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Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology
Thi-Nguyen-Ny Tran, Gérard Aboudharam, Didier Raoult, and Michel Drancourt
Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), Faculté de Médecine, Université de la Méditerranée, Marseille, France
BioTechniques, Vol. 50, No. 6, June 2011, pp. 370–380
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Identifying the causes of past epidemics depends on the specific detection of pathogens in buried individuals; this field of research is known as paleomicrobiology, an emerging field that has benefited from technological advances in microbiology. For almost 15 years, the detection, identification, and characterization of microbes in ancient environmental and human specimens emerged on the basis of ancient DNA (aDNA) analyses. aDNA limitations due to potential contamination by modern DNA and altered aDNA led to the development of alternative methods for the detection and characterization of nonnucleotidic biomolecules, including mycolic acids (of ancient mycobacteria) and proteins. Accordingly, immunohistochemistry, immunochromatography, and enzyme-linked immunosorbent assay techniques have been developed for the specific detection of microbes from ancient human and environmental specimens. Protein analysis by mass spectrometry, a standard for ancient animal identification, has also recently emerged as a technique for ancient mycobacteria detection, while immuno-PCR is yet another promising technique. As with aDNA, strict protocols must be enforced to ensure authenticity of the data. Here we review the analysis of nonnucleotidic biomolecules from ancient microbes and the ability of these analyses to complement aDNA analyses, which opens new opportunities for identification of ancient microbes as well as new avenues to potentially resolve controversies regarding the cause of some historical pandemics and study the coevolution of microbes and hosts.

Paleomicrobiology, the identification of microbes in ancient environmental and human remains, is an emerging field of research that has benefited from technological advances in microbiology. The majority of past studies have relied molecular biology techniques to detect and analyze microbial ancient DNA (aDNA) (1). This aDNA-based approach is prone to contamination of the ancient material by environmental DNA, including aDNA that was previously PCR-amplified in the laboratory (2), leading to false-positive results. Moreover, chemical modifications and fragmentation during the natural decaying of DNA, along with the presence of poorly characterized PCR inhibitors in ancient specimens (3), may further limit the aDNA-based identification of micro-organisms in some ancient specimens (4). A theoretical limit of the ratio of the D- and L-enantiomers (D/L) of aspartic acid of >0.08 has been calculated, beyond which aDNA could be nondetectable (5). However, experimental study did not confirm this hypothesis, and the ratio cannot be used as a useful marker for bones and teeth (6). For example, it was shown that Treponema aDNA was not preserved in human bone specimens dating back to the period between the 9th century and the 19th century (7). The extent of DNA mycobacterial in ancient bones has also been challenged by the observation of negative PCR-based detection of Treponema pallidum and Mycobacterium tuberculosis in the bones of 18th–19th century individuals suspected of syphilis and tuberculosis, respectively (8). For these reasons, the interpretation of data derived from some initial aDNA studies has been questioned (9). Strict rules governing the experimental processes and the interpretation of data need to be enforced to ensure the authenticity of the data (1).

After findings from a seminal study suggested that ancient proteins retain some integrity and antigenic specificity (10), a demonstration of detectable proteins in insects preserved in amber (5,11) paved the way toward the analysis of nonnucleotidic ancient biomolecules (12). Although it has been proposed that diagenesis may alter the antigenicity of ancient proteins (13), paleo-pathologists have used two approaches to demonstrate the presence of microbial proteins and immunoglobulins in archaeological tissues (14), namely, immunological methods that detect antigenic epitopes of selected microbial proteins and analytical methods that identify protein sequences.

Accordingly, the immunodetection of microbial antigens was used as early as the 1980s in paleomicrobiology (15,16). These approaches are part of the framework that extensively demonstrates how ancient proteins, including plant and animal proteins, can persist across geological time (17-22). Several studies have further indicated that proteins may be more resistant than aDNA to taphonomic decay (12,19,23), as demonstrated by the higher ratio of detection of the Yersinia pestis protein F1 than of Y. pestis aDNA fragments in some ancient plague burial sites (24).

These observations led to new perspectives on the detection and characterization of microbial nonnucleotidic biomolecules in ancient remains. Here we review the methods and initial results of the detection of such nonnucleotidic microbial biomolecules in ancient remains.

Literature review methods

The papers analyzed in this review were obtained from the PubMed and Medline databases and from the database of the Journal of Archaeological Science (journal homepage: and the database of the American Journal of Physical Anthropology [journal homepage:] using the following combined keywords: ancient, past, virus, parasite, bacteria, infection, pathogen, proteome, proteomics, DNA contamination, paleomicrobiology, quantification protein, immunodetection, immunohistochemistry, immunological method, late serology, mass spectrometry, MALDI-TOF, mycolic acid, and dental pulp. References from a previous review (1) and references from selected papers were also reviewed. The selected papers were evaluated for strength and quality of evidence based on a previous publication (1) (Box 1 and Table 1). This bibliometry analysis indicated a shift in the methods used in paleomicrobiology from aDNA analysis toward nonnucleotidic biomolecule analyses. Excluding reviews, we found a total of 87 papers involving paleomicrobiology published between January 1986 and November 2010, including 60 papers (69%) based on aDNA analysis, 17 papers (19.5%) based on nonnucleotidic biomolecule analyses, and 10 papers (11.5%) based on both aDNA and nonnucleotidic biomolecule analyses (Figure 1).

Box 1. Criteria used in this review to evaluate the authenticity of the methods.

Collection of papers:

  • Published in English

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