G Academic des sciences / Elsevier, Paris

Anthropology / Anfhropologie

Identification of Mycobacferium DNA

in an Egyptian Pott’s disease of 5 400 years old
Identificdtion dilDN de Mycobacterium dims un mul de POE &-yptien
de 5400 ans

Eric Cru b6zya*, Bertrand Ludes”, Jean-Dominique Povedac, John Claytond, Brigitte Crouau-Royd,
Daniel Montagnone

a Fkderation d’anthropologie, UMR 150 du CNRS, universite Toulouse-Ill, 39, allee Jules-Guesdes, 31000 Toulouse, France

b lnstitut de medecine Iegale, 11, rue Humann, 67085 Strasbourg cedex, France
’ Centre de biologie medicale specialisee, lnstitut Pasteur, 28, rue du Docteur-Roux, 75724 Paris cedex 15, France
d CNRS, CIGH UPR 8291, CHU Purpan, 31300 Toulouse, France
e lnstitut d’embryologie, 1 1, rue Humann, 67085 Strasbourg cedex, France

(Received 27 March 1997; accepted after revision 12 October 1998)

Abstract - The antiquity of tuberculosis in the Old World is controversial because the
morphology of the lesion in skeletal remains is non-specific. We report the recovery of
a DNA fragment from a 5 400-year-old Predynastic Egyptian skeleton that exhibits a
kyphotic, ‘hunchback’ spinal deformity consistent with Pott’s disease and suggestive of
tuberculous vertebral involvement. The recovered DNA fragment was sequenced and
is consistent with an original Mycobacterium sequence. We cannot prove that it is
M. tuberculosis, M. bovis or an ancient mycobacteria resembling the two current forms
because the observed modifications in the sequence could be attributed to the antiquity
of Mycobacterium and/or to the effects of Taq polymerase. This provides the most
specific evidence for the antiquity of human Mycobacterium disease in the world.
(0 Academic des sciences / Elsevier, Paris.)

ancient DNA / paleopathology / tuberculosis / anthropology

Resume - L’anciennete de la tuberculose dans le Vieux Monde est controversee car la morphologie
des lesions osseuses est non specifique. Nous presentons la dkouverte de fragments d’ADN prove-
nant d’un squelette egyptien predynastique date de 5400 ans dont la colonne vertebrale presente
des lesions et des deformations hocatrices dun ma1 de Pott et de tuberculose osseuse. Le sequen-
Gage des fragments d’ADN est compatible avec celle d’une sequence de Mycobacterium. Nous ne
pouvons pas savoir si c’est M. tuberculosis, M. bovis ou une ancienne mycobacterie qui leur ressem-
blerait car les modifications de la sequence pourraient tout aussi bien etre attribuees a l’anciennete
de la mycobacterie etlou aux effets de la Taq polymhase. Cela apporte la preuve formelle de
l’anciennete des atteintes osseuses par Mycobacterium dans le monde. (0 Academic des sciences /
Elsevier, Paris.)

ADN an&n I p&opatbologie I tuberculose / anthropologic

Note communicated by Yves Coppens

*Correspondence and reprints
E-mail: crubezyeric@wanadoo.fr

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321,941-951
6. Crubky et al.

Version abrCgCe grfce au kit Dye Deoxy Terminator d’Applied Biosystem@ et

1. Introduction
Alors que la tuberculose est restee d’actualite, ses origines
font toujours I’objet de discussions car aucune l&ion osseuse
n’esr pathognomonique de la maladie, Toutefois, son dia-
gnostic par amplification de fragments de 1’ADN de la myco-
bacterie extraits des l&ions osseuses esr possible. Nous
prCsentons I’amplification et le sequenqage de 1’ADN d’une
mycobacr&ie extrait d’un ma1 de Pott - tuberculose osseuse
vertebrale d’un squelette d’enfant egyptien, d’epoque predy-
nastique (Nagada IId2, environ 3400 ans av. J.-C.).
2. Mat&iel et mCthodes
au sequenceur Applied Biosystem 373A selon les recomman-
dations du fabricant. Pour tviter tout risque de contamina-
tion par de I’ADN moderne, de multiples p&cautions ont et6
prises et des contrales negatifs (sans ADN) ont eW realists B
chaque &tape. Les sequences obrenues furent comparees g cel-
les des souches de rt!f&ence de M. tuberculosis H37Rv,
M. bovis var. BCG et celles du gene de 65 kD disponible pour
d’autres mycobact&ies ainsi qu’8 celles des plus proches, chez
les champignons, les batteries et les hommes (HSP 70).
L’idenrite des sequences proteiques fut aussi recherchee.
L’ensemble des arbres phylog&&iques racines possibles a iti
examine et, pour chacun, la sequence ancestrale, qui mini-
mise le nombre de mutations et la variance du nombre de
mutation, a et6 dCfinie.

2.1. Extraction de 1’ADN 3. Rkltats

Au total, 1,45 g de cBte et 2,5 g de vertPbre provenant du Au total, 50 ng d’ADN ont et& extraits du fragment de c&e
mal de Pott furent abras& en surface puis Ccrases en petits et 10 ng de celui de la vertkbre. L’klectrophorPse des ampli-
fragments (0,2-0,3 cm3) et pilts dans un mortier en utilisant ficats rtalises avec les amorces TBlITB2, pour le gtne de
de l’azote liquide comme solution de refroidissement. La l’antigtne 65 kD des mycobact&ies, montre des fragments de
poudre d’os a Ctk peste sur un papier aluminium et placCe la longueur attendue (343 bp) mais le signal apres hybrida-
dans un tube sttrile etiquete de 50 mL en polycarbonate avec tion avec la sonde TBlO est faible. Sur les neuf PCR nichees
un bouchon visse. Les tchantillons furent incubts dans rPali&es B partir de la msme PCR avec les amorces internes
40 mL de tampon (100 mM Tris-HCl, pH 7,6, 5 mM TB28/TB29,4 (no 7,9, 10, 17) prCsentent un signal B I’&?-
EDTA, 2 % SDS, 0,3 M acetate de sodium, 800 pg pro&- trophorkse (exemple 2c de lajgure 2) de la longueur atten-
nase K/mL ) g 42 “C durant 21 h. AprPs dialyse au Tris- due (133 bp) mais l’hybridation avec la sonde TBlO n’a
EDTA (0,l M, pH 8) durant 29 h, les echantillons furent don& aucun rCsultat. L’ADN amplifie fut s&quenc& Le frag-
centrifuges (3 000 tours/min pendant 10 min) puis precipi- ment de la PCR no 7 est celui qui ressemble le plus aux
t& dans l’bthanol absolu a -20 “C pendant 12 h. Le pr&ipitC sCquences Mycobacterium de refkrence, pour la longueur
fut remis en solution dans l’eau distillee et centrifugt (I33 bp), comme pour le nombre de transitions et de trans-
g 13 000 tours/min durant 45 min, purifie dans une solution versions Les arbres racinis prCsentant le nombre minimum
organique (phenol/ chloroforme/ isoamyl alcool, 25/24: 1, de mutations (73) et une variance faible (1,2) moncrent que
v/v) puis precipite avec 0,l vol de 3 M d’ac&ate de sodium B I’ADN provenant de la PCR no 7 se regroupe avec les autres
pH 8 et 3 vol d’ethanol absolu. Sur 10 % de l’extrait obtenu, sCquences de type mycobacteries, la sbquence H37 &ant tou-
une Clectrophorbe fut rtalisie dans un gel contenant 1 % jours plus tloignte. Les arbres suivants presentent essentiel-
SeaKem et des fragments d’ADN de tailles diffbrentes qui ser- lement un changemenr au niveau du regroupement BCG,
vaienc de marqueurs et du bromide d’tthidium afin de pou- 65K et H37RV.
voir visualiser 1’ADN sous U.V. Seul I’ADN extrait de la vertebre a permit une amplifica-
Deux sCquences furent amplifiees : a) celle codant pour le tion detectable sur gel avec les amorces ISTBZ/ISTB-/. 11
gene de l’antigene 65 kD des mycobacteries ; b) l’&ment s’hybride avec la sonde IS2 mais le signal est plus petit que ce
d’insertion IS6110, dont lo-15 copies existent chez qui etait attendu. La PCR rCalis&e sur 1’ADN extrait de la ver-
M. tuberculosis et I-4 copies chez M. bovis. Les amorces tebre avec les amorces IS 1 /IS2 et la PCR nichee utilisant J/K
TBl/TB2 furent utilisCes pour l’amplification du gene et les amorces ISI/IS2 a fournit un fragment de la longueur
65 kD. Celle-ci fut suivie d’une hybridation avec une sonde attendue, mais il ne s’est pas hybrid6 avec la sonde BKl et son
interne oligonu&otide marquee au gamma- 32P. Une ampli- sCquen$age tchoua.
fication nichee fut aussi rtalisee avec comme amorces exter-
nes TBl/TB2, comme amorces internes TB28/TB29, et avec 4. Discussion
TBlO comme sonde interne Pour l’amplification de l’&-
merit d’insertion IS 6110, trois types d’amorces et de sondes Le fragment provenant de la PCR no 7 a une sequence pro-
furent utilists : a) les amorces ISTB2/ISTB7 suivit d’une the de celle attendue. Les diffirences et nommment les tran-
hybridation avec une sonde oligonuclCotide marquee IS2, b) sitions entre AT er GC pourraient Ctre reliCes a) aux effets de
les amorces ISl/IS2 suivit d’une hybridation avec une sonde la Taq polym&ase lors de son utilisation avec de 1’ADN
oligonucleotide BKl marquee au gamma-32P et c) une PCR ancien, done degrade mais il faudrait alors cloner, b) etlou B
nichee dans un seul tube en utilisant comme amorces exter- l’anciennete de la mycobactCrie qui pourrait avoir CtC une
nes J et K comme amorces internes ISI /IS2 et comme sonde espece intermediaire en M bovis et Ad. tuberculosis. 11 en est
interne pour l’hybridation BKl. Le sequenqage fut r&&se de m?me pour l’impossibilite d’hybrider correclement la

942 C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321,941-951
 5 400-year-old Mycobacferium DNA

sonde TBlO aux fragments amplifies. MCme en augmentant et la fin de sa maladie. Un autre cas de ma1 de Pott, cicatrise
le nombre de cycles de I’amplification, lors de la PCR nichee celui la, a et6 signal6 dans la necropole. Precedemment, un
avec les amorces TB28/TB29, le nombre de fragments ampli- bacille tuberculeux avait et-6 reconnu histologiquement dans
fies visibles sur le gel de l’electrophoreseest plus important un poumon de momie tgyptienne da&e entre 1000 B.C. et
mais aucune hybridation n’est obtenue. 11 faut signaler AD 400 et la sequence codant pour le gene de l’antigirne
qu’apres amplification avec les amorces TBl/TB2 la sonde 65 kD a et& amplifiee dans I’ADN extrait d’un autre poumon
oligonucltotide TBlO utilisee dans l’hybridation peut diffe- de momie date de 1550 a 1080 BC. Le cas decrit ici est done
rencier des molecules d’ADN de mycobacteries d’especes t&s le plus vieux cas de tuberculose formellement identifit, il est
proches done les sequences ne different que de quelques le resultat, soit d’une infection a M. bovis ou M. tuberculosis,
nuclCotides. soit d’une infection par une mycobacterie plus proche de
L’aspect anatomique des pieces est tout B fait similaire a M. tuberculosis que de M. bovis. Cette dtcouverte est en
celui des tuberculoses modernes d’avant l’ere des antibioti- accord avec les differences genetiques entre les mycobacteries,
ques et il signe mCme la presence d’un ou deux abets para- qui suggerent que la tuberculose aurait pu apparaitre il y a
vertebraux et une survie assez longue du sujet entre le debut plus de 15 000 ans.

1. Introduction showing classical morphological indications suggestive of

tuberculous involvement. The precise identification of
Tuberculosis not only remains a major cause of mor- tuberculosis in the skeletal record is hampered by the
ambiguous nature of the morphological diagnostic crite-
bidity and mortality in developing countries, but recent
years have witnessed a resurgence in the incidence of ria available in the archaeological record. For example,
tuberculosis in the United States and Europe with recent the spinal deformity called ‘Pott’s disease’, secondary to
tuberculous vertebral involvement, which has typically
strains highly resistant to drug treatment [l]. It is a chronic
been used to identify the most ancient cases of skeletal
infectious disease caused by Mycobacterium tuberculosis
tuberculosis in the Old World (circa 4 500 BC) [81 can be
of the human or bovine type, with visceral and sometimes
caused by a compression fracture, brucellosis, osteomy-
skeletal lesions. Tuberculosis is associated with high
elitis or by a variety of fungi, such as Coccidioides immitis
morbidity and mortality, particularly among populations
[3, 91. Causal can only be ascertained by direct evidence
suffering from malnutrition and immunosuppression,
of the presence of Mycobacterium.
characteristics of a large percentage of the third world. It is
a leading cause of death due to an infectious agent world-
wide and estimates suggest that as much as one third of
the world’s population may be infected [I] 2. Materials and methods
The origin of tuberculosis as a human disease has been
2.1. Skeleton remains
the topic of speculation [2]. Reliable reconstruction of
infectious disease patterns in antiquity depends on the In the present study, we had the opportunity to examine
recovery and identification of the infectious agent directly the nearly complete skeleton of a 12-14-year-old child
from human remains 131. This has only recently been showing classical evidence of Pott’s disease (figure 7).
made possible, modern methods ]4] based on DNA This individual was exhumed from a tomb (T 35) in the
amplification by polymerase chain reaction [5] permitting Predynastic necropolis of Ada’ima, located in the district
the effective diagnosis of tuberculosis directly from of Esna (Upper-Egypt), where more than 200 graves have
archaeological tissues [3]. While DNA normally degrades been excavated since 1990 [lo]. Associated with the skel-
rapidly after death [6] owing to the action of DNAses eton was pottery typical of Nagada IID2, dating between
released at this time, the cell walls of mycobacteria pro- 3400 and 3300 BC. This period was a time of increasing
tect the bacterial DNA until these enzymes become inac- urbanization, as the settlement centres of Upper-Egypt,
tivated. Salo et al. [3] have recently been successful in such as Ada’ima, expanded into large towns or villages
extracting, amplifying and sequencing ancient DNA of with considerable use of mud-brick architecture. Three
Mycobacterium tuberculosis from the lung tissue of a asymmetrically collapsed vertebral bodies of the eighth to
spontaneously mummified pre-Columbian Peruvian body tenth thoracic vertebrae and the proximal part of the
about 1 000 years old. Using identical primers, Spigel- eighth left rib with periosteal new bone formation were
man and Lemma [7] were able to extract DNA from the focus of the study.
apparently tuberculosis-infected bones from medieval
Europe, Turkey and pre-European-contact Borneo, but 2.2. DNA Extraction
were unable to sequence these fragments. Samples of 1.45 g of rib and 2.5 g of the vertebral frag-
We had the opportunity to attempt the recovery of DNA ment containing the lesion were cleaned with fine emery
from a 5 400-year-old skeleton of Predynastic Egypt paper to remove as much material as possible from the

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 943

1998.321,941-951
E. Crubky et al

samples were centrifuged (3 000 rpm/lO min) and pre-

cipitated in absolute ethanol at -20 “C for 12 h. The pre-
cipitate was resuspended in distilled water and
centrifuged at 13 000 rpm for 45 min, and then purified
with an organic procedure (phenol/chloroform/isoamyl
alcohol, 25:24: 1, v/v) followed by precipitation with 0.1
volume of 3 M sodium acetate pH 8 and 3 volumes of
absolute ethanol. Ten per cent of the extracts were elec-
trophoresed through 1 % SeaKem gel with DNA size
marker gels and stained with ethidium bromide to visu-
alize the DNA under UV light. In addition to fragments of
expected size, cf. infra, two other observations were nota-
ble: a) infrequently, a blue fluorescence was noted in
ethidium bromide gels examined under UV transillumi-
nation, thought to be caused by soil-derived humic acids
in the bone DNA extracts [l 11; b) light weight molecular
DNA (I 14 bp) is observed on the electrophoresis gels of
bone extracts. These have been attributed to micro-organ-
isms [12] and the specificity of the sequence was per-
formed with the sequencing.

2.3. PCR

The DNA extract was then subjected to PCR. Two

mycobacterial sequences were targeted: i) the sequence
coding for the mycobacterial 65 kD antigen [13]
(figure 2), present in a single copy in the mycobacterial
genome; ii) the insertion element IS61 10 [14], present as
1 O-l 5 copies in M. tuberculosis and 1-4 copies per cell
in M. bovis [I 51 (figure 3).
Figure 1. The vertebral lesion is centred between T8 (at the top of the
For the amplification of the 65 kD protein gene, T&l/
photograph) and Tl 0, with fusion at their apophyses.
Severe kyphosis and scoliosis is evident along the vertebral column. TB2 primers [16] were used, followed by hybridization
Despite these lesions, the neural canal is not affected. The eighth to with an internal gamma-32P labelled oligonucleotide
tenth thoracic vertebrae exhibit significant destruction. The body of probe TBlO. In addition, nested amplification was also
T8 is collapsed with erosion of the pedicles. The superior plateau is used with TBl/TB2 as external primers and TB28/TB29 as
intact and has a normal vertical anatomical orientation. There is a internal primers, with TBlO as an internal probe [17]
complete loss of the body ofT9 with only the left pedicle and lamina
(figure 4).
remaining intact. Finally, there is massive destruction of the body of
TlO with assymetric collapse of the anterior part, more pronounced For the amplification of the IS61 10 insertion element,
on the left side than on the right. Evidence of lesions are found three sets of primers and probes were used: a) ISTB2/
throughout the vertebrae from T6 to L4. These include an anterior ISTB7 primers followed by hybridization with a peroxi-
cavitation of T7, an extensive destruction without collapse, of the dase labelled IS2 oligonucleotide probe [4], b) ISl/lS2
body of Tl 1 and T12. In T6 and Ll, periosteal new bone formation
primers followed by hybridization with a gamma-32P
covers part of the anterior and lateral faces of the bodies including
labelled oligonucleotide BKl probe 1181, and c) nested
the proximal part of the twelth right rib. Finally, a periosteal reaction
on the proximal inferior part of the twelth right rib is evident. PCR in a single tube [I 91 using J and K as external primers
[201, ISl/lS2 as internal primers and BKl as an internal
probe for hybridization.

outside surface of the bones to minimize contamination. 2.4. Sequencing

After cleaning, the specimens were pulverized into small
PCR products were bidirectionally sequenced using an
fragments (0.2-0.3 cm3) and ground in a mortar using liq-
automatic sequencer (Applied Biosystem 3734) and Taq
uid nitrogen as a freezing solution to avoid melting any
Dye Deoxy TM terminator cycle sequencing and protocols
DNA that might be present. The bone powder was
from Perkin Elmer. The primers used for the sequencing
weighed on disposable aluminium foil and placed in a
are those used for the amplification reaction. Templates
labelled, sterile, 50 mL polycarbonate tube with a screw-
were prepared from gel purified fragments and were sin-
cap. The samples were incubated in 40 mL of lysis buffer
gle stranded. The nested PCR products as well as the cor-
(100 mM Tris-HCl, pH 7.6, 5 mM EDTA, 2 % SDS, 0.3 M
responding sequences from &CC and H37 reference
sodium acetate, 800 pg proteinase K/mL ) at 42 “C for 21
strains were directly sequenced using the two primers
h. After dialysis with Tris-EDTA (0.1 M, pH: 8) for 29 h, the
TB28 and TB29 separately (each sequencing was per-

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998. 321,941-951
 5 400.year-old Mycobacterium DNA

2a 2b 2c 2d
1
1 1 2
7
2 3
; 4
4 3 5
5 6
4 4
6 7
7 5
5 8
8
9
9 6 6
10
10
7 11 7
11
12
12 8 8
13
13
14 9 9
15
10 10

11 11

12 12

13 13

14

15
Figure 2. Amplification of the gene encoding the 65 kD antigen
PCR was performed in a 100 pL reaction mixture containing 100 pg/mL bovine serum albumine per pL, 10 mM Tris-HCI (pH 8.31, 1.5 mM M&I,,
1 pL:mL gelatin, 0.250 nM of TBl/TBZ primers, 2.5 units of Taq polymerase (Perkin-Elmer) and 200 PM of each dNTP.
The 40 cycle programme was performed at 95 “C for 120 s., 60 “C for 120 s., 72 “C for 120 s on a Perkin-Elmer DNA Thermal cycler. For nested
PCR 12~ and 2dl, internal primers were used to re-amplify 1 pL of the first stage reaction with the same programme conditions. Fragments were
visualized after electrophoresis (2a and 2c) and hybridization (2b and 2d) with the “P labelled T&l 0 probe.
2a (Fragments after electrophoresis) and 2b (fragment aiter hybridization for 7 h). Lane 4: vertebra fragment DNA, lane 1: positive control M. bovis
BCG DNA, lanes 2, 3, 5, 6, 7: 8, 9, 10, 11, 12, 13, 14, 15: negative PCR controls.
2c (Nested PCR, fragment after electrophoresis) and 2d (nested PCR, fragment after hybridization). The high concentrations of primer-dimers can
be seen (according to the 200 uM of each dNTPi. Length fragment on the gel is 133 bp. Lanes 3, 4, 9 and 10: vertebra iragment DNA (lane 3:
PCR no. 10; lane 4: PCR no. 9; lane 9: PCR no. 8; lane 10: PCR no. 7), lanes 1, 2, 6, 7, 12 and 13: positive controls, M. bovis BCC or ,%1. tuber-
culosis H37Rv DNA, lanes 5, 8 and 1 1: nested PCR from negative controls.

formed twice). The obtained sequences were compared 2.5. Phylogenetic analyses
using the BESTFIT and PILEUP programs (KG Software
Package) and aligned with the region nt. 650-786 from To further investigate the relationship of the studied
the sequence register at the bank EMBL (Ml 5467), cor- DNA sequence to contemporary Mycobacterium DNA
responding to a portion of cDNA of the 65 kD antigen variations, phylogenetic tree reconstructions were per-
from M. tuberculosis (figure 4). We took care to avoid formed {figure 5). All possible rooted trees were examined
contamination of the lanes with the positive controls and, for each, the ancestral sequence was defined that

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321.941-951
i. Crub&y et al

3a 3b 3c 3d

1 1 1
1
2 2 2 2
3 3
3 3
4 4
5 4 5 4
6 6
5 5
7 7
8 6 8 6
9 9
7 7
10 10
11 8 11 8
12 12
9
13 9 13
14 14 10
10
15 15
11
11
II 12
12
13
13
14
14
15
15

Figure 3. Amplification of the insertion element IS61 10.

PCR (3a and 3b, DNA of the vertebra) was performed with ISTB2/lSTB7 primers. For nested PCR (3c and 3d! J/K were used as external primers
and ISl/lS2 as internal primers and reactions were conducted in a single tube with a drop of mineral oil separating the two reaction mixtures [18].
PCR programmes were as previously described. Fragments were visualized after electrophoresis (3a and 3~) and hybridization with the peroxidase
labelled IS2 probe (3b) or the 32P labelled BKl probe (3d).
3a and 3b. Lanes 2 and 5: positive controls: M. bovis BCC DNA, lanes 10 and 11: vertebra fragment DNA, lanes 1, 3, 4, 6, 7, 8, 9, 12, 13, 14,
15: negative controls.
3c and 3d. Lane 2: positive control: M. bovis BCG DNA, lanes 9 and 10: vertebra fragment DKA (5 and 10 ng), other lanes: negative controls.

minimized both the number of mutational events and the treated (Millipore) water, wrapped in aluminium foil and
variance of the number of mutations between the ances- baked at 180 “C for 12 h; d) sterile disposable labware
tral sequence and the observed sequences. The programs was used whenever possible, including positive displace-
to perform this analysis were written in C++. ment pipettes; e) various components of the extraction
buffers were autoclaved and blank extractions and rea-
2.6. General laboratory procedures gent blanks (negative controls) were run alongside the tis-
sue extracts. The PCR was handled in two separate
Several precautions were taken to guard against con- stations: one for the set up, one for the PCR itself,
tamination from contemporary DNA: a) the sampling dis- equipped with a hood for the work-up of the reactions.
sections were carried out in a room with no previous Each station was equipped with its own set of essential
exposure to mycobacteria; b) the extractions were per- equipment and these were not interchanged. Gloves were
formed in another separate room with similar history; c) worn at all times and always changed before going from
all reusable labware (mortar and pestle) were soaked one area to the next. Contemporary M. tuberculosis
overnight in 0.6 M HCI, thoroughly rinsed with Milli Q H37Rv and M. bovis var. BCG were prepared in a sepa-

C. R Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321,941-951
 5 400.year-old Mycobacterium DNA

Figure 4. Definition of the sequences studied on the sequence of cDNA of the 65 kD antigen of M. tuberculosis.
The amplified fragments BCG, H37, 7, 9, 10 and 17, obtained with the primers TB28 et TB29 have been sequenced directly using the two primers
separately (each sequencing was effected in double in different series). The sequences were compared using the programs BESTFIT and PILEUC
(GCC Software Package! and aligned with the zone from the registered sequence of the bank EMBL (Ml 54671, corresponding to a portion of cDNA
of the antigen of 65K of Mycobacterium tuberculosis.

rate laboratory and station, where ancient DNA had not and 10 ng DNA from the vertebra. We will present the
been amplified, and diluted to 100 fg/pL before used in results of the two mycobacterial sequences which were
the PCR. targeted.
- Amplification of the 65 kD antigen region (figure 2)
was performed with external primers TBl/TB2. For both
3. Results bone samples (vertebra and rib), gel electrophoresis (fig-
ure 2a, example of the rib fragment) showed expected
As estimated from gels, the extraction procedure fragment lengths (343 bp). These amplifications were per-
resulted in 60 ng of DNA: 50 ng DNA from the rib sample formed twice and gave the same results.

32
H37

5 7

2
BCG

27
2
65K

L 1
H37RV

Figure 5. Phylogenetic tree minimizing both the number of mutational events (73) and the variance of the number of mutations (1.2) between the
ancestral sequence and the observed sequences.
- H37RV: M. tuberculosis H37Rv.
- 65K: reference sequence from EMBL: MP 15989 (65 kDa antigen - Mycobacterium paratuberculosis) from nucleotid 673.
- BCG: M. bovis BCG.
- 7: nested PCR no. 7.
- H37: mycobacterial sequence close to human heat shock protein.

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

19X321,941-951
i. Crubhy et al.

Table I. The DNA sequences.

ANCES. CC AAC GAG GTC TAG TCC AAG GAGGAG ATC CCC CCC CCC GAC CCC ATT TCC GCGGGT CAC TAG KG ACC GGCGAC
65 K CC AAG GAG GTC GAG ACC AAG GAG CAG ATT GCCGCC ACC GCA CCC ATT TCG GCC GGT GAC CAG TCC ATC GCT GAC
H37RV AAG GAG GTC GAG ACC AAG GAG CAG ATT CCC CCC ACC CGA CCC ATT TCG CCC GGT GAC CAG TCC ATC GGT GAC
BCG CC TAG GAG GTC GAG ACC AAG GAG CAG ATT CCC CCC ACC CAA GCG ATT TCG GCG GGT GAC CAG TCC ATC GGT CAC
7 C CCC CCC ACC GCA CCC ATC TCC CCC GTA GAC CAG TCG ATC GCT GAC
9 TC GGGGGG GTC GAG TCC CGA CGG GAG ATC GTC CCC ACC CCC TCC ATC TCC CCC CCC CAT CCC CCC ATC CCC GAC
10 C ACG GAG CTC GAG ACC GAGGGG GAG ATC GCT GGC ACC GCT TCC ATC TCC CCC GCC CAT CCC GGG ATC CAT CAC
17 CC CAC GAC GTC GAG TCC AAG GAG CAG ATC GCT CGC ACC CCC TCC ATC TCC GGCGGC CAT CCC GAG ATC CAT GAG
H37 CC GGG CTG ATG TAC TCC AAG GGG GAC ATC GCC GCG CAC GAC ACC AAG TCC GCG GGT CAG TAC GGG ACG CCC GGC

ANCES. CTT ATC TCT GTG TCG CTG CAC AAG CTC GTC AAC GTG GTL: !ZIC BI(1 ACf ATC !ZLCU.Ci II;
65 K CTG ATC CCC GAG GCG ATG GAC AAG GTG GGC AAC GAG GGCGIC: 81% AX !Z.lImGBG II:
H37RV CTG ATC CCC GAG CCC ATG GAC AAG GTG CCC AAC GAG GGT c;Ic && &xX GI(: !Z,!&GBG Ic
BCG CTG ATC CCC GAG CCC ATG GAC AAG GTG GGC AAC GAG GGC GIc ATC A= GII: GAG GAG Iz
7 CTG ATC CCC GAG GCG ATG GAC GAG GTC GGC AAC GAG CCC fJ.C AIC 81l; GIG GAG&&Z III
9 CTC ATC GCC GAT TCC ATA GAG AAC GTG GGCAAG GAGGGCCJ..C BIG: ACC GICf,AfC& IC
10 CTC ATC CCC GAG TCG ATG AAG GAG GTC GGC AAG GAG CC&&ICC ATC ACC GICGA.Cf&Z IC
17 CTC ATT TCC GAA GCA CCT GAC AAG GTG GGCAAG GAGGGTm 81% &X!X.C&UXI&Z II;
H37 CTT TTC TTT TTC TCT CAG CAG AAG CTC ATC ATC GTC GTC GTI: 9u; &X Au; &IC &I& I&

65 K is a refeence sequence from EMBL: MP 15989 (65 kDa antigen - Mycobacterium paratubercu/osis - nucleotid 673. The Ancest sequence cor-
responds to the predicted ancestal sequence from phylogenetic analysis.

- Hybridization with internal gamma-32P labelled oli- labelled TBI 0 gave no result (figure 24. Then the products
gonucleotide probe TBlO gave a very weak signal only on of amplification were sequenced. These sequences
the amplified DNA from the vertebra fragment (figure 2b, (table I) were compared to the sequences of M. tuberculo-
80 “C for 7 h). No hybridization was observed on the sis H37Rv, M. bovk var. BCC and 65 kD gene sequences
amplified DNA from the rib sample. The DNA extracted, available for other mycobacteria from EMBL (table II). They
not used for the hybridization, was divided into nine aliq- were also compared to the most closely related sequences
uots in order to be submitted to PCR. in fungi, bacteria and humans (HSP 70) and to the proteic
- Four (nos 7, 9, 10, 17) of the nine nested PCR (realized sequence of BCG, Mttcwpa and H37RV (tables 111and Iv).
from a PCR with external primers TBlTTB2) with the prim- The amplified DNA of the nested PCR no. 7 was found to
ers TB28/TB29 gave a positive signal (figure 2c) visualized be more similar than those obtained from PCR nos 9, 10
after electrophoresis. The hybridization with the 32P and 17 to each of the comparative sequences.

Table II. Percentage of identity between the four fragments from the nested PCR nos 7, 9, 10 and 17 of the 65 kD gene target and the corresponding
sequences of M. tuberculosis H37Rv, M. bovis var. BCG and the 65 kD gene sequence available for other mycobacteria.

Amplified fragment M. bovis var. BCG M. tuberculosid M. gordonae M. kansaii M. xenopi M. segmatis
from PCR no. reference strain H37Rv

7 89.5 92.4 88.6 87.6 90.7 85.5

9 72.2 73.2 74.4 79.2 79.2 76.0
10 75.7 78.5 78.2 80.6 80.6 75.8
17 75.9 78.8 78.6 79.4 79.4 76.9

Table Ill. The proteic sequences.

BCC EVETKEQIAATEAISAGDQSICDIIAEAMDKVGNEGVITVEE
Mttcwpa KEVETKEQIAATAAISAGDQSICDllAEAMDKVGNECVITVEE
H37RV KEVETKEQIAATGAISACDQSICDllAEAMDJVGNECVITVEE
Amplified AATAAISAVDQSIGDIIAEAMDEVGNEGVITVEE
frag. no. 7
Amplified CGVESRREIVATASISAGDPGIGDIIADCIEKVRKAGVITVEE
frag. no. 9
Amplified REVETEGEIAGTASISCADPGIDHllAESMKEVGKEGVITVEE
frag. no. 10
Amplified CDVESKEQIAGTASISGGDPEIDEIISEAPDKVGKEGVITGEE
frag. no. 17

948 C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321,941-951
 5 4OOyear-old Mycobacterium DNA

Table IV. Percentages of identity between the different proteic sequences.

BCG Mttcwpa H37RV Amplified Amplified Amplified Amplified

frag. no. 7 frag. no. 9 frag. no. 10 frag. no. 17

BCC
Mttcwpa 97.6
H37RV 97.6 97.7
Amplified frag. no. 7 91.2 91.2 94.1
Amplified frag. no. 9 64.30 62.8 65.1 67.6
Amplified frag. no. 10 66.70 65.1 62.8 70.6 60.5
Amplified frag. no. 17 71.40 69.8 67.4 64.7 55.8 65.1

- For IS61 10 amplification using ISTB2/1STB7 primers 4. Discussion

on the DNA extracted from the rib fragment, no results
were obtained (only one PCR). DNA extracted from the
vertebra fragments (one PCR) gave a positive result detect- The distribution and morphology of the vertebral
able on gel (figure 3a) and hybridizing with IS2 probe, but lesions are identical to modern skeletons known to have
smaller than expected (figure 3b). One of the nine PCR had spinal tuberculosis (figure I).The massive destruction
performed on the vertebral fragment DNA with ISl/lS2 and collapse of the vertebral bodies, often asymmetric
primers and nested PCR using J/K and ISl/lS2 primers with preferential destruction of the anterior aspect,
yielded an amplified fragment of expected length (figure together with the absence of osteophytic spurring and
3~). These, however, were unable to hybridize to the BKI non-involvement of the spinous processes are character-
probe (figure 3$) and the subsequent sequencing of these istic signs of tuberculosis [21]. The fusion of the vertebrae
fragments failed. at their apophyses between T8 and TIO and a remodelling
of the left inferior articular surface of the apophyses of T7,
3.1. Phylogenetic analysis
indicate long-term disease in the subject. The periosteal
We only discuss (figure 5) the rooted tree with the new bone formation and the lack of massive bony fusion
amplified fragment no. 7 which is the tree where the pre- may indicate that this was a terminal infection. The peri-
dicta1 ancestral sequence could be close to the real ances- osteal new bone formation on T6 and Ll and the lesions
tral sequence. The rooted tree with the four amplified on the eighth left rib and twelfth right rib could indicate
fragments shows that the amplified fragments nos 9, 10 the presence of one or two paravertebral abscesses, which
and 17 are not close to no. 7 or to BCC, 6.5K or to H37RV may have been a source of contact infection. Thus, it is
(on a cluster with H37 but on a different sub-cluster). likely that this individual experienced long-standing
Several rooted trees with the minimum number of muta- mycobacterial disease that was an infectious source and
tions (73) and variance (1.2) were observed. The phylo- eventually led to the death of the individual.
genetic tree displaying the higher maximum likelihood is
shown in figure 5 with the predicted ancestral sequence Differences in the lengths and sequences of the ampli-
in table V. Trees with slightly larger variances or number of fied experimental fragments by comparison with the
mutations varied by changes in the arrangements of the Mycobacterium sequences are likely to be related to the
BCG-65K-H37RV group. The relationships between the significant effects of degradation in ancient DNA samples.
different groups can therefore be considered to be rela- It can be suggested that the DNA amplified in PCR no. 7 is
tively well determined, but not the details within the the least degraded. It can be seen that the number of tran-
groups. sitions and transversions occurring in the experimental

Table V. Number of transitions and transversions occurring in the four amplified fragments from the nested amplification nos 7, 9, 10, 17 of the
65 kD and M. tuberculosis 65 kD antigen gene.

Amplified fragment A <---->T G<---->C A, T-->G, C G, C-->A, T

from the PCR no.

7 1 6 3 1
(0.95 %j (5.70 %) (2.86 %j (0.95 “/o)
9 1 14 (9.77 %)2 8
10.75 %j (1 0.52 %) (6.01 %)
15 9 6
10 (0.7; %j (1 1.36 “1’) (6.82 %I (4.54 %)
17 1 12 10 11
io.75 %) (9.02 %) (7.52 %) (8.27 %)

C. R. Acad. Sci. Paris, Sciences de la vie / Life Sciences

1998.321.941-951
i. Crubky et al.

amplification samples in comparison with the Mycobac- - b) This case is 5 400 years old. At this period Myco-
terium 65 kDa antigen gene are also minimized in this bacterial sequences are supposed to be more close to
amplified DNA from PCR no. 7 (table v). This sequence atypical mycobacteria than to present day M. tuberculosis
from PCR no. 7 appears to have been best preserved, or M. bovis [23]. In fact, the obtained sequence from PCR
yielding a PCR product with the closest approximation to no. 7 is closer to M. tuberculosis than to M. bovis. This
an original Mycobacterium sequence and more than last result is also consistent with the following.
94 % homology with the proteic sequence of H37RV. The a) The speculation that the agent of human tuberculosis
observed modifications can be attributed to the antiquity arose from a very closely related cattle pathogen M. bovis
of the Mycobacterium and/or to the effects of Taq by host specialization [23] several thousand years before
polymerase which propagates transitions, particularly the predynastic case of Ada’ima 121. At this period, the
between AT and GC [3, 221. The inability to correctly evolution of the host specialization could have been
hybridize to TBlO probe of the amplified fragments closer to M. tuberculosis than to M. bovis. Even at the
(figure 2) can also be explained by the same reasons. This present time, in vivo, an important number of species
is even clearer after more cycles of amplification with between M. bovis and M. tuberculosis exist, especially in
nested PCR using TB28rTB29 primers. The amount of Africa [24].
amplified fragment, as seen on electrophoresis gel b) The healed cases of bone tuberculosis (Pott’s disease)
(figure 2), is higher, but no more hybridization is obtained. in this necropolis could prove that one part of the pop-
The high stringency conditions used in the hybridization ulation was already immunized against Mycobacterium
reaction with an oligonucleotide probe such as TBlO can and that the disease already had a long evolution.
differentiate mycobacterial DNA molecules from closely Our extraction and identification of mycobacterial
related species after amplification with TBl/TBZ primers DNA from the bone lesions of this 5 400-year-old Predy-
even though their sequences contain only a few nucle- nastic burial is the oldest evidence at the present time for
otides differences [16]. These differences in sequence Mycobacterium as a human disease. Previously, an acid-
were higher here after amplification of ancient DNA and fast bacilii had been identified histologically from lung tis-
did not allow a correct hybridization. This lack of hybricl- sue of a mummified specimen dated to circa 1 000 BC to
ization of amplified fragment also eliminates the possi- AD 400 [25] and 65 kD antigen has been amplified in the
bility of a laboratory contamination with contemporary DNA extracted from another lung tissue of a mummified
DNA, which would have strongly hybridized with the specimen dated to circa 1 550 to 1 080 BC [261. The tech-
probe used. nological advances in recent years in molecular biology
allow the further investigation of this disease from skeletal
Two hypotheses can be invoked to explain the unusual
remains alone, and the more specific, genetic identifica-
diversity of this DNA sequence of Mycobacteria.
tion of disease agents. Our results are intriguing regarding
- a) The large amount of damage present in ancient the origins of human-hosted tuberculosis: we cannot be
DNA, and the presence of modified nucleotides at ran- sure whether the lesions in the present individual were
dom positions increases the error rate during PCR due to M. tuberculosis, M. bovis or an ancient Mycobac-
because the natural polymerase errors are supplemented terium resembling the present two. It is possible, that if
by misreadings. In order to increase the statistical likeli- more of the original DNA were recoverable, the sequence
hood of overcoming random error in direct sequencing, a similarities could be clarified. Our results agree with
larger number of fragments must be sequenced when recent research in molecular biology 121 that tuberculosis
employing ancient DNA. This last step was not performed is a human disease likely to be not less than 15 000 years
in our experiment. old.

Acknowledgements: This work was supported by the lnstitut frangais d’archbologie orientale. We thank C. Chureau, B. Gicquel, M. Lampl,
8. Midant-Reynes, S. Cole, T. Janin, N. Crimal and S. Vicaire for their generous intellectual and material support of this work. We thank A. Leclerc
for photography.

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