Abstract
There are many reports of ancient DNA from bacteria of the Mycobacterium tuberculosis complex (MTBC) being present in skeletons with and without osteological indications of tuberculosis. A possible complication in these studies is that extracts might also contain DNA from the microbiome of the individual whose remains are being analysed and/or from environmental bacteria that have colonised the skeleton after death. These contaminants might include ‘mycobacteria other than tuberculosis’ (MOTT), which are common in the environment, but which are not normally associated with clinical cases of tuberculosis. In this paper we show that MOTT of various types, as well as bacteria of related genera, are present in most if not all archaeological remains. Our results emphasise the complications inherent in the biomolecular study of archaeological human tuberculosis. The specificity of any polymerase chain reaction directed at the MTBC cannot be assumed and, to confirm that an amplification is authentic, a sequencing strategy must be applied that allows characterisation of the PCR product. Any variations from the reference MTBC sequence must then be checked against sequence data for MOTT and other species to ensure that the product does actually derive from MTBC. Our results also illustrate the challenges faced when assembling MTBC genome sequences from ancient DNA samples, as misidentification of MOTT sequence reads as MTBC would lead to errors in the assembly. Identifying such errors would be particularly difficult, if not impossible, if the MOTT DNA content is greater than that of the authentic MTBC. The difficulty in identifying and excluding MOTT sequences is exacerbated by the fact that many MOTT are still uncharacterized and hence their sequence features are unknown.
Highlights
Tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC) of bacteria
We applied a genus-specific PCR directed at the heat shock protein gene hsp65 (Khan and Yadav, 2004) to ten samples, cloned the PCR products, and sequenced 2e6 clones per sample in order to identify the mycobacterial species that were present
A maximum likelihood tree constructed from the clone sequences and the equivalent hsp65 regions of 168 mycobacterial species displayed the expected division of Mycobacterium into slow and rapid growers, with the exception of a few species which did not fall into their respective clusters (Fig. 1)
Summary
Tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC) of bacteria. More non-specific bone changes have been identified as potentially related to TB in archaeological skeletons, such as granular impressions and new bone formation on the endocranial surface of the skull, new bone formation on the visceral surfaces of the ribs, hypertrophic pulmonary osteoarthropathy and dactylitis (see Roberts and Buikstra, 2003:99e109 for a summary). These non-specific lesions can be recognized in archaeological skeletons, and not specific for TB provide indications of the possible prevalence of the disease in past societies
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