tetani strain. A total of 4103 genes were identified. At left a dendrogram, in red the two strains of this study. Right: heatmap of the core genes each row shows the gene profile of each strain's gene presence are in blue and gene absence in white matrix.
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case_images: GCA_949,357,675), and a 2 857 805-pb genome with a 28.6 % GC content for C. tetani CSUR Q7452 isolated from Ind-44 (accession number: GCA_949,357,665). Genomic analysis confirmed C. tetani identification with an 87.9 % DDH value with reference genome (GCF_000762305.1) for ancient isolates, both encoding a 3948-bp, 27 % GC content- tetanus neurotoxin tetX gene exhibiting 99.34 % sequence similarity with homologous reference gene. Genomic comparisons revealed 2268 core genes (present in 100 % of studied genomes) forming 55.28 % of the 4103-gene pangenome, 1094 shell genes (present in 15-95 % of genomes) forming 26.67 % and 741 cloud genes (present in < 15 % of genomes) forming 18.05 % of pangenome (Fig. 4).