Paleopathology

Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5–15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268–14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.

Undoubtedly, one of the major challenges that S. mutans had to overcome as the carbohydrate content of the human diet increased was surviving at low pH. Although S. mutans does not constitute a significant proportion of the oral flora colonizing healthy dentition, it can become numerically significant when there is repeated and sustained acidification of the biofilms associated with excess dietary carbohydrates or impaired salivary function (Burne 1998).

Highlights

• Carbonate δ13C was measured in tooth enamel and bone of Ancient Egyptians.

• δ13C remains largely constant from 5500 to 2000 BP and indicates very low C4-intake.

• High δ15N of mummy hair is indicative of aridity and not of trophic level.

• δ13C of hair indicates <50% of dietary protein came from animals.

• Sulfur isotopes suggest that fish, such as the Nile Perch, was not regularly consumed.

Abstract

Carbon, nitrogen and sulfur stable isotope compositions were measured in hard and soft tissues from Egyptian mummies of humans and animals in order to track the diet of ancient Egyptians from 5500 to 1500 years B.P. The carbon isotope ratios of bone apatite (δ13Cbo = −14.3 ± 0.9‰) and hair protein (δ13Ch = −19.9‰) are compatible with a diet based almost exclusively on C3-derived food (proportion of C4 < 10%). Less negative carbon isotope ratios of enamel (δ13Cen = −11.6 ± 0.7‰) relative to bones from the same mummies could be the result of differences in the chemical microenvironment in which mineralization occurred, as well as of differences in diet between children and adults, in particular through the consumption of milk or millet gruel during infancy and childhood. High values of nitrogen isotope ratios for hair protein (δ15Nh = 9.1‰–15.5‰) are ascribed to aridity rather than fish consumption because the δ34S values of human hair are lower than those measured in Nile perch scales. Except for Coptic mummies, the constancy of δ13Cbo and δ13Cen over a duration of ∼3000 years is striking considering the various political, technological, and cultural changes that impacted the Egyptian civilization during this time interval.

Carbon isotope ratios were measured in enamel, bone, and hair of ancient Egyptians.

 A significant offset (+2.5‰) is observed between the 13C values of teeth and bones that

 cannot be ascribed to the weaning effect. Following Warinner and Tuross (2009), this isotopic

offset rather may be caused by differences in mineralization conditions of the two types of

tissue. Using tissue-specific equations, the 13C value of the reconstructed diet is comparable and close to the average value of C3-plants (-25‰). 13C values of hair from ancient

with previous studies (Iacumin et al., 1996; Thompson et al., 2005).

Egyptians also suggest that C4-derived foods were rare in the diet (<10%), a result consistent

proportion of protein of animal origin may have reached 50%. Both estimates are lower than
Sulfur isotope ratios of mummy hairs further indicate that freshwater fish, such as the Nile

Carbon isotope ratios in mineralized tissues are constant throughout the studied period, indicating a preference for C3-derived food throughout the investigated time span. This is a surprising result given that C4 plants are better suited to arid environments, and that the climate became increasingly arid during this period (Touzeau et al., 2013). Coptic mummies have 13C values slightly lower than other mummies, possibly as a result of the introduction of olive oil during the Roman Period.

Assessing the consumption of animal products is difficult because the 15N of soft tissues, such as hair, is controlled by parameters other than diet, and in particular by the prevailing hydric stress. Using the carbon isotope ratios of mummy hairs, the contribution of animal protein to the total dietary protein was estimated here at 29±19%, corresponding to an ovo-lacto-vegetarian diet. Taking into account potential biases in the diet reconstruction, the

the average value of 64% characterizing modern omnivorous Europeans (Petzke et al., 2005).

perch, was not consumed in significant proportions.

Summary

Background 

Atherosclerosis is thought to be a disease of modern human beings and related to contemporary lifestyles. However, its prevalence before the modern era is unknown. We aimed to evaluate preindustrial populations for atherosclerosis.

Methods 

We obtained whole body CT scans of 137 mummies from four different geographical regions or populations spanning more than 4000 years. Individuals from ancient Egypt, ancient Peru, the Ancestral Puebloans of southwest America, and the Unangan of the Aleutian Islands were imaged. Atherosclerosis was regarded as definite if a calcified plaque was seen in the wall of an artery and probable if calcifications were seen along the expected course of an artery.

Findings 

Probable or definite atherosclerosis was noted in 47 (34%) of 137 mummies and in all four geographical populations: 29 (38%) of 76 ancient Egyptians, 13 (25%) of 51 ancient Peruvians, two (40%) of five Ancestral Puebloans, and three (60%) of five Unangan hunter gatherers (p=NS). Atherosclerosis was present in the aorta in 28 (20%) mummies, iliac or femoral arteries in 25 (18%), popliteal or tibial arteries in 25 (18%), carotid arteries in 17 (12%), and coronary arteries in six (4%). Of the five vascular beds examined, atherosclerosis was present in one to two beds in 34 (25%) mummies, in three to four beds in 11 (8%), and in all five vascular beds in two (1%). Age at time of death was positively correlated with atherosclerosis (mean age at death was 43 [SD 10] years for mummies with atherosclerosis vs 32 [15] years for those without; p<0·0001) and with the number of arterial beds involved (mean age was 32 [SD 15] years for mummies with no atherosclerosis, 42 [10] years for those with atherosclerosis in one or two beds, and 44 [8] years for those with atherosclerosis in three to five beds; p < 0.0001).

Interpretation 

Atherosclerosis was common in four preindustrial populations including preagricultural hunter- gatherers. Although commonly assumed to be a modern disease, the presence of atherosclerosis in premodern human beings raises the possibility of a more basic predisposition to the disease.

37 mummies from populations of four disparate geo- graphic regions were studied by whole body CT scanning: 76 ancient Egyptians (predynastic era, ca 3100 BCE, to the end of the Roman era, 364 CE, 13 excavation sites), 51 early intermediate to late horizon peoples in present day Peru (ca 200–1500 CE, five excavation sites), five Ancestral Puebloan of the Archaic and Basketmaker II cultures living in southwest America (ca 1500 BCE to 1500 CE, five excavation sites), and five Unangan people living in the Aleutian Islands of modern day Alaska (ca 1756–1930 CE,

one excavation site). These geographical areas were selected because of access to mummies with appropriate age and varied cultural attributes. Mummies were selected for imaging on the basis of their good state of preservation and the likelihood of being adults. Mummies were not selected for study in a random fashion.

The classic pattern of cardiac pain--radiation to the left arm--was so well known that the ancient Egyptians and Copts even identified the left ring finger as the "heart" finger.

Altogether, ancient Egyptians were aware of a variety of abnormal cardiac conditions, particularly of angina pectoris and sudden death, arrhythmia, aneurysm, congestive heart failure, and venous insufficiency. Numerous remedies for afflicitions of the heart are found throughout the Ebers payrus. 

There were a range of them using different foods, some even including carbohydrates like dates or honey and dough, but interesting, there is another combination of "fat flesh, incense, garlic, and writing fluid".

Extensive histologic analysis of mummies began, however; well before the development of the scanning electron microscope. In 1912, Shattock' made sections of the calcified aorta of Pharaoh Merneptah; and the work of Sir Marc Armand Rufer, published posthumously in 1921, is our most valuable early source of information about vascular disease in ancient Egyptians. Ruffer was able to study a relatively large number of tissue specimens from mummies, mainly from New Kingdom (1600-1100 BC) burials, but covering a wide period of time. In a mummy of the 28th to 30th Dynasty (404-343 BC), he observed atheromas in the common carotids and calcific atheromas in the left subclavian, common iliac, and more peripheral arteries. Ruffer concluded from the state of the costal cartilage that this mummy was not that of an old person. A mummy of a man of the Greek period (ca. 300 to 30 BC), who died at not over 50 years of age, showed atheromas of the aorta and brachial arteries. Since the discoveries of Rufer, numerous other mummies, whose ages at death ranged from the 4th to the 8th decade, have shown similar vascular changes (Fig.4).

In 1931, Long described a female mummy of the 21st Dynasty (1070-945 BC), found at Deir-el- Bahari-that of the lady Teye, who died at about 50 years of age. The heart showed calcification of one mitral cusp, and thickening and calcification of the coronary arteries. The myocardium is said to have had patchy fibrosis, and the aorta "nodular arteriosclerosis." The renal capsule was thickened, many of the glomeruli were fibrosed, and the medium-sized renal vessels were sclerotic. The condition appears to be that of hypertensive arteriosclerotic disease associated with atheromatous change. In the 1960s, Sandison examined and photographed mummy arteries using modern histologic methods (Fig.5). Arteries in the mummy tissues were described as tape-like, but could be dissected easily, whereupon arteriosclerosis, atheroma with lipid depositions, reduplication of the internal elastic lamina, and medial calcification were readily visible under microscopy.

Still more recently, one of the most extensively studied Egyptian mummies has been PUMIL from the Pennsylvania University Museum(hence its initials), now on loan to the National Museum of Natural History at the Smithsonian. It is believed to be from the later Ptolemaic period, circa 170BC. The heart and portions of an atherosclerotic aorta were found in the abdominal cavity. Histologically, large and small arterioles and arteries from other organs showed areas of intimal fibrous thickening typical of sclerosis. These findings are particularly striking since the estimated age of PUM I at time of death was between 35 and 40 years.