1 Centre for Molecular Microbiology and Infection, Flowers Building, Armstrong Road, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
2 Museums of The Royal College of Surgeons of England, 3543 Lincoln's Inn Fields, London WC2A 3PE, UK
3 Department of Histopathology, Imperial College London, St Mary's Campus, London W2 1PG, UK
4 TB Diagnostic Department, Veterinary Laboratory Agency, Addlestone, New Haw, Weybridge, Surrey KT15 3NB, UK
Correspondence
G. M. Taylor
gm.taylor{at}imperial.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Koch was in London in July of 1901 for the British Congress on Tuberculosis. It was at this meeting that he voiced the opinion that the bacteria causing the human and bovine forms of tuberculosis were different. Whilst contentious at the time, his view was eventually proven correct. However, his belief, voiced at the congress, that humans were rarely if ever susceptible to bovine tuberculosis (Koch, 1901) was certainly not correct. It was at the end of this meeting that Koch presented the curator of the museum of the congress, otolaryngologist Walter Jobson Horne, with a souvenir of his earlier work into the cause of human tuberculosis. This consisted of a boxed set of five glass culture tubes containing plentiful bacterial colonies on slopes of solid growth media (Fig. 1
). Onto these slopes in June of that year had been inoculated material from the original case of miliary tuberculosis from which Koch had identified the rod-like bacilli as the cause of tuberculosis some 20 years previously in August 1881. According to a label on the presentation case housing the tubes, the original inoculum had undergone 435 subcultures during the intervening period, without again having been passed through the body of an animal.
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At the time of Koch's discovery, one in seven members of the population died of tuberculosis (Koch, 1882). The following century witnessed a steady decline in tuberculosis due to mass X-ray screening, the development of drug therapy, BCG immunization programmes, improvements in socio-economic factors and possibly to the gradual acquisition of resistance to infection by the population as a whole. Pasteurization of milk and herd control measures also played a part in markedly reducing the risk from bovine tuberculosis (Hardie & Watson, 1992
). However, the latter part of the 20th century witnessed a steady resurgence of the disease, which remains a global problem (Dye et al., 1999
). Recent years have seen the completion of whole-genome sequencing projects for a strain of Mycobacterium bovis (Garnier et al., 2003
) and two strains of M. tuberculosis, H37Rv and CDC1551 (Cole et al., 1998
; Fleischman et al., 2002
). With these projects has come a growing awareness of the molecular basis of pathogenesis and the rational development of genotyping methods. In the light of these advances we have examined material from this unique historical relic to assess if the cultures remained viable and to determine what a molecular approach might reveal in the context of recent examination of the evolution of the M. tuberculosis complex.
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METHODS |
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Light microscopy.
To confirm the presence of mycobacteria, acid-fast staining with carbolfuchsin was performed using the TB stain kit K from Becton Dickinson.
Electron microscopy.
Colonies scraped from the exhibit were suspended in melted growth medium (Middlebrook 7H11) and centrifuged at 14 000 g as the medium cooled. To compare the preservation and morphology of the Koch isolate with a modern strain, live M. tuberculosis, reference strain H37Rv, was fixed in 4 % neutral buffered formalin for 2 h and then pelleted by centrifugation at 14 000 g for 10 min. The residue was then resuspended in cooling growth medium as described for the Koch material. The pellets were post-fixed in osmium tetroxide and stained en bloc in 2 % uranyl acetate, dehydrated, cleared in Inhibisol and embedded in Taab 815 resin (Taab Laboratories Equipment). The block was polymerized at 70 °C. Using a glass knife, semi-thin (0·5 µm) sections were cut and stained with Azure II to ascertain presence of mycobacteria. Then, using a diamond knife, ultrathin sections were cut and picked up on a copper grid. The grid was stained with Reynolds' lead citrate and examined using a Philips 400 Transmission Line microscope.
DNA extraction.
For safety reasons it was assumed that the bacteria could still be viable and all samples were taken within a HEPA filtered cabinet housed inside a containment level three laboratory. This precaution was also necessary to prevent possible contamination of the media with modern bacteria. Colonies were removed from the tubes using sterile disposable plastic loops and the material transferred immediately to 0·9 ml guanidinium lysis buffer, part of the NucliSens commercial kit (Biomérieux). The tubes were heated to 100 °C for 5 min to inactivate any surviving mycobacteria (Bemer-Melchior, 1999) and thereafter partially purified DNA was prepared according to the manufacturer's instructions. Four extracts were prepared in this way. Two additional samples were collected into 100 mM Tris/EDTA (TE) buffer, pH 7·4, heat treated as above and a conventional extraction procedure with lysozyme-proteinase K/phenol-chloroform was performed, followed by ethanol precipitation (Goyal et al., 1997
). Residues after precipitation were taken up in 50100 µl TE buffer.
PCR.
Hot-start PCR was performed on a Hybaid Express thermal cycler in a final volume of 25 µl using the Excite Core kit (BioGene) according to the manufacturer's instructions. The sequences of primers used in the individual methods and other details of the PCR cycling conditions are shown in Table 1. Many of these methods were developed for use with archaeological samples and were designed to amplify small DNA fragments, which can persist in archival material. As some of these methods appeared originally in archaeological journals (Taylor et al., 1996
; Mays et al., 2001
) or have since been modified (Fletcher et al., 2003
), they are brought together here for convenience. Generally, 43 cycles of amplification were performed. On some occasions when sequencing of products was required and with the katG463 PCR, a hemi-nested method was adopted using an initial 41 cycles followed by an additional maximum of 35 cycles.
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Gel electrophoresis.
Routine gel electrophoretic analysis of products was performed on 3 % (w/v) agarose gels as described previously (Taylor et al., 1996). Products for sequencing were subsequently purified on either 1 or 2 % LMP agarose (Gibco-BRL). Bands were excised from the gel with a sterile scalpel blade and purified using the NucleiClean DNA isolation kit (Sigma-Aldrich).
Spoligotyping.
Spoligotyping was performed on two of the guanidinium thiocyanate (GuSCN)/silica extracts and on two of the lysozyme-proteinase K/phenol-chloroform extracts as described by Kamerbeek et al. (1997) but with 45 cycles of amplification. Hybridization analysis of one of each type of extract was also performed at VLA, Weybridge.
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RESULTS AND DISCUSSION |
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Culture
Attempts to culture Koch's isolate in various media for periods of up to one year were unproductive and this was ascribed either to the age of the specimen or to the possibility that it had been fixed by chemical means to make it safe for exhibition.
Light and electron microscopy
Light and electron microscopy of material from representative colonies from the exhibit revealed abundant rod-shaped bacilli consistent with mycobacteria. Fig. 2 shows the appearance of the organisms visualized after carbolfuchsin staining. Fig. 3
(a) shows a high-power electron microscopic enlargement (x28 000) of one of the fields. Whilst the majority of organisms appeared intact, morphological preservation was poor and detail of the cell wall membranes and internal components was unclear. Many of the mycobacteria showed a dark mottling, almost certainly due to osmium tetroxide staining of exposed lipid moieties in the mycobacterial cell wall. As there was anecdotal evidence to suggest that the cultures may have been previously fixed, we compared the morphology of the archival material with modern M. tuberculosis strain H37Rv which had been fixed using 4 % formalin. This exhibited a well-preserved trilaminate wall and cell membrane and internal components could be more readily discerned (Fig. 3b
).
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PCR and genotyping
A series of extracts of the bacterial DNA from the cultures were prepared and exposed to PCR amplification and automated sequencing to provide genotyping information of the strain. Data were obtained for a set of polymorphic loci but PCR required high cycle numbers and methods targeting shorter templates were preferred, supporting the suggestion that the samples may previously have been inactivated in some way for exhibition. Generally, the GuSCN/silica extraction method was found to be superior to the lysozyme-proteinase K/phenol-chloroform method. The data gained from PCR analysis of the exhibit are summarized in Table 2.
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Corroboration of the species, M. tuberculosis, came from amplification and sequencing of the species-specific polymorphisms at codon 57 of the pyrazinamidase gene pncA57 (CAC, His) and also of nucleotide 285 (G) in the oxyR pseudogene (Sreevatsan et al., 1996; Scorpio et al., 1997
). We also tested for the presence of the mtp40 element (Del Portillo et al., 1991
). Loss of this element is associated with the RD5 deletion event, which is 8964 bp in size and located between genome positions 2 626 067 and 2 635 031 on the H37Rv reference strain (Gordon et al., 1999
). The region includes a membrane-associated phospholipase C 1 (mtp40 antigen, Rv2351c). This locus is present in the vast majority of M. tuberculosis isolates and absent from the great majority of M. bovis isolates (Liébana et al., 1996
; Weil et al., 1996
), although this alone is not definitive corroboration of species (Weil et al., 1996
). In this case, mtp40 was present, consistent with the Koch strain being an M. tuberculosis isolate.
Attempts to classify the genetic group of this strain of M. tuberculosis according to the Musser system (Sreevatsan et al., 1997) were only partially successful. A PCR product was obtained for the gyrA95 codon. Sequencing of this indicated that the Koch strain belonged to either genotypic group 1 or 2. Amplification of the katG203 locus, which further subtypes group 1 organisms into either group 1a or group 1b (Frothingham et al., 1999
) showed an ACC (Thr) sequence at this position, indicative of group 1b. Therefore the Koch's strain can be assigned to either group 1b or group 2 in this scheme. However, repeated attempts to narrow down the genotypic group by amplifying the katG463 region with different primer combinations (Table 1
) were unsuccessful.
As all other PCR methods were successful with equivalent DNA target lengths, generally without the need to resort to second rounds of amplification, we speculate there may have been either a partial deletion or truncation of the catalase-peroxidase gene in this strain of M. tuberculosis which prevented amplification. The amplification of codon 203 within katG and of regions of DNA both upstream (furA) and downstream of katG (Rv1907c) rules out the possibility of complete deletion of this region an observation which has been recorded in a relatively small percentage of strains with isoniazid resistance (Zhang & Young, 1994; Rouse & Morris, 1995
). A careful examination of all previously reported katG polymorphisms (Scior, 2002
, and references cited therein) showed only one mutation, involving generation of a stop codon at codon 477 that might have impeded PCR amplification of this region by introducing a mismatch at the 5' end of primer R2 (Table 1
). However, repetition of the katG PCR with primers F1 and R2 at low stringency (56 versus 62 °C) also failed to generate any product. It is therefore unlikely that this mutation was the cause of our inability to amplify this region.
However, we cannot be certain that a deletion or truncation was responsible for failure of katG463 PCRs. Previous experience with historical material has indicated that not all PCR methods will work on degraded DNA, which we have interpreted as inconsistency in survival of different regions of the mycobacterial genome in such samples (Fletcher et al., 2003).
The katG gene is a virulence factor in M. tuberculosis strains, conferring the ability to replicate and persist within macrophages in both mice and guinea pig models of infection (Li et al., 1998). This is due to the capacity of strains with intact katG genes and catalase-peroxidase activity to withstand the reactive oxygen intermediates produced by the host in response to infection (Beaman & Beaman, 1984
). Numerous reports attest to the variable nature of the katG locus. Single nucleotide polymorphisms (Scior et al., 2002
), small deletions (Rouse & Morris, 1995
; Rouse et al., 1995
; Pretorius et al., 1995
), larger truncations (Rouse & Morris, 1995
), insertions (Rouse et al., 1995
) as well as complete loss of the gene (Zhang & Young, 1994
; Rouse & Morris, 1995
) have all been recorded. A number of the mutations are associated with loss or reduction of catalase-peroxidase activity and resistance to the anti-tuberculous drug isoniazid (Wei et al., 2003
). Whilst the Koch strain could not have been exposed to isoniazid, a drug that first became available only after 1952, it is conceivable that a mutation or partial deletion may have occurred in the katG gene during the 20 year period during which it was maintained in culture, resulting in our inability to amplify the region around codon 463. The label on the case states that between 1881 and 1901 a total number of 435 passages had been performed, indicating that on average the original isolate was subcultured every 16 days. Loss of a functional katG gene may not be disadvantageous to a strain maintained in vitro for a prolonged period.
The TB D1 deletion was first described by Brosch et al. (2002) in their paper on the evolution of the M. tuberculosis complex. It is considered to distinguish ancestral from modern strains of M. tuberculosis. PCR primers spanning the deletion site yielded a short product of the expected size (139 bp). Therefore, the Koch's isolate, in common with the majority of contemporary strains of M. tuberculosis, is derived from a precursor strain in which this deletion has occurred. Such isolates are probably deficient in the transport of complex lipids to the mycobacterial cell wall. Sequencing of the PCR product was undertaken to confirm its identity. Unexpectedly, this showed that the Koch strain possessed a mutation in the mmpL6 region, at nt 1 761 752. This would in theory have resulted in an Ile (ATT) to Thr (ACT) mutation in mmpL6, but as this region is already shortened by the TB D1 deletion it is unlikely that this polymorphism is of any significance.
Spoligotyping resulted in amplification of spacers within the DR region. However, obvious differences were seen between the two extraction methods, i.e. the standard lysozyme-proteinase K/phenol-chloroform method and the use of guanidinium buffer with silica to trap DNA fragments. The latter was clearly more suited to recovery of DNA fragments as judged by the greater number of spacers which amplified in the same assay (Fig. 4).
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In summary, we have studied Robert Koch's original isolate of M. tuberculosis cultured in 1881 from a case of miliary tuberculosis. The genotyping methods used provided consistent phylogenetic data that this was indeed a strain of M. tuberculosis and that it could be grouped with the modern isolates of this species based on loss of the TB D1 region. Work on late 18th century Hungarian mummies with tuberculosis has shown that by this time the deletion had occurred in at least some European isolates (H. D. Donoghue & G. M. Taylor, unpublished observations). The findings in the Koch strain are therefore consistent with recent theories for the evolution of the M. tuberculosis complex (Brosch et al., 2002; Sreevatsan et al., 1997
) and with the limited number of observations emerging from bioarchaeological studies (Fletcher et al., 2003
).
Of the Koch cultures it has been written: The bacteria may be beyond resuscitation but the information in their DNA lives on (Watts, 2001). Some of the technical hurdles in unlocking that information may have been overcome, but many pieces of the puzzle remain to challenge scientists of the future.
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ACKNOWLEDGEMENTS |
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Received 18 July 2003;
revised 18 August 2003;
accepted 28 August 2003.
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