Widespread occurrence of botulinum and tetanus neurotoxin genes in ancient DNA

Ancient DNA collected from archaeological specimens not only provides a window into ancient human genetic diversity but also contains a rich mixture of associated microbial DNA including potential pathogens. In recent work, we identified C. tetani and tetanus neurotoxin (TeNT) genes in ancient DNA datasets collected from human archaeological specimens. However, the reasons underlying the occurrence of these toxin genes and the extent to which other toxin genes are present in ancient DNA is unclear.

Here, we performed a large-scale analysis of 6435 ancient DNA (aDNA) sequencing datasets including human and non-human sources, searching for 49 clostridial neurotoxin types and subtypes, and 3 additional unrelated toxins.

Our search identified a total of 105 ancient DNA datasets (1.6 %) containing significant matches to one or more neurotoxin genes. Consistent with our earlier work, TeNT genes were most common, found in 50 ancient DNA datasets. In addition, we identified sequences encoding diverse botulinum neurotoxins including BoNT/C (40 samples), BoNT/D (6 samples), BoNT/B (4 samples), BoNT/E (1 sample), and the Enterococcus-associated BoNT/En (10 samples). TeNT genes were detected in a broad range of ancient samples including human and animal (horse, wild bear, chimpanzee, gorilla, dog) remains, whereas the largest diversity of toxins was detected in aDNA from Egyptian mummies. Phylogenetic and sequence analysis of the identified matches revealed close identity to modern forms of these toxins. Damage analysis revealed several toxin genes with hallmarks of ancient DNA associated damage, indicative of an ancient origin.

Our work reveals that clostridial neurotoxin genes occur frequently in aDNA samples, including human and animal-associated toxin variants. We conclude that the frequent association of these genes with aDNA likely reflects a strong ecological association of pathogenic clostridia with decaying human and animal remains and possible post-mortem colonization of these samples.