2000-year-old pathogen genomes reconstructed from metagenomic analysis of Egyptian mummified individuals.

BACKGROUND: Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. RESULTS: The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus' microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-year-old human hepatitis B virus, were successfully reconstructed. CONCLUSIONS: The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA.

The microbiomes of a XVIII century mummy from the castle of Krásna Hôrka (Slovakia) and its surrounding environment.

This microbiological survey was performed to determine the conservation state of a mummy in the Slovak castle of Krásna Hôrka and its surrounding environment. Culture-dependent identification was coupled with biodegradation assays on keratin, gelatin and cellulose. Next Generation Sequencing (NGS) using Illumina platform was used for a deeper microbial investigation. Three environmental samples were collected: from the glass of the sarcophagus, from the air inside it, and from the air of the chapel where the mummy is located. Seven different samples were taken from mummy's surface: from the left ear, left-hand palm, left-hand nail, left instep, right hand, abdomen and mineral crystals embedded within the skin. Three internal organ samples, from the lung, pleura and stomach, were also included in this study. Together, the culture-dependent and culture-independent analyses revealed that the bacterial communities present had fewer taxa than the fungal ones. The mycobiome showed the largest variability and included Epicoccum nigrum, Penicillium spp., Alternaria spp., Aspergillus spp., Cladosporium spp. and Aureobasidium pullulans; many other Ascomycota and Basidiomycota genera were detected by NGS. The most interesting results came from the skin mineral crystals and the internal organs. The hydrolytic assays revealed those microorganisms which might be considered dangerous 'mummy pathogens'. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Halophilic bacteria are colonizing the exhibition areas of the Capuchin Catacombs in Palermo, Italy.

The Capuchin Catacombs of Palermo, Italy, contain over 1800 mummies dating from the 16th to 20th centuries AD. Their environment is not conducive to the conservation of the remains due to, among other factors, water infiltration, which is producing salt efflorescences on the walls. A multiphasic approach was applied to investigate the halophilic microbiota present in the Catacombs. Enrichment cultures were conducted on media containing different NaCl concentrations, ranging from 3 to 20 %. For screening of the strains, the following two PCR-based methods were used and compared: fluorescence internal transcribed spacer PCR (f-ITS) and random amplification of polymorphic DNA (RAPD) analyses. Results derived from RAPD profiles were shown to be slightly more discriminative than those derived from f-ITS. In addition, the proteolytic and cellulolytic abilities were screened through the use of plate assays, gelatin agar and Ostazin Brilliant Red H-3B (OBR-HEC), respectively. Many of the strains isolated from the wall samples displayed proteolytic activities, such as all strains belonging to the genera Bacillus, Virgibacillus and Arthrobacter, as well as some strains related to the genera Oceanobacillus, Halobacillus and Idiomarina. In addition, many of the strains isolated from materials employed to stuff the mummies showed cellulolytic activities, such as those related to species of the genera Chromohalobacter and Nesterenkonia, as well as those identified as Staphylococcus equorum and Halomonas sp. Furthermore, many of the strains were pigmented ranging from yellow to a strong pink color, being directly related to the discoloration displayed by the materials.

[Decomposition of corpses--a microbial degradation process with special reference to mummification, formation of adipocere and incomplete putrified corpes]. / Leichenzersetzung--ein mikrobieller Abbauprozess (unter besonderer Berücksichtigung der Entstehung von Mumien, Fettwachs und Faulleichen).

Decomposition of the human body is a microbial process. It is influenced by the environmental situation and it depends to a high degree on the exchange of substances between the corpse and the environment. Mummification occurs at low humidity or frost. Adipocere arises from lack of oxygen, incomplete putrified corpses develop when there is no exchange of air or water between the corpse and the environment.

Microbiological analysis of the mummy of St. Marcian.

The paper provides the results of the microbiological analysis of samples taken from the mummified remains of St. Marcian. Microorganisms found belong to bacteria and fungi of the genera Aspergillus, Bacillus, Penicillium, Sarcina, and Shewanella which are all apart of the common air, soil, and human microbial flora and therefore present no health hazard.

Verification of tuberculosis infection among Vác mummies (18th century CE, Hungary) based on lipid biomarker profiling with a new HPLC-HESI-MS approach.

Tuberculosis (TB) was a large burden of infections that peaked during the 19th century in Europe. Mummies from the 18th century CE, discovered in the crypt of a church at Vác, Hungary, had high TB prevalence, as revealed by amplification of key fragments of TB DNA and genome-wide TB analysis. Complementary methods are needed to confirm these diagnoses and one approach uses the identification of specific lipid biomarkers, such as TB mycocerosic acids (MCs). Previously, MC derivatives were profiled by specialised gas chromatography-mass spectrometry (GC-MS), so an alternative more direct approach has been developed. Underivatized MCs are extracted and analysed by high-performance liquid chromatography linked to a mass spectrometer, in heated electrospray ionisation mode (HPLC-HESI-MS). The method was validated using representatives of the Mycobacterium tuberculosis complex and other mycobacteria and tested on six Vác mummy cases, previously considered positive for TB infection. Analysing both rib and soft tissue samples, four out of six cases gave profiles of main C32 and major C29 and C39 mycocerosates correlating well with those of M. tuberculosis. Multidisciplinary methods are needed in the diagnosis of ancient tuberculosis; this new protocol accesses important confirmatory evidence, as demonstrated by the confirmation of TB in the Vác mummies.

Chronic active non-lethal human-type tuberculosis in a high royal Bavarian officer of Napoleonic times-a mummy study.

In paleopathology, morphological and molecular evidence for infection by mycobacteria of the M. tuberculosis complex (MTC) is frequently associated with early death. In the present report, we describe a multidisciplinary study of a well-preserved mummy from Napoleonic times with a long-standing tuberculous infection by M. tuberculosis senso stricto who died at the age of 88 years of focal and non-MTB related bronchopneumonia. The well-preserved natural mummy of the Royal Bavarian General, Count Heinrich LII Reuss-Köstritz (1763-1851 CE), was extensively investigated by macro- and histomorphology, whole body CT scans and organ radiography, various molecular tissue analyses, including stable isotope analysis and molecular genetic tests. We identified signs for a long-standing, but terminally inactive pulmonary tuberculosis, tuberculous destruction of the second lumbar vertebral body, and a large tuberculous abscess in the right (retroperitoneal) psoas region (a cold abscess). This cold abscess harboured an active tuberculous infection as evidenced by histological and molecular tests. Radiological and histological analysis further revealed extensive arteriosclerosis with (non-obliterating) coronary and significant carotid arteriosclerosis, healthy bone tissue without evidence of age-related osteopenia, evidence for diffuse idiopathic skeletal hyperostosis and mild osteoarthrosis of few joints. This suggests excellent living conditions correlating well with his diet indicated by stable isotope results and literary evidence. Despite the clear evidence of a tuberculous cold abscess with bacterioscopic and molecular proof for a persisting MTC infection of a human-type M. tuberculosis strain, we can exclude the chronic MTC infection as cause of death. The detection of MTC in historic individuals should therefore be interpreted with great caution and include further data, such as their nutritional status.

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.

Microbiological analysis of a mummy from the archeological museum in Zagreb.

In this paper we report the results of the microbiological analysis of the samples taken from the mummy from the collection of the Archaeological museum in Zagreb, Croatia. Samples were taken from specific places such as oral, orbital, abdominal cavity and bandages surrounding the mummy, and analyzed in Department of Microbiology and Hospital Infections in University Hospital "Dubrava" in Zagreb and in National Reference Laboratory for systemic mycoses of Croatian National Institute of Public Health in Zagreb. The analysis indicated that all of the found organisms were non-primary pathogenic and are not harmful for healthy humans. Isolated microorganisms mainly belonged to the group of saprophytic fungi as listed: Monilia spp., Penicillium spp., Alternaria spp., Aspergillus fumigatus, Aspergillus nidulans, Rhizopus spp. and Chrysosporium spp. and to the genus of saprophytic bacteria, Bacillus spp.

The past, present and future of ancient bacterial DNA.

Groundbreaking studies conducted in the mid-1980s demonstrated the possibility of sequencing ancient DNA (aDNA), which has allowed us to answer fundamental questions about the human past. Microbiologists were thus given a powerful tool to glimpse directly into inscrutable bacterial history, hitherto inaccessible due to a poor fossil record. Initially plagued by concerns regarding contamination, the field has grown alongside technical progress, with the advent of high-throughput sequencing being a breakthrough in sequence output and authentication. Albeit burdened with challenges unique to the analysis of bacteria, a growing number of viable sources for aDNA has opened multiple avenues of microbial research. Ancient pathogens have been extracted from bones, dental pulp, mummies and historical medical specimens and have answered focal historical questions such as identifying the aetiological agent of the black death as Yersinia pestis. Furthermore, ancient human microbiomes from fossilized faeces, mummies and dental plaque have shown shifts in human commensals through the Neolithic demographic transition and industrial revolution, whereas environmental isolates stemming from permafrost samples have revealed signs of ancient antimicrobial resistance. Culminating in an ever-growing repertoire of ancient genomes, the quickly expanding body of bacterial aDNA studies has also enabled comparisons of ancient genomes to their extant counterparts, illuminating the evolutionary history of bacteria. In this review we summarize the present avenues of research and contextualize them in the past of the field whilst also pointing towards questions still to be answered.

Presence of Helicobacter pylori in a Mexican Pre-Columbian Mummy.

BACKGROUND: Recent studies showed that Helicobacter pylori existed in the New World prior to the arrival of Columbus. The purpose of the present study was to detect the presence of Helicobacter pylori in pre-Columbian mummies from Northern Mexico. METHODS: Six samples were studied (four samples of gastric remains, tongue-soft palate, and brain remained as negative controls) from two of the six naturally mummified corpses studied (adult male and infant male). Samples were taken from tissues suitable for DNA amplification by Polymerase chain reaction (PCR). DNA was extracted and H. pylori detection was carried out by PCR and hybridized with the pHp probe from 16S rRNA gene. The purified PCR products were cloned and sequenced in both directions. DNA sequences were analyzed with ALIGN and BLAST software. A second amplification was performed using ureB gene by real-time PCR. RESULTS: From four samples of gastric remnant, only two were H. pylori-positive for amplification of a 109 bp DNA fragment; the remaining two were negative, as were the tongue-soft palate and the brain biopsies as well. These PCR products were hybridized with a pHp probe. Nucleotide sequence analysis showed homology with H. pylori in 98 of 99% when compared with the gene bank nucleotide sequence. Only one sample of gastric remnant H. pylori-positive with 16S rRNA gene was also positive for ureB gene from H. pylori. CONCLUSION: This data supported infection with H. pylori in Mexican pre-Columbian mummies dating from approximately 1,350 AC.

Attempts to revive Mycobacterium tuberculosis from 300-year-old human mummies.

Environmental persistence of Mycobacterium tuberculosis is subject to speculation. However, the reality that infected postmortem tissues can be a danger to pathologists and embalmers has worrisome implications. A few experimental studies have demonstrated the organism's ability to withstand exposure to embalming fluid and formalin. Recently, a failure was reported in an attempt to resuscitate an original isolate of Robert Koch to determine the lifetime of the tubercle bacillus. The present study also considers a historical approach to determine persistence under favorable environmental conditions. It asks whether acid-fast forms observed in tissues of 300-year-old Hungarian mummies can be resuscitated. Finding organisms before the advent of antibiotics and pasteurization may yield valuable genetic information. Using various media modifications, as well as guinea pig inoculation, an attempt was made to culture these tissues for M. tuberculosis. In addition, a resuscitation-promoting factor, known to increase colony counts in high G+C bacteria, was applied to the cultures. Although an occasional PCR-positive sample was detected, no colonies of M. tuberculosis were obtained. Our results may indicate that the life span of the tubercle bacillus is less than a few hundred years, even though in the short run it can survive harsh chemical treatment.

Helicobacter pylori DNA obtained from the stomach specimens of two 17th century Korean mummies.

ABSTRACT: Helicobacter pylori is a bacterium that grows in the stomach mucosal epithelium, and can induce gastric diseases. Although many studies on modern H. pylori genomes have been reported from all over the world, a comprehensive picture of H. pylori is still lacking. Therefore, there is a pressing need to obtain archaeological specimens and to subject the ancient DNA (aDNA) extracted therefrom to analysis. Considering the typically excellent state of preservation of Joseon mummies discovered in Korea, we thus tried to isolate ancient H. pylori DNA from their mummified stomach specimens. After screening Korean mummy stomachs containing remnant H. pylori DNA, vacA (s- and m-region) alleles were successfully identified in the stomach isolates of two samples. The H. pylori strains identified had vacA s1/m2 (Cheongdo mummy) and s1 (Dangjin mummy) alleles. This paper is significant in that it is the first report of presumptive ancient H. pylori DNA obtained from East Asian archaeological specimens. However, full characterization and exploitation of ancient H. pylori DNA remnant in Joseon mummy specimens will require subsequent investigations utilizing the most cutting-edge techniques established for the analysis of ancient intestinal-content samples, such as next-generation sequencing (NGS).

[Effectiveness of imazalil to control the effect of fungal deterioration on mummies at the Mexico City Museum "El Carmen"]. / Efectividad del imazalil en el control del deterioro por hongos de momias del museo de El Carmen, Ciudad de México.

We present a study on the control and elimination of the fungi affecting the mummies specifically at the museum "El Carmen", in San Angel, Mexico City. Twelve analysed mummies presented an important deterioration attributed to colonizing fungi. The degree of fungal contamination and the efficacy of imazalil were evaluated. Two samplings were performed in order to isolate and identify the fungal genera, one for control and the other after the treatment. Isolation was done by the carpet-square technique and identification was performed by morphological features. Each sampling gave a total of 100 samples as follows: 17 from the air, 23 from the walls and 60 from the mummies. Samples were cultured on Sabouraud dextrose agar. From the first sampling a total of 649 colonies corresponding to 24 genera were obtained being the most frequent Penicillium, Cladophialophora and Aspergillus. From the second sampling, after the imazalil treatment, which was applied by means of lit candles containing the antifungal drug, 57 colonies were recovered, representing a 91.2% fungal reduction; 18 genera were eliminated. In spite of resistance showed by many Penicillium strains, the imazalil is an alternative drug for the control of fungal colonization on these studied materials.

[Tuberculosis in ancient Egypt]. / La tuberculose en ancienne Egypte.

INTRODUCTION: Did Tuberculosis plague Ancient Egypt five millennia ago? STATE OF THE ART: Some medical papyri appear to evoke tuberculosis. Egyptian physicians did not individualize it, but they seem to have noticed some of its clinical expressions, such as cough, cervical adenitis, and cold abscesses. In Egyptian iconography, some cases of hump-backs were probably due to Pott's disease of the spine Descriptive paleopathology, born with the 20th century, has identified pulmonary and especially spinal lesions compatible with tuberculosis. PERSPECTIVES: Progress of molecular biology has made a decisive contribution with the diagnosis of tuberculosis on ancient samples. Tuberculosis has been identified using PCR in nearly a third of the Egyptian mummies recently examined. Spoligotyping has made it possible to re-evaluate the phylogenic tree of the Mycobacterium tuberculosis complex in Ancient Egypt. CONCLUSIONS: Tuberculosis certainly plagued the Nile Valley and appears to have been an important cause of mortality in Ancient Egypt.

What pre-Columbian mummies could teach us about South American leishmaniases?

A recent report on the taxonomic profile of the human gut microbiome in pre-Columbian mummies (Santiago-Rodriguez et al. 2016) gives for the first time evidence of the presence of Leishmania DNA (sequences similar to Leishmania donovani according to the authors) that can be reminiscent of visceral leishmaniasis during the pre-Columbian era. It is commonly assumed that Leishmania infantum, the etiological agent of American visceral leishmaniasis (AVL) was introduced into the New World by the Iberian conquest. This finding is really surprising and must be put into perspective with what is known from an AVL epidemiological and historical point of view. Beside L. infantum, there are other species that are occasionally reported to cause AVL in the New World. Among these, L. colombiensis is present in the region of pre-Columbian mummies studied. Other explanations for these findings include a more ancient introduction of a visceral species of Leishmania from the Old World or the existence of a yet unidentified endemic species causing visceral leishmaniasis in South America. Unfortunately, very few molecular data are known about this very long pre-Columbian period concerning the circulating species of Leishmania and their diversity in America.

Ancient bacteria of the Ötzi's microbiome: a genomic tale from the Copper Age.

BACKGROUND: Ancient microbiota information represents an important resource to evaluate bacterial evolution and to explore the biological spread of infectious diseases in history. The soft tissue of frozen mummified humans, such as the Tyrolean Iceman, has been shown to contain bacterial DNA that is suitable for population profiling of the prehistoric bacteria that colonized such ancient human hosts. RESULTS: Here, we performed a microbial cataloging of the distal gut microbiota of the Tyrolean Iceman, which highlights a predominant abundance of Clostridium and Pseudomonas species. Furthermore, in silico analyses allowed the reconstruction of the genome sequences of five ancient bacterial genomes, including apparent pathogenic ancestor strains of Clostridium perfringens and Pseudomonas veronii species present in the gut of the Tyrolean Iceman. CONCLUSIONS: Genomic analyses of the reconstructed C. perfringens chromosome clearly support the occurrence of a pathogenic profile consisting of virulence genes already existing in the ancient strain, thereby reinforcing the notion of a very early speciation of this taxon towards a pathogenic phenotype. In contrast, the evolutionary development of P. veronii appears to be characterized by the acquisition of antibiotic resistance genes in more recent times as well as an evolution towards an ecological niche outside of the (human) gastrointestinal tract.