A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade.

Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species-Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14th-century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14th CE and suggest that its divergence was due to human and ruminant co-migration.

Examining pathogen DNA recovery across the remains of a 14th century Italian friar (Blessed Sante) infected with Brucella melitensis.

OBJECTIVE: To investigate variation in ancient DNA recovery of Brucella melitensis, the causative agent of brucellosis, from multiple tissues belonging to one individual MATERIALS: 14 samples were analyzed from the mummified remains of the Blessed Sante, a 14 th century Franciscan friar from central Italy, with macroscopic diagnosis of probable brucellosis. METHODS: Shotgun sequencing data from was examined to determine the presence of Brucella DNA. RESULTS: Three of the 14 samples contained authentic ancient DNA, identified as belonging to B. melitensis. A genome (23.81X depth coverage, 0.98 breadth coverage) was recovered from a kidney stone. Nine of the samples contained reads classified as B. melitensis (7-169), but for many the data quality was insufficient to withstand our identification and authentication criteria. CONCLUSIONS: We identified significant variation in the preservation and abundance of B. melitensis DNA present across multiple tissues, with calcified nodules yielding the highest number of authenticated reads. This shows how greatly sample selection can impact pathogen identification. SIGNIFICANCE: Our results demonstrate variation in the preservation and recovery of pathogen DNA across tissues. This study highlights the importance of sample selection in the reconstruction of infectious disease burden and highlights the importance of a holistic approach to identifying disease. LIMITATIONS: Study focuses on pathogen recovery in a single individual. SUGGESTIONS FOR FURTHER RESEARCH: Further analysis of how sampling impacts aDNA recovery will improve pathogen aDNA recovery and advance our understanding of disease in past peoples.

A 16th century Escherichia coli draft genome associated with an opportunistic bile infection.

Escherichia coli - one of the most characterized bacteria and a major public health concern - remains invisible across the temporal landscape. Here, we present the meticulous reconstruction of the first ancient E. coli genome from a 16th century gallstone from an Italian mummy with chronic cholecystitis. We isolated ancient DNA and reconstructed the ancient E. coli genome. It consisted of one chromosome of 4446 genes and two putative plasmids with 52 genes. The E. coli strain belonged to the phylogroup A and an exceptionally rare sequence type 4995. The type VI secretion system component genes appears to be horizontally acquired from Klebsiella aerogenes, however we could not identify any pathovar specific genes nor any acquired antibiotic resistances. A sepsis mouse assay showed that a closely related contemporary E. coli strain was avirulent. Our reconstruction of this ancient E. coli helps paint a more complete picture of the burden of opportunistic infections of the past.

A human MMTV-like betaretrovirus linked to breast cancer has been present in humans at least since the copper age.

The betaretrovirus Mouse Mammary Tumor Virus (MMTV) is the well characterized etiological agent of mammary tumors in mice. In contrast, the etiology of sporadic human breast cancer (BC) is unknown, but accumulating data indicate a possible viral origin also for these malignancies. The presence of MMTVenv-like sequences (MMTVels) in the human salivary glands and saliva supports the latter as possible route of inter-human dissemination. In the absence of the demonstration of a mouse-man transmission of MMTV, we considered the possibility that a cross-species transmission could have occurred in ancient times. Therefore, we investigated MMTVels in the ancient dental calculus, which originates from saliva and is an excellent material for paleovirology. The calculus was collected from 36 ancient human skulls, excluding any possible mouse contamination. MMTV-like sequences were identified in the calculus of 6 individuals dated from the Copper Age to the 17th century. The MMTV-like sequences were compared with known human endogenous betaretroviruses and with animal exogenous betaretroviruses, confirming their exogenous origin and relation to MMTV. These data reveal that a human exogenous betaretrovirus similar to MMTV has existed at least since 4,500 years ago and indirectly support the hypothesis that it could play a role in human breast cancer.

Identification of Italian Renaissance noble women through a bioarchaeological study of skeletal remains.

Three female skeletons, dated back to the beginning of the 15th century were recovered during an archaeological excavation in the Guinigi Chapel in Lucca (Italy). Archaeological and historical sources indicated that the remains might be those of Paolo Guinigi's wives. At the beginning of the 15th century Paolo Guinigi ruled Lucca for nearly 30 years (1400-1429), providing a long period of wealth and peace to the city. In those years of power Paolo Guinigi married four women from different countries. This study is aimed at identifying the three skeletons of Paolo's wives found in the Guinigi Chapel. The three skeletons were identified using a multidisciplinary approach, by integrating historical, archaeological, anthropological, and isotopic data. The anthropological studies evidenced the biological profile of two adult women and of one adolescent female, in agreement with the historical descriptions of three of the four wives of Paolo Guinigi. The isotopic analysis supported the identification of each individual.

Commensal and Pathogenic Members of the Dental Calculus Microbiome of Badia Pozzeveri Individuals from the 11th to 19th Centuries.

The concept of the human oral microbiome was applied to understand health and disease, lifestyles, and dietary habits throughout part of human history. In the present study, we augment the understanding of ancient oral microbiomes by characterizing human dental calculus samples recovered from the ancient Abbey of Badia Pozzeveri (central Italy), with differences in socioeconomic status, time period, burial type, and sex. Samples dating from the Middle Ages (11th century) to the Industrial Revolution era (19th century) were characterized using high-throughput sequencing of the 16S ribosomal RNA (rRNA) gene V4 region. Consistent with previous studies, individuals from Badia Pozzeveri possessed commensal oral bacteria that resembled modern oral microbiomes. These results suggest that members of the oral microbiome are ubiquitous despite differences in geographical regions, time period, sex, and socioeconomic status. The presence of fecal bacteria could be in agreement with poor hygiene practices, consistent with the time period. Respiratory tract, nosocomial, and other rare pathogens detected in the dental calculus samples are intriguing and could suggest subject-specific comorbidities that could be reflected in the oral microbiome.

A case of erosive polyarthropathy from Medieval northern Italy (12th-13th centuries).

OBJECTIVE: To evaluate and differentially diagnose erosive skeletal lesions located on multiple joints of an individual archaeologically recovered in 2017. MATERIALS: Skeletal remains of a well-preserved skeleton dating to the 12th-13th centuries from the medieval church of San Biagio in Cittiglio (Varese, northern Italy). METHODS: Macroscopic and radiographic imaging. RESULTS: Erosive marginal symmetrical lesions are present on the metatarsophalangeal, metacarpophalangeal and interphalangeal joints of an adult male, aged 55-75 years. Osteolytic changes, in the form of pocket erosions, surface resorptions and pseudocyst formations, are also macroscopically observed on some carpal and tarsal bones and on several large peripheral joints. CONCLUSIONS: A careful differential diagnosis of the lesions and their macroscopic and radiological appearance is suggestive of a case of rheumatoid arthritis-like polyarthropathy. SIGNIFICANCE: This case contributes to the debate regarding the antiquity of erosive polyarthropathies, providing additional evidence for the existence of these diseases in the Old World prior to the European discovery of the Americas. LIMITATIONS: Small sample size limits discussion of the scope of the disease in antiquity. SUGGESTIONS FOR FURTHER RESEARCH: This case highlights the need for further macroscopic, radiographic, and biomolecular studies of pre-modern European skeletal samples to investigate the hypothesized pre-existence of these pathological conditions in Europe prior to 1492.

Multiple myeloma in paleopathology: A critical review.

This paper provides a critical literature review concerning paleopathological evidence of multiple myeloma discovered both in the Old and in the New World. A critical assessment of the bioarchaeological and paleopathological documentary sources permitted to identify a total of 25 ascertained cases of multiple myeloma from different geographical areas in the world ranging from Prehistoric times up to the Contemporary age. The distribution of multiple myeloma findings in past times shows that the majority of cases have been discovered in the Old World (n = 18) and extend back to the Middle Ages, while the evidences in the New World (n = 7) seems to date back to the pre-Columbian era. The demographic profile suggests a sex ratio of 1.3:1, while 91.7% of affected individuals (n = 24) are adults. This critical review also discusses the diagnostic criteria and methodological issues commonly attempted in paleo-oncological research, with particular regard to the differential diagnosis of multiple myeloma. As such, the main focus of this work is to present a comprehensive and exhaustive scrutiny of the skeletal manifestations identified as multiple myeloma in order to improve the accuracy of diagnoses within the field of paleopathology.

Metastatic Prostate Carcinoma from Imperial Rome (1st to 2nd Centuries AD).

OBJECTIVE: This study describes and discusses a rare case of metastatic carcinoma that affected the skeleton of an adult male recovered in the necropolis of Casal Bertone in Rome (Italy). The necropolis, which dates back to the Imperial Age (1st to 2nd century AD), is located near some residential structures and a large place identified as a fullery (fullonica). METHODS: Anthropological and paleopathological studies of the skeletal remains were performed via careful macroscopic, microscopic, radiological (X-ray and CT scan), and histological investigation. RESULTS: The skeleton displayed mixed osteoclastic and osteoblastic lesions that mainly involved the axial bones, in particular the sternum, the ribs, the spine, and the scapular and pelvic girdles. The anatomical distribution and the destructive and proliferative nature of the lesions suggested diffuse metastases arising from a soft-tissue primary cancer. The age and sex of the individual, as well as radiographic and histological pictures, allowed diagnosis of an advanced prostate cancer with extensively diffused bone metastases. CONCLUSION: At present, this is the only case of prostate cancer from the Imperial Age recovered in Rome.

Histology of ancient soft tissue tumors: A review.

Ancient neoplasms diagnosed in the soft tissues of mummies are limited to 18 cases so far, with only 5 malignant tumors. The apparent paucity of neoplasms in ancient populations is sometimes attributed to shorter life spans and fewer oncogenic substances in the environment. However, this paucity may also be a result of the scarcity of autopsies of mummies, together with technical difficulties in detecting neoplastic lesions in mummified tissues. An exception, and example of the benefits of thorough systematic analysis, is the small sample of 10 Renaissance mummies from Naples (15th-16th centuries), in which 3 cases of cancer were found. In order to increase detection of soft tissue tumors, it is imperative that mummies undergo systematic autopsies and histological examinations performed by skilled paleopathologists. This review of the known ancient soft tissue neoplasms demonstrates the state of histology of malignant and benign soft tissue neoplasms in mummies, and the potential for further study. The limitations of paleopathological diagnosis will be discussed and an argument will be made for the use of autopsies and histological analysis on mummified human remains.

Correction: The paradox of HBV evolution as revealed from a 16th century mummy.

[This corrects the article DOI: 10.1371/journal.ppat.1006750.].

The paradox of HBV evolution as revealed from a 16th century mummy.

Hepatitis B virus (HBV) is a ubiquitous viral pathogen associated with large-scale morbidity and mortality in humans. However, there is considerable uncertainty over the time-scale of its origin and evolution. Initial shotgun data from a mid-16th century Italian child mummy, that was previously paleopathologically identified as having been infected with Variola virus (VARV, the agent of smallpox), showed no DNA reads for VARV yet did for hepatitis B virus (HBV). Previously, electron microscopy provided evidence for the presence of VARV in this sample, although similar analyses conducted here did not reveal any VARV particles. We attempted to enrich and sequence for both VARV and HBV DNA. Although we did not recover any reads identified as VARV, we were successful in reconstructing an HBV genome at 163.8X coverage. Strikingly, both the HBV sequence and that of the associated host mitochondrial DNA displayed a nearly identical cytosine deamination pattern near the termini of DNA fragments, characteristic of an ancient origin. In contrast, phylogenetic analyses revealed a close relationship between the putative ancient virus and contemporary HBV strains (of genotype D), at first suggesting contamination. In addressing this paradox we demonstrate that HBV evolution is characterized by a marked lack of temporal structure. This confounds attempts to use molecular clock-based methods to date the origin of this virus over the time-frame sampled so far, and means that phylogenetic measures alone cannot yet be used to determine HBV sequence authenticity. If genuine, this phylogenetic pattern indicates that the genotypes of HBV diversified long before the 16th century, and enables comparison of potential pathogenic similarities between modern and ancient HBV. These results have important implications for our understanding of the emergence and evolution of this common viral pathogen.

Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.