FEMS Microbiology Letters 213 (2002) 141^147

www.fems-microbiology.org

MiniReview

Molecular analysis of ancient microbial infections

Albert R. Zink a , Udo Reischl b , Hans Wolf b , Andreas G. Nerlich a;

a
Division of Paleopathology, Institute of Pathology, Academic Teaching Hospital Mu«nchen-Bogenhausen, Engelschalkingerstrasse 77,
D-81925 Munich, Germany
b
Institute of Medical Microbiology and Hygiene, University of Regensburg, Franz-Josef-StrauM-Allee 11, D-93053 Regensburg, Germany

Received 10 June 2002; received in revised form 10 June 2002; accepted 11 June 2002

First published online 15 July 2002

Abstract

The detection of ancient microbial DNA offers a new approach for the study of infectious diseases, their occurrence, frequency and
host^pathogen interaction in historic times and populations. Moreover, data obtained from skeletal and mummified tissue may represent
an important completion of contemporary phylogenetic analyses of pathogens. In the last few years, a variety of bacterial, protozoal and
viral infections have been detected in ancient tissue samples by amplification and characterization of specific DNA fragments. This holds
particularly true for the identification of the Mycobacterium tuberculosis complex, which seems to be more robust than other microbes due
to its waxy, hydrophobic and lipid-rich cell wall. These observations provided useful information about the occurrence, but also the
frequency of tuberculosis in former populations. Moreover, these studies suggest new evolutionary models and indicate the route of
transmission between human and animals. Until now, other pathogens, such as Mycobacterium leprae, Yersinia pestis, Plasmodium
falciparum and others, have occasionally been identified ^ mostly in single case studies or small sample sizes ^ as well, although much less
information is available on these pathogens in ancient settings. The main reason therefore seems to be the degradation and modification
of ancient DNA by progressive oxidative damage. Furthermore, the constant risk of contamination by recent DNA forces to take time
and cost effective measures and renders the analysis of ancient microbes difficult. Nevertheless, the study of microbial ancient DNA
significantly contributes to the understanding of transmission and spread of infectious diseases, and potentially to the evolution and
phylogenetic pathways of pathogens. 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All
rights reserved.

Keywords : Ancient DNA ; Microbial infection; Tuberculosis ; Host^pathogen interaction

1. Introduction 1990s on a 1000 year old South American mummy [1]

The molecular detection of microbial infections in an-
cient human and animal remains represents an emerging
¢eld of research. This research developed rapidly within
the last few years, from isolated reports on diagnosis of
infectious diseases to much more profound studies on dis-
ease frequencies in ancient populations and evolutionary
aspects of host^pathogen interaction. In the beginning of
this new scienti¢c branch modern molecular methods, like
PCR-based ampli¢cation of speci¢c DNA fragments, were
used to amplify ancient DNA (aDNA) residues in human
remains to provide evidence for a presumed infectious dis-
ease. The ¢rst successful studies were performed in the
* Corresponding author. Tel. : +49 (89) 92 70 23 10;
Fax : +49 (89) 92 70 20 67.
E-mail address : andreas.nerlich@extern.lrz-muenchen.de
(A.G. Nerlich).
0378-1097 / 02 / $22.00 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 1 4 - 5
and an Egyptian mummy from the New Kingdom
(1550^1080)[2], in which Mycobacterium tuberculosis
DNA was detected, leading to the con¢rmation of a tuber-
culosis infection in these mummies. In the following years,
the diagnostic ability of this approach was widened by the
detection of several other microbes including Mycobacte-
rium leprae [3], Yersinia pestis [4], Plasmodium falciparum
[5], Trypanosoma cruzi [6], Treponema pallidum [7], Esche-
richia coli [8] and Corynebacterium spp. [9]. These studies
were mostly based on single ¢ndings or on small series of
selected samples. In more recent studies on the molecular
detection of M. tuberculosis complex DNA, larger series
were analyzed including specimens with non-speci¢c or
even without morphological alterations, suggesting infec-
tion with tuberculosis [10]. Beside the bene¢t of a more
precise diagnosis in ancient material, this approach paved
the way to investigate both the frequency and the distri-
bution of infectious diseases in various ancient popula-

FEMSLE 10566 5-8-02 Cyaan Magenta Geel Zwart

142 A.R. Zink et al. / FEMS Microbiology Letters 213 (2002) 141^147
Fig. 1. Pleural adhesions in an ancient Egypt mummy (1500^500 BC). The torso of this approximately 6^8 year old female mummy presented with ma-
jor connective tissue adhesions extended to the left body wall (arrow), suggesting chronic pleural infection.
tions. This new approach, recently termed ‘molecular pa-
leoepidemiology’, rises above the satisfaction of a purely
historic interest, and o¡ers the possibility to compare
modern data on tuberculosis epidemiology with the situa-
tion prevalent in previous times. In particular, direct in-
sights into the evolution of pathogens and the interaction
with their hosts may be obtained which are crucial to
understand the transmission and spread of particular in-
fectious diseases. Nevertheless, the extensive degradation
and chemical modi¢cation of double stranded DNA in
ancient samples clearly reduces the analytical potential
of this approach and engenders the risk of producing
non-speci¢c or even false positive results. The usually
low amount of authentic DNA in samples of up to several
thousands years of age signi¢cantly increases the suscepti-
bility of the technique to contamination with modern
DNA. Therefore strict precautions to avoid contamination
have to be imposed and there exists a general consensus on
the clear necessity of strictly controlled conditions by all
researchers working with aDNA.
2. Molecular identi¢cation of pathogens
2.1. The analysis of tuberculosis
Most of the molecular research on ancient microbial
infections has been performed on the Mycobacterium tu-
berculosis complex (M. tuberculosis, Mycobacterium bovis,
Mycobacterium africanum, Mycobacterium microti, Myco-
bacterium canettii), which causes human and animal tuber-
culosis. It can be assumed that mycobacteria are better
preserved than other bacteria due to their special cell
wall structure. They di¡er from most other bacteria due
to the high content of lipids in the cell wall, which renders
them highly resistant against environmental destruction.
FEMSLE 10566 5-8-02
Since tuberculosis involves the skeletal system to a consid-
erable extent and leads to characteristic macromorpholog-
ical alterations (such as the severe angular kyphosis called
gibbus), this disease can be identi¢ed with some certainty
even in skeletal material (which represents by far the most
abundant human biomaterial from historic populations).
In addition, in mummi¢ed human remains, adhesions of
the lung to the chest wall can provide evidence for pulmo-
nary tuberculosis (Fig. 1). Such alterations are easily de-
tected in mummies and represented the starting point for
further molecular investigations. In most studies, a 123-bp
fragment of the insertion sequence IS6110 [11] was ampli-
¢ed by PCR and the speci¢city of the reaction was sub-
sequently con¢rmed by direct sequencing or restriction
enzyme digestion (Fig. 2). Occasionally, the speci¢city of
IS6110 has been questioned, but in several studies it has
been shown that the insertion sequence is highly speci¢c
for the detection of M. tuberculosis complex DNA, com-
bined with an adequate sensitivity. Thereby, this molecular
approach meets the requirements to study successfully an-
cient DNA, since the aDNA is degraded and therefore
only small fragments of DNA can be ampli¢ed.
The molecular con¢rmation of supposed tuberculosis
infections in human remains was complemented by more
recent studies on material with non-speci¢c or without
morphological alterations (Fig. 3). This o¡ers potential
insight into the frequency of tuberculosis in ancient pop-
ulations and generally allows evaluating the impact of in-
fectious diseases on historic populations. In particular, the
high mortality rate of young adults observed in a wide
range of studies on e.g. ancient Egyptian, medieval and
later populations may be explained by high incidences of
infectious diseases, such as tuberculosis.
A further important point in analyzing ancient myco-
bacterial DNA is the di¡erentiation of sub-types of the M.
tuberculosis complex. First, studies using the spoligotyping
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 A.R. Zink et al. / FEMS Microbiology Letters 213 (2002) 141^147
Fig. 2. PCR ampli¢cation of M. tuberculosis complex DNA (IS6110) in
ancient tissue samples. Lanes: 1, 50-bp standard ladder; 2^4, ampli¢ca-
tion products of selected tissue samples; 5^6, blank controls. In two out
of the three presented samples (lanes 3 and 4) a positive band of the ex-
pected size (123 bp) was ampli¢ed, the ¢rst sample (lane 1) provided no
signal. The extraction (lane 5) and PCR (lane 6) controls were negative.
technique [12] have been performed successfully on ancient
tissue samples. This technique is based on the variation of
the DR (direct repeat) region in M. tuberculosis complex
members and allows di¡erentiation of strains up to a sub-
species level. Recently, this approach has widely been used
in diagnostic medical microbiology for the initial genotyp-
ing of the M. tuberculosis complex at a populational level.
Moreover, spoligotyping seems to be the most suitable
method for ancient material, since only small amounts of
an even highly fragmented mycobacterial aDNA can be
Fig. 3. Ancient Egyptian skeletal sample with non-speci¢c morphological alterations probably due to tuberculosis. This lumbar vertebra from a New
Kingdom tomb (1500^500 BC) shows slight destructive changes of the ventral margin of the vertebral body, suggesting an in£ammatory reaction possi-
bly caused by a tuberculosis infection.
FEMSLE 10566 5-8-02 Cyaan Magenta Geel Zwart
143
retrieved in the samples under investigation. Other recent
methods, such as IS6110 RFLP [13] or VNTR [14] require
high molecular bacterial DNA and are therefore not ap-
plicable to ancient tissue material. Spoligotyping can be
used for investigating evolutionary aspects of human tu-
berculosis and may help to clarify the origin and trans-
mission of the disease in humans of various time periods
and populations. In combination with other data it is suit-
able for constructing phylogenetic trees drawing back to
the beginning of pathogenesis and spread of the disease in
humans and animals.
Currently, there is still an open debate regarding the
origins of tuberculosis in human and animal species. The
classical hypothesis suggests that M. bovis is the probable
ancestor which was transmitted from cattle to humans
during domestication [15]. This theory, however, is not
supported by the as yet published spoligotyping results
on ancient material from di¡erent time periods. In all
studies, spoligotyping revealed no typical M. bovis type.
In di¡erent studies on medieval bone samples [16,17], on
bone samples ranging from 17 400 years of age to those of
barely a hundred years [18] and on naturally mummi¢ed
Hungarians from the 18th to the 19th century [19], the
patterns were found to be very similar to present day M.
tuberculosis isolates. The as yet oldest successful spoligo-
typing pattern has been obtained from an isolated fossil-
ized bison sample, dating back to ca. 17 000 years before
present [20]. Interestingly, in this study the spoligotyping
pattern, which does not match with any known spoligo-
typing signatures, was di¡erent from M. bovis patterns. In
our own recent study on ancient Egyptian material with
molecularly proven tuberculosis, dating back to 4000 years
before present, no M. bovis speci¢c pattern was detected
by spoligotyping. In contrast, M. africanum and M. tuber-
culosis speci¢c patterns could be obtained while, quite in-
terestingly, the M. africanum ¢ndings were detected in the
oldest samples included in this study (Middle Kingdom,
144 A.R. Zink et al. / FEMS Microbiology Letters 213 (2002) 141^147
2050^1650 BC). This supports the theory that a M. tuber-
culosis complex precursor evolved from M. africanum and
that the present day M. tuberculosis and M. bovis may
have developed in parallel. This theory is also substanti-
ated by nucleotide sequence analyses of current M. tuber-
culosis isolates, revealing the absence of allelic variation.
Therefore, the evolutionary origin of human pathogenic
mycobacteria was suggested to be 15 000^20 000 years
ago [21].
This clearly demonstrates that only the investigation of
ancient biomaterial can provide a direct insight into the
situation up to several thousand years ago. With this ap-
proach it will be possible to evaluate evolutionary models
which are usually developed on data obtained from recent
strains by comparing their frequencies, the occurrence of
certain polymorphisms and their genetic di¡erences. Since
such statistical models are restricted to present day data
and cannot take into account possible particular events of
molecular evolution (such as bottlenecks, environmental
disasters etc.), only the investigation of authentic historic
material can gain access to the real story of molecular
evolution and the development and spread of mycobacte-
rial disease.
2.2. Identi¢cation of other bacterial infections
The molecular identi¢cation of other pathogens such as
M. leprae, Y. pestis, P. falciparum and others has also been
performed successfully on ancient skeletal and mummi¢ed
soft tissue material. They were, however, mainly based on
isolated ¢ndings or small series. Nevertheless, these studies
demonstrate the capability of molecular methods to detect
pathogens even without having a morphological pendant.
This is particularly true for the detection of Y. pestis in
skeletons excavated from 16th and 18th century French
mass graves of probable victims of the plague [4]. The
successful ampli¢cation of Y. pestis DNA in six out of
12 plague victim teeth con¢rmed the diagnosis of ancient
septicemia, which had been assumed on the basis of his-
torical data. This molecular approach represents the only
possibility to prove the ancient infection, as manifestations
of bone lesions usually do not occur in septicemic plague.
In addition to the application to speci¢c primer sequen-
ces for the pointed detection of infectious organisms, the
ampli¢cation of a segment of the eubacterial 16S rDNA
with primer pairs recognizing conserved regions but £ank-
ing hypervariable species-speci¢c regions allows the iden-
ti¢cation of unknown bacteria in a given sample (‘broad-
range’ eubacterial PCR). This neat experimental strategy
for pathogen identi¢cation is methodically restricted to the
detection of predominant species within a complex mix-
ture of bacterial organisms usually observed with ancient
samples.
To overcome this limitation, PCR amplicons obtained
with broad-range 16S rDNA primers from a mixture of
bacterial species are cloned into appropriate vectors and at
FEMSLE 10566 5-8-02
least 20 randomly selected clones from each experiment
are sequenced. The comparison of the obtained sequences
with all GenBank entries and other eubacterial 16S rDNA
databases reveals scores of homology leading to a list of
candidate bacteria (Fig. 4). This approach allows the de-
termination of a variety of bacterial species simultaneously
present in ancient tissue samples without targeting a single
species. Thereby, pathogens or potential pathogenic bac-
teria may be detected in individuals without morphologi-
cal evidence for a certain disease. Recently, we have ap-
plied this technique successfully in two case studies on an
infant bone and a tooth sample of two di¡erent mummies
from pharaonic Egypt. The analysis of the 16S rDNA
revealed a probable septicemic infection by E. coli in the
infant mummy [22] and provided evidence for Coryne-
bacterium spp. in the tooth sample, suggesting the pres-
ence of diphtheria in pharaonic Egypt [9].
2.3. Protozoal infections
Besides the identi¢cation of bacteria, these more general
approaches can also be applied to detect protozoal infec-
tions. Likewise, malaria infections caused by one of the
four di¡erent human pathogen Plasmodium species (P.
falciparum, Plasmodium vivax, Plasmodium malarie and
Plasmodium ovale) can be identi¢ed. This diagnosis may
be observed particularly in those individuals with evidence
for chronic anemia which in turn manifests itself in the
skeleton as porotic hyperostosis of the orbital vault (‘cri-
bra orbitalia’) and/or of the parietal skull bones [23]. In
fact, porotic hyperostosis as a bone reaction to chronic
anemia may occur during many di¡erent pathological con-
ditions, such as malnutrition, iron de¢ciency, other infec-
tions, etc. A clear diagnosis of malaria infections is there-
fore only possible by molecular detection of ancient
plasmodial DNA. The existing studies already demon-
strated the capability of PCR based identi¢cation of plas-
modia [5], but PCR systems need to be improved in order
to provide a su⁄ciently powerful approach to evaluate
ancient malaria.
2.4. Detection of viral infections
There exist only a few reports on the detection of viral
infections in ancient tissue specimens. The most spectac-
ular case was the molecular identi¢cation of a previously
unknown strain of human T-cell lymphotropic virus type I
(HTLV-I) provirus DNA in an Andean mummy dated
approximately 1500 years old [24]. Sequence comparison
to those in contemporary Andeans and Japanese provided
evidence that HTLV-I was carried into the New World
during the ancestral migrations of humans from Asia
across the Bering Strait. However, this conclusion was in
contrast to the most molecular phylogenetic analyses that
indicate a more recent origin for HTLV-I within South
America and therefore led to a controversial scienti¢c de-
Cyaan Magenta Geel Zwart
 A.R. Zink et al. / FEMS Microbiology Letters 213 (2002) 141^147
Fig. 4. Schematic presentation of the extraction, preparation and analytical procedure for the species-speci¢c ampli¢cation of the bacterial 16S rDNA.
Following extraction of the ancient DNA a fragment of the eubacterial 16S rRNA gene is ampli¢ed with primer pairs recognizing conserved regions
and £ank hypervariable species-speci¢c regions. Subsequently, the PCR amplicons are cloned into vectors and sequenced. The results are compared with
GenBank entries and eubacterial 16S rDNA databases.
bate. Although there is still a lack of extended and reliable
studies on ancient viral infections, the capability of this
approach was demonstrated by the successful ampli¢ca-
tion of RNA from the 1918 ‘Spanish’ in£uenza virus
[25]. The in£uenza RNA was isolated from two para⁄n-
embedded and one permafrost lung tissue sample from
victims of the in£uenza pandemic, which killed over 20
million people in 1918 and 1919. The complete sequence
of the hemagglutinin gene of the 1918 virus could be de-
tected and subsequently used for phylogenetic analyses. It
can be hoped that further molecular studies on ancient
viral infections will help to determine the genetic basis
for such an exceptional virulence.
3. Limitations of ancient pathogen retrieval
The work with ancient DNA bears some major limita-
tions, which reduces the success rate of detecting ancient
pathogens. One major point is the stability of the aDNA.
Normally, DNA degrades rapidly after death. Several pa-
rameters such as high temperature, UV radiation, humid-
ity and a low pH value signi¢cantly accelerate this DNA
decay. On the other hand, a dry and cool climate or a
rapid desiccation of an organism due to arti¢cial or natu-
ral mummi¢cation processes may slow down the degrada-
tion and increases the probability of ancient DNA retriev-
al. In any case it has to be taken into account that the
amount of authentic DNA in ancient tissue samples may
be very low or even may have disappeared completely. In
most instances, the aDNA molecules are signi¢cantly frag-
mented and destabilized by deamination and depurination.
These factors can severely disturb the PCR ampli¢cation,
leading to non-speci¢c products or incorrect sequences
[26]. Therefore, an adequate primer design for ancient
FEMSLE 10566 5-8-02
145
DNA purposes is necessary. This can lead to di⁄culties
in ¢nding the optimal PCR system, as in most instances
only small DNA fragments of not more than 200 bp
should be targeted and at the same time the ampli¢ed
fragment should provide a high speci¢city for the wanted
pathogen. Most approaches of molecular clinical diagnos-
tics do not ful¢ll these criteria and cannot directly be used
for ancient pathogen retrieval.
Another major limitation is the risk of contaminating
the specimens with DNA of recent origin. This is a major
problem when working with human DNA, since human
DNA is prevalent in the environment and every person in
contact with the material is a probable source of contam-
ination. The danger of contamination with modern bacte-
rial DNA is far less, since the researcher usually is not a
contamination source and most pathogens are not present
ubiquitously in the environment, but require living organ-
isms or particular conditions to survive. Nevertheless,
cross-contamination and over£ow of modern microbial
DNA have always to be taken into account and should
strictly be avoided. Therefore, ancient DNA analyses
should always be performed in laboratories which are ex-
clusively used for these purposes. The identity of PCR
ampli¢cation products should always be veri¢ed by addi-
tional molecular methods, e.g. determination of the se-
quence or restriction enzyme digestion of the PCR prod-
ucts. With regard to the properties of the ancient DNA,
cloning of the PCR products may be very helpful to dis-
tinguish non-speci¢c or nonsense sequences from the true
microbial targets. Moreover, overlying sequences of e.g.
fungal or modern bacterial DNA may thereby be ex-
cluded.
Despite the reduced susceptibility to contamination in
detecting ancient DNA of pathogens, all authenticity cri-
teria necessary to determine ancient DNA sequences [27]
Cyaan Magenta Geel Zwart
146 A.R. Zink et al. / FEMS Microbiology Letters 213 (2002) 141^147
should be considered, to avoid as far as possible any false
positive results. It is important to produce reliable data
which are comparable to other studies.
4. Conclusions and outlook
In the beginning of the search for ancient microbial
infections, much work was focused on the detection of
pathogens which produce typical morphological altera-
tions in bone or soft tissues. Some interesting ¢ndings
on single individuals or small series have been published
in which a presumed diagnosis was con¢rmed by molec-
ular methods. Nevertheless, this already demonstrated the
far-reaching capacity of the detection of ancient microbial
DNA. The technical development for particularly adopted
extraction and ampli¢cation procedures provided the suc-
cessful detection of di¡erent pathogens in skeletal and
mummi¢ed material up to several thousand years of age.
Most molecular investigations have been made on the M.
tuberculosis complex, which seems to be more appropriate
for analysis due to the protective cell wall properties of the
mycobacteria. The studies on ancient tuberculosis infec-
tions are no longer restricted to the diagnosis of the infec-
tious disease, but have presented interesting data about
the occurrence and frequency of tuberculosis in ancient
populations. The recent introduction of the spoligotyping
technique in this ¢eld o¡ered the possibility to di¡erentiate
between the members of the M. tuberculosis complex and
therefore allowed to investigate evolutionary aspects of
mycobacterial infections. Along with these evaluations
theories are emerging as to the origin and transmission
of the disease. This represents an important contribution
to modern microbiological research as the comparison of
recent mycobacterial species can only lead to a theoretical
or statistical model without direct evidence from the past.
Theoretically, any fungal, bacterial or viral infection can
probably be detected, depending primarily on the preser-
vation state of the specimens and the availability of suit-
able PCR systems. The most limiting factor is the suscep-
tibility of DNA to degradation and modi¢cation in
ancient tissue samples in£uenced by several factors such
as temperature, humidity, pH value, etc.
There exists a lot of historical and archaeological evi-
dence of other infectious diseases, such as leprosy, plague,
malaria and others, which were already detected in ancient
specimens by the successful ampli¢cation of the corre-
sponding pathogens. However, there is still a lack of ex-
tended studies to gain more information about the fre-
quency, transmission and probable evolution of these
pathogens. Keeping in mind the clinical and populational
signi¢cance of these infectious diseases, this holds partic-
ularly true for malaria and increasingly for tuberculosis.
The ongoing research in this relatively new scienti¢c ¢eld
has the potential to contribute signi¢cantly to a better
understanding of host^pathogen interaction, transmission
FEMSLE 10566 5-8-02
and spread of infectious diseases in historic and modern
times.
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