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Genotyping of Mycobacterium tuberculosis and Mycobacterium leprae ancient DNA

  • Kerudin, Ammielle
Publication Date
Dec 14, 2020
Manchester eScholar
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The overall aim of this study was to employ a biomolecular technique – ancient DNA (aDNA) – to study two ancient diseases that were endemic in Europe (and therefore Britain) during the medieval period: tuberculosis and leprosy. In humans, the diseases are caused by M. tuberculosis and M. leprae, respectively – both of which are members of the M. tuberculosis complex (MTBC). Skeletal manifestations of both diseases may develop in bone remains, which can be recognized using osteological analysis. In some cases, however, the skeletal changes are ambiguous. Ancient DNA methods are used for case confirmation and to answer historical questions such as the spread, origin and evolution of disease. The first objective of this thesis was to determine whether the MTBC aDNA detection frequency is high enough to plan a larger study to test hypotheses such as possible strain differences in urban and rural areas, as it has been suggested that urbanization assists the spread of tuberculosis, enhancing its virulence. To meet this objective, 60 skeletal remains from 16 different locations in Yorkshire, England were studied. All samples were screened for MTBC aDNA presence and 8 samples were selected for next-generation sequencing (NGS). In the PCR assay screening, only 1 sample produced a positive MTBC amplification. However, when subjected to NGS, this sample together with the other 7 samples did not produce enough sequence reads to allow genome comparisons. An attempt to compare metagenomic content between urban and rural sites was also performed. There was no specific difference in metagenomic content between urban and rural samples. Based on the PCR analysis, the sample St Andrew Fishergate 6, dated to the early 14th century AD, showed evidence of possible tuberculosis infection. NGS analysis further revealed a possible M. tuberculosis and M. leprae mixed infection, albeit with insufficient read coverage to determine genome sequence polymorphism. The second objective was to use NGS to determine the genotype of the M. leprae strains present in skeletons from two mediaeval sites, at Chichester and Raund Furnells, both in England. This study served as a continuation for the previous confirmation by PCR of leprosy in these skeletons. The samples were further subjected to whole M. leprae genome target enrichment before subsequent high-throughput sequencing. For all 3 historical M. leprae isolates, at least 70% genome sequence coverage was obtained, with a mean read depth of 4-10x. The near-complete genome sequences that were obtained allowed subtype identification for each of the ancient M. leprae isolates. Two mediaeval samples from Chichester belonged to the 3I subtype, which is typical of ancient Northern European and contemporary North American isolates. Meanwhile, an M. leprae isolate from Raunds was identified as belonging to the 3K subtype – the first example of this subtype identified in Britain. Transmission of the M. leprae 3K subtype to Britain is suggested to have been associated with the travels of crusaders and pilgrims to the Holy Land during the mediaeval period. The overall conclusion of the work is that although M. leprae aDNA is well preserved in skeletal remains showing osteological signs of leprosy, the same is not true for MTBC preservation in skeletons showing indications of tuberculosis. To test hypotheses such as the effect of urbanisation on tuberculosis, a high frequency of MTBC detection must be achieved, but this is complicated by the very nature of ancient DNA itself – highly fragmented, low endogenous DNA copy, presence of environmental contaminants – and by the possibility of low bacterial load in skeletons at the time of death. In projects where the testing of a high number of samples is required, more stringent selection criteria must be imposed to minimize the impact of destructive analysis.

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