1 Department of Medical Microbiology, University College London, London W1T 4JF, UK
2 Imperial College London, Centre for Microbiology and Infection, Flowers Building, Armstrong Rd, London SW7 2AZ, UK
3 Medical Microbiology and Infectious Diseases, Location Lukas, Gelre hospitals, Albert Schweitzerlaan 31, 7300 DS Apeldoorn, The Netherlands
Correspondence
Helen D. Donoghue
h.donoghue{at}ucl.ac.uk
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ABSTRACT |
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Abbreviations: MTB, Mycobacterium tuberculosis
Present address: Molecular Immunology (Hill Group), Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK.
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INTRODUCTION |
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Contemporary written records were available for many of the individuals found in the crypt, including date of death, age, sex, family name, relatives and, in some cases, a brief description of the cause of death. These records enabled several bodies to be placed into family groups. Of particular interest in this respect was a small family group consisting of a mother and two daughters, all of whom had died over a four year period between 1793 and 1797. Body 28 was that of a 55-year-old woman who died on 16 December 1793, the mother of the two other individuals reported here. Her height was noted as 1·45 m. Soft tissue was taken from the tracheal region and abdomen of body 28, using Stortz endoscopes and an aseptic technique as precautions against cross-contamination. Body 68 was the daughter of the above, and she died on 25 December 1797 aged 28 years. No abnormalities were detected in a chest radiograph of body 68 but she was noticeably small for her age and of cachectic appearance. A tissue sample from the chest was examined. Body 72 was the younger daughter of body 28 and sister to body 68, and she died on 2 March 1795. Although the archive stated that body 72 was aged 14 years at death, when examined she was initially believed to be aged only eight or nine years due to her very small size and cachectic appearance. No abnormalities were seen in a chest radiograph. Samples of body 72 were taken from the abdomen, chest and from possible calcified pleura.
Several studies have examined skeletal and mummified remains for the presence of mycobacterial DNA (Spigelman & Lemma, 1993; Salo et al., 1994
; Baron et al., 1996
; Faerman et al., 1997
; Nerlich et al., 1997
; Braun et al., 1998
; Crubézy et al., 1998
; Donoghue et al., 1998
; Taylor et al., 1996
, 1999
, 2001
; Haas et al., 2000
; Mays et al., 2001
; Zink et al., 2001
). However, the state of surviving DNA has only seldomly permitted detailed molecular examination of samples (Mays et al., 2001
). Consequently, few population-based studies have been reported for ancient tuberculosis (Zink et al., 2001
). However, the completion of the sequencing of the MTB genome (Cole et al., 1998
) and comparative genomic studies of members of the MTB complex have revealed an increasing number of targets for the study of historical cases of tuberculosis. In the present study a number of PCR-based methods were applied to the MTB complex strains from the family group described above. The aim was to evaluate the use of these markers for the analysis of archaeological specimens and to assess the extent to which such studies may contribute to increasing our understanding of the evolution and molecular epidemiology of tuberculosis.
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METHODS |
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Reproducibility.
Data were verified by repeating PCRs on the same and on fresh extracts of samples. In addition, in several cases duplicate reactions were carried out independently in one of the other collaborating laboratories.
DNA extraction.
DNA extraction in laboratory one was performed using an adaptation of the Boom method (Boom et al., 1990) as described previously (Donoghue et al., 1998
). Repeat extracts were performed using the Qiagen DNeasy Tissue Kit with the following adaptations: 25 mg of sample was added to a 1·5 ml tube containing 1·52 mm glass beads and incubated overnight at 56 °C with 200 µl digestion buffer [0·5 M EDTA, pH 8·0 (Promega), 0·4 mg proteinase K ml-1 (Finnzymes)]. Each sample was homogenized for 50 s at medium speed with a Mini Bead Beater (Stratech Scientific) and transferred to a column where it was further processed according to the manufacturer's instructions. In laboratory two DNA was extracted using a silica-based method as described previously (Taylor et al., 1999
).
Screening methods.
Primer sequences, amplification conditions and details of where the work was carried out are given in Table 2. Samples were screened for an MTB-complex-specific region of the insertion sequence IS6110 (Eisenach et al., 1990
) using a two-tube nested PCR as described previously (Taylor et al., 1996
; Donoghue et al., 1998
). Thereafter, target sequences in the 19 kDa antigen gene (Mustafa et al., 1995
) and the dnaAdnaN region were examined.
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RD7 PCR.
As a further screening test to refute the presence of M. bovis DNA in the samples, flanking primers were used in a PCR to detect deletion region 7 (RD7). This deletion, of around 12·7 kb, extends from base pair 2 208 003 to base pair 2 220 721 in the MTB genome (Zumárraga et al., 1999; Gordon et al., 1999
). A product of 211 bp is amplified from M. bovis isolates but under the conditions used no product is expected from MTB, particularly in fragmented archaeological material (Mays et al., 2001
).
Molecular fingerprinting.
Spoligotyping (Kamerbeek et al., 1997) was carried out in laboratories two (Taylor et al., 1999
) and three (van der Zanden et al., 1998
).
Detection and analysis of amplified DNA.
In laboratories one and two, PCR products were electrophoresed in a 3·0 % (w/v) NuSieve (Flowgen) or agarose gel, respectively. DNA was visualized by ethidium bromide staining plus UV light. Images were recorded with a digital image capture system. Products for sequencing were run subsequently on 0·8 % low-melting-point agarose gels. DNA was purified and sequenced using in-house sequencing services or MWG-Biotech.
Quantification and amplification efficiency.
In laboratory one, the amount of amplified product obtained from the 18th century samples was determined in a single experiment by gel densiometric analysis using a DNA mass marker (Gibco) and LABWORKS software (UltraViolet Products). This was compared with DNA from an M. bovis culture, strain AN-5, which was prepared and set up in an isolated, totally separate laboratory, which was under negative air pressure. Four target sequences were used for this comparison, IS6110 using the inner primers (92 bp), the 19 kDa antigen gene (131 bp), the dnaAdnaN region (159 bp) and the MPB70 antigen gene (372 bp). The quantity of amplicon was converted to the number of copies per microlitre using Avogadro's constant. Modern DNA was used to ascertain whether differences in yield were due to variations in amplification efficiency between the PCRs. A dilution curve (106 to 101) of each product was subjected to real-time PCR amplification (ABI 7000 SDS; Applied Biosystems) using Qiagen Quantitect master mix.
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RESULTS |
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Genotypic grouping
Fragments of DNA spanning the regions of polymorphisms in the katG and gyrA genes (Table 3) were amplified and sequenced in laboratories one and two. Body 28 classified as group 2 with katG codon 463 CGG, katG 203 ACC and gyrA 95 ACC. Bodies 68 and 72 classified as group 3 with katG 463 CGG, katG 203 ACC and gyrA 95 AGC (Table 4
).
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mtp40 and plcD-cutinase PCR
Samples from all three individuals were positive for the mtp40 fragment, which strongly indicated that the DNA was from MTB rather than M. bovis (Table 4). Samples from bodies 28 (abdomen and trachea) and 68 (chest) were consistently positive for plcD but body 72 yielded a negative result with single-stage PCR based on the larger target sequence (177 bp; Fig. 1
a). However, in laboratory one, with single-stage PCR of the 123 bp target region, some positive results were obtained from body 72, although samples from the chest, pleura and abdomen differed in the quantity of amplifiable MTB DNA, and repeated DNA extracts also yielded differing amounts. Nested PCR of apparently negative PCR products of either the 177 or 123 bp target regions was required to give clear positive results from body 72 (Fig. 1b
).
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Screening for IS6110 in the dnaAdnaN region
A 159 bp product was amplified from the Vác samples with primers Pr1 and Pr2 and no product was obtained with primers Pr2 and Pr3 (Table 4), indicating that there was no insertion sequence in this region specific for the Beijing family of multi-drug-resistant MTB (Kurepina et al., 1998
).
Spoligotyping
The spoligotyping patterns obtained from the extracts from bodies 28, 68 and 72 were remarkably complete (Fig. 2a). Two spoligotype patterns were observed for the three bodies, patterns 50 and 53 (Sola et al., 1999
), which can also be described by an octal code as 777777777730771 and 777777777760771, respectively (Dale et al., 2001
). Body 28 (mother) had pattern 53 and bodies 68 and 72 (daughters) had pattern 50. To confirm these results, spoligotyping was repeated in laboratory two using separate extracts from the bodies. In addition, a third set of extracts from the same bodies was taken to laboratory three for spoligotyping. Fig. 2(b)
is a schematic representation of the results obtained from the two laboratories. The extracts from body 72 prepared in laboratory one and amplified in laboratory two gave extremely faint hybridization in spacers 29, 30 and 32, although these spacers were negative when the DNA was amplified according to the normal protocol used in laboratory three.
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DISCUSSION |
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Bodies 68 and 72 were physically small in comparison to the majority of the remains found in the crypt, some of which were of individuals who lived to a great age and who showed no signs of malnourishment or wasting (Fletcher et al., 2003). Indeed, it is unlikely that the physical appearance of bodies 68 and 72 was due to malnutrition as it is known that only wealthy, middle class families were buried in the crypt. Therefore, the cachectic appearance of these bodies, in association with the age of death and ease of detection of MTB DNA, strongly suggests that death was as a result of active tuberculosis infection. Therefore, it can be concluded that the MTB characterized here was virulent.
Due to the close association of humans with domesticated animals, it was believed that many cases of tuberculosis in antiquity were due to M. bovis (Dankner et al., 1993). In addition, it has been suggested that as M. bovis can be transmitted via the consumption of dairy products it may be found infecting the abdomen. The abdomen of body 28 was found to be strongly positive for MTB; therefore, five different tests were performed that can differentiate between the DNA of MTB and M. bovis. All the results indicate that the DNA was from MTB and not M. bovis. M. bovis is a member of the MTB complex genotype 1, yet the strains we have analysed classify as genotypes 2 and 3. Finally, the presence of spacers 3743 on the spoligotype confirm that these genotype 2 and 3 strains are MTB and not M. bovis. Initially, the failure to detect evidence of M. bovis infection, also reported by others (Mays et al., 2001
), was surprising. However, M. bovis infections are zoonotic and person-to-person spread is thought to occur only in exceptional circumstances (Grange, 2001
). Therefore, in a population with endemic MTB infection, M. bovis infections are always likely to comprise a small proportion of the total number of cases.
The findings from the dnaAdnaN spacer region are consistent with both the genotyping and spoligotyping data. Beijing strains are of genotype 1 and have a unique spoligotype. In addition, the multi-drug-resistant strain W has a characteristic IS6110 insertion in this target region. The Beijing/W group of strains is believed to be of recent origin (Glynn et al., 2002), which is supported by the failure to detect any members of this group in the mummified material from Vác examined so far (H. D. Donoghue, unpublished observations).
The spoligotypes obtained from the Hungarian MTB DNA samples were remarkably reproducible. Patterns 50 and 53 are the patterns most commonly found across the globe today and are non-discriminatory. Sola et al. (1999) have proposed a phylogenetic tree, the root of which is pattern 53. Although tuberculosis occurred in antiquity (Sola et al., 1999
), it is thought that the modern tuberculosis epidemic began in Europe in the 1700s, then moved to the New World and Africa (Stead et al., 1995
). Consistent with the above theories, we have demonstrated that patterns 50 and 53 were present in Europe in the 18th century. Spoligotypes 50 and 53 differ by the absence of one spacer (spacer 31). Recent studies have demonstrated that in some cases the absence of spacer 31 in spoligotype 50 may be due to the presence of IS6110 in this region (Filliol et al., 2000
; Legrand et al., 2001
). In an extensive genotyping study it has been shown that strains with spoligotype 53 are groups 2 or 3 and that strains with spoligotype 50 are group 2 (Soini et al., 2000
). However, our spoligotype pattern 50 strains are group 3 rather than group 2. It is possible that the strains we are observing are indeed spoligotype 53 with an IS6110 element in spacer 31 as opposed to a true type 50 (Legrand et al., 2001
). Screening for the presence of IS6110 in spacer 31 of the direct repeat (DR) region should determine if this is the case, although so far we have been unsuccessful in obtaining sequence data from this region.
The present study, based on multiple targets in the MTB genome, has confirmed that the amplified DNA from the Hungarian samples was that of MTB. The two-centre system used to analyse the Hungarian samples confirms the inter- and intra-laboratory reproducibility of the results. Using MTB-specific primers followed by sequencing or probing, DNA has been amplified from eight loci on the MTB genome, including both single-copy and multiple sites. The detection of small-scale genomic deletions has been shown to be a useful technique for exploring the molecular epidemiology, microbial evolution and pathogenesis of tuberculosis (Ho et al., 2000; Kato-Maeda et al., 2001
). Using a novel multiplex PCR technique we have demonstrated the use of screening historical cases of tuberculosis for such deletions. Spoligotyping revealed a difference between the strain infecting the mother and the strain infecting her two daughters; the mother typing as type 53 and the daughters as type 50. Using multiplex PCR of the mtp40 and plcD loci we initially concluded that there was a further difference between the strains infecting the two daughters, the isolate from the younger daughter having undergone a deletion in the plcD-oxidoreductase region that was detected using the larger of the two target sequences used (Fig. 1a
, lane 6). About one-third of clinical isolates of MTB studied appear to have undergone a deletion in this region (Ho et al., 2000
). However, the use of a shorter target sequence and nested PCR demonstrated (Fig. 1b
) that this initial assumption was incorrect and that the material from the younger daughter was less-well preserved, shown by the difficulty in obtaining positive results when larger target sequences were used. The spoligotyping data also suggest poor preservation of part of the DR spacer regions from MTB in this individual. As determination of deletion subsets in this field is likely to be increasingly attempted for phylogenetic purposes, it will be important, as a check on DNA preservation, to multiplex PCRs such as plcD with primers to another target region that produce a slightly longer product.
In conclusion, it appears that the three members of the family group studied here were infected with two distinct strains of MTB. This is the first demonstration of spoligotyping combined with screening of small-scale deletions for determining the molecular epidemiology of MTB from archaeological material. In comparison with the strain infecting the mother, there was one point mutation and one deletion event, the loss of spacer 31, detected in the MTB from the two daughters. This supports the theory that MTB undergoes more deletion events as it evolves (Ho et al., 2000; Kato-Maeda et al., 2001
; Brosch et al., 2002
). However, screening of the entire genomes of these strains would be needed to confirm this. The findings are also consistent with the major genotypic grouping described by Sreevatsan et al. (1997)
, which is thought to broadly represent an evolutionary scenario for the MTB complex. Genotypes 2 and 3 are believed to be younger in evolutionary terms than genotype 1 organisms, so their demonstration in 18th century Europe supports the latest hypothesis of MTB evolution based on deletion analysis of the genomes of MTB and M. bovis (Brosch et al., 2002
). Future studies adopting the methods described here should contribute further to our knowledge of the evolution and epidemiology of tuberculosis.
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ACKNOWLEDGEMENTS |
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Received 23 August 2002;
revised 9 October 2002;
accepted 10 October 2002.