A nontuberculous mycobacterium could solve the mystery of the lady from the Franciscan church in Basel, Switzerland

Abstract

Background: In 1975, the mummified body of a female has been found in the Franciscan church in Basel, Switzerland. Molecular and genealogic analyses unveiled her identity as Anna Catharina Bischoff (ACB), a member of the upper class of post-reformed Basel, who died at the age of 68 years, in 1787. The reason behind her death is still a mystery, especially that toxicological analyses revealed high levels of mercury, a common treatment against infections at that time, in different body organs. The computed tomography (CT) and histological analysis showed bone lesions in the femurs, the rib cage, and the skull, which refers to a potential syphilis case.

Results: Although we could not detect any molecular signs of the syphilis-causing pathogen Treponema pallidum subsp. pallidum, we realized high prevalence of a nontuberculous mycobacterium (NTM) species in brain tissue sample. The genome analysis of this NTM displayed richness of virulence genes and toxins, and similarity to other infectious NTM, known to infect immunocompromised patients. In addition, it displayed potential resistance to mercury compounds, which might indicate a selective advantage against the applied treatment. This suggests that ACB might have suffered from an atypical mycobacteriosis during her life, which could explain the mummy's bone lesion and high mercury concentrations.

Conclusions: The study of this mummy exemplifies the importance of employing differential diagnostic approaches in paleopathological analysis, by combining classical anthropological, radiological, histological, and toxicological observations with molecular analysis. It represents a proof-of-concept for the discovery of not-yet-described ancient pathogens in well-preserved specimens, using de novo metagenomic assembly.

Keywords: Ancient DNA (aDNA); Anna Catharina Bischoff (ACB); Bacteriophage; Brain infections; De novo assembly; Franciscan church mummy; Mycobacteriosis; Nontuberculous mycobacteria (NTM); Syphilis.

Fig. 1

Description of the mummy’s finding site. A Map of Europe with zoom-in on Basel in Switzerland. B The Franciscan church during the renovation in 1975. C Photograph showing the first glance on the mummy tomb; recognizable are the overlapped hands. D The location of the burial chamber (indicated by red arrow) inside the Franciscan church (© Archäologische Bodenforschung, 1975/6 – Plan “Grabkammer und Grabschächte (G564)”, modified by H. Eichin 1981)

Fig. 2

Overview on the radiological, toxicological, and microbiological characteristics of the ACB mummy. A Computed tomography (CT)-based three-dimensional reconstruction of the skull. Notice the darker colors which represent lower x-ray densities than healthy bone. Copyright: Holger Wittig, Institute of Forensic Medicine, University of Basel. B Concentration of elemental mercury in different body samples, where the error bars refer to the standard errors. C Relative abundances of the top 12 microbial families on different body tissues as inferred by number of shotgun-metagenomic reads compared against the nr-database (please refer to the “Methods” section for details). Numbers in parentheses refer to sample IDs (please refer to Additional file 1: Table S1 for further details)

Fig. 3

Genome-level taxonomic assignment of the brain bacterium. A Taxonomic assignment of the brain NTM contigs as assigned by searching against the NCBI-nt database, using BLASTn. The number of the assigned contigs is shown next the taxon names based on the lowest common ancestor (LCA) as determined by MEGAN (please refer to the “Methods” section for further details). B Unrooted phylogenetic tree of the family Mycobacteriaceae, including a single representative genome of each species, based on PhyloPhlAn marker genes. The background colors of the clades refer to: red, Mycobacterium spp.; yellow, Mycolicibacter spp.; green, Mycobacteroides spp.; and blue, Mycolicibacterium spp. C Heatmap-based on MASH distances of all characterized species’ genomes within the genus Mycolicibacterium including the genome of the brain bacterium (highlighted in bold red font). The heatmap annotations to the left of the heatmap refers to whether the microbe was isolated from a host or was reported as a human pathogen. For further details on the isolation sources, please refer to the Additional file 1: Table S6. D Damage plots of human DNA of different tissues as well as the brain NTM. The damage plots were generated considering the mapped reads of the indicated tissues different body tissues (i.e., tooth, intestinal tissues, skull, dura mater, and brain) against human genome (hg19) while the brain NTM was generated considering the brain metagenomic reads mapped against the brain NTM assembled genome. Ancient DNA damage represented by the terminal substitution of Cytosine to Thymine at the 5′ ends of the DNA fragments. The labels in parenthesis refer to sample ID and the mercury concentrations ± standard error. For further information on the read lengths distribution please refer to Additional file 2: Figure S3 [14, 15]. Note: The human DNA are showing variable levels of ancient DNA damages, despite of being of the same individual. The lowest levels of the human DNA damages are in the brain and dura mater samples, which goes with the abundance of the brain NTM and the mercury concentrations as well

Fig. 4

Genetic map of the brain bacterium genome. A The heatmap shows distribution of the DNA of the brain NTM in the mummy’s samples as well as other control samples; The numbers to the left refer to the sample ID as referred to in Additional file 1: Table S1, while the letters refer to the samples group as follows: S, the mummy’s tissue samples; C1, control samples that were in contact with the mummy; C2, samples that were taken from other individuals from the same burial site; C3, soil sample that was collected from inside the coffin of the mummy (please refer to Additional file 1: Table S1 and Additional file 2: Figure S4 for further details). The presence is shown by breadth values with minimum coverage of 3x per site. The contigs are arranged in a descending order from left to right (for details on the contigs, please refer to Additional file 1: Table S5). The loci referred to in bold red fonts are gene clusters/operons and are further detailed in Additional file 2: Figure S5, while those in black bold are genes that are known to be involved in crossing blood brain barrier (BBB) and brain invasion. B The genetic map of the contig 38, which is assumed to be a phage genome, as inferred by the tool PHASTER (for further details on the phage annotation, please refer to Additional file 1: Table S7)