7000 éves dél-magyarországi tuberkulózis esetek oszteológiai és molekuláris biológiai vizsgálata

This study derives from the macroscopic analysis of a Late Neolithic population from southern Hungary. Remains were recovered from a tell settlement at Hódmezővásárhely-Gorzsa from graves within the settlement as well as pits, ditches, houses and as stray finds. Pathological analysis of the 71 individuals revealed numerous cases of infections and non-specific stress indicators, metabolic diseases, and evidence of trauma and mechanical changes. Several cases showed potential signs of tuberculosis and further analyses were undertaken, including biomolecular studies. The five individuals were all very young adults and included a striking case of hypertrophic pulmonary osteopathy. The initial macroscopic diagnosis of these five cases was confirmed by lipid biomarker analyses, and three of them were corroborated by DNA analysis. At present, these 7000-year-old individuals are among the oldest palaeopathological and palaeomicrobiological cases of tuberculosis worldwide

Mapping the phylogeny and lineage history of geographically distinct BCG vaccine strains

The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in sigK, plaA, mmaA3 and eccC5 were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature

Osteological and Biomolecular Evidence of a 7000-Year Old Case of Hypertrophic Pulmonary Osteopathy Secondary to Tuberculosis from Neolithic Hungary

Seventy-one individuals from the late Neolithic population of the 7000-year-old site of Hódmezővásárhely-Gorzsa were examined for their skeletal palaeopathology. This revealed numerous cases of infections and non-specific stress indicators in juveniles and adults, metabolic diseases in juveniles, and evidence of trauma and mechanical changes in adults. Several cases showed potential signs of tuberculosis, particularly the remains of the individual HGO-53. This is an important finding that has significant implications for our understanding of this community. The aim of the present study was to seek biomolecular evidence to confirm this diagnosis. HGO-53 was a young male with a striking case of hypertrophic pulmonary osteopathy (HPO), revealing rib changes and cavitations in the vertebral bodies. The initial macroscopic diagnosis of HPO secondary to tuberculosis was confirmed by analysis of Mycobacterium tuberculosis complex specific cell wall lipid biomarkers and corroborated by ancient DNA (aDNA) analysis. This case is the earliest known classical case of HPO on an adult human skeleton and is one of the oldest palaeopathological and palaeomicrobiological tuberculosis cases to date

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis

‘Dr Granville's mummy’ was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783–1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid–pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu

Mycobacterium tuberculosis Complex Lipid Virulence Factors Preserved in the 17,000-Year-Old Skeleton of an Extinct Bison, Bison antiquus

Tracing the evolution of ancient diseases depends on the availability and accessibility of suitable biomarkers in archaeological specimens. DNA is potentially information-rich but it depends on a favourable environment for preservation. In the case of the major mycobacterial pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, robust lipid biomarkers are established as alternatives or complements to DNA analyses. A DNA report, a decade ago, suggested that a 17,000-year-old skeleton of extinct Bison antiquus, from Natural Trap Cave, Wyoming, was the oldest known case of tuberculosis. In the current study, key mycobacterial lipid virulence factor biomarkers were detected in the same two samples from this bison. Fluorescence high-performance liquid chromatography (HPLC) indicated the presence of mycolic acids of the mycobacterial type, but they were degraded and could not be precisely correlated with tuberculosis. However, pristine profiles of C29, C30 and C32 mycocerosates and C27 mycolipenates, typical of the Mycobacterium tuberculosis complex, were recorded by negative ion chemical ionization gas chromatography mass spectrometry of pentafluorobenzyl ester derivatives. These findings were supported by the detection of C34 and C36 phthiocerols, which are usually esterified to the mycocerosates. The existence of Pleistocene tuberculosis in the Americas is confirmed and there are many even older animal bones with well-characterised tuberculous lesions similar to those on the analysed sample. In the absence of any evidence of tuberculosis in human skeletons older than 9,000 years BP, the hypothesis that this disease evolved as a zoonosis, before transfer to humans, is given detailed consideration and discussion.The study was supported by Leverhulme Trust Project Grant F/00 094/BL (GSB, DEM, OY-CL) and Wellcome Trust Grant 080988/Z/06/Z (GSB, OY-CL). A grant from the UK Natural and Environmental Research Council (DEM, GSB) provided access to the Mass Spectrometry Unit at the University of Bristol, UK

Pathophysiological Implications of Cell Envelope Structure in Mycobacterium tuberculosis and Related Taxa

Mycobacterium tuberculosis has a cell envelope incorporating a peptidoglycan-linked arabinogalactan esterified by long-chain mycolic acids. A range of “free” lipids are associated with the “bound” mycolic acids, producing an effective envelope outer membrane. The distribution of these lipids is discontinuous among mycobacteria and such lipids have proven potential for biomarker use in tracing the evolution of tuberculosis. A plausible evolutionary scenario involves progression from an environmental organism, such as Mycobacterium kansasii, through intermediate “smooth” tubercle bacilli, labelled “Mycobacterium canettii”; cell envelope lipid composition possibly correlates with such a progression. M. kansasii and “M. canettii” have characteristic lipooligosaccharides, associated with motility and biofilms, and glycosyl phenolphthiocerol dimycocerosates (“phenolic glycolipids”). Both these lipid classes are absent in modern M. tuberculosis sensu stricto, though simplified phenolic glycolipids remain in certain current biotypes. Dimycocerosates of the phthiocerol family are restricted to smaller phthiodiolone diesters in M. kansasii. Diacyl and pentaacyl trehaloses are present in “M. canettii” and M. tuberculosis, where they are accompanied by related sulfated acyl trehaloses. In comparison with environmental mycobacteria, subtle modifications in mycolic acid structures in “M. canettii” and M. tuberculosis are notable. The probability of essential tuberculosis evolution taking place in Pleistocene megafauna, rather than Homo sapiens, is reemphasised

Evolutionary changes in the genome of Mycobacterium tuberculosis and the human genome from 9000 years BP until modern times

The demonstration of Mycobacterium tuberculosis DNA in ancient skeletons gives researchers an insight into its evolution. Findings of the last two decades sketched the biological relationships between the various species of tubercle bacilli, the time scale involved, their possible origin and dispersal. This paper includes the available evidence and on-going research. In the submerged Eastern Mediterranean Neolithic village of Atlit Yam (9000 BP), a human lineage of M. tuberculosis, defined by the TbD1 deletion in its genome, was demonstrated. An infected infant at the site provides an example of active tuberculosis in a human with a naïve immune system. Over 4000 years later tuberculosis was found in Jericho. Urbanization increases population density encouraging M. tuberculosis/human co-evolution. As susceptible humans die of tuberculosis, survivors develop genetic resistance to disease. Thus in 18th century Hungarian mummies from V ac, 65% were positive for tuberculosis yet a 95-year-old woman had clearly survived a childhood Ghon lesion. Whole genome studies are in progress, to detect changes over the millennia both in bacterial virulence and also host susceptibility/resistance genes that determine the NRAMP protein and Killer Cell Immunoglobulin-like Receptors (KIRs). This paper surveys present evidence and includes initial findings.The contribution made by our many collaborators, researchers and students is gratefully acknowledged. Special acknowledgement is due to Dr Angela Gernaey (deceased) who helped pioneer the early mycolic acid work on the bison bone