Identification of bacterial isolates obtained from intestinal contents associated with 12,000-year-old mastodon remains

Abstract

Mastodon (Mammut americanum) remains unearthed during excavation of ancient sediments usually consist only of skeletal material, due to postmortem decomposition of soft tissues by microorganisms. Two recent excavations of skeletal remains in anoxic sediments in Ohio and Michigan, however, have uncovered organic masses which appear to be remnants of the small and large intestines, respectively. Macrobotanical examinations of the composition of these masses revealed assemblages of plant material radiocarbon dated to approximately 11,500 years before the present and thought to be incompletely digested food remains from this extinct mammal. We attempted to cultivate and identify bacteria from the intestinal contents, bone-associated sediments, and sediments not in proximity to the remains using a variety of general and selective media. In all, 295 isolates were cultivated, and 38 individual taxa were identified by fatty acid-methyl ester (FAME) profiles and biochemical characteristics (API-20E). The taxonomic positions of selected enteric and obligately anaerobic bacteria were confirmed by 16S ribosomal DNA (rDNA) sequencing. Results indicate that the intestinal and bone-associated samples contained the greatest diversity of bacterial taxa and that members of the family Enterobacteriaceae represented 41% of all isolates and were predominant in the intestinal masses and sediments in proximity to the skeleton but were uncommon in the background sediments. Enterobacter cloacae was the most commonly identified isolate, and partial rDNA sequencing revealed that Rahnella aquatilis was the correct identity of strains suggested by FAME profiles to be Yersinia enterocolitica. No Bacteroides spp. or expected intestinal anaerobes were recovered. The only obligate anaerobes recovered were clostridia, and these were not recovered from the small intestinal masses. Microbiological evidence from this study supports other, macrobotanical data indicating the intestinal origin of these masses. Whether these organisms are direct descendants of the original intestinal microbiota, however, cannot be established.

FIG. 1

Schematic vertical cross sections through features inferred as preserved intestinal contents (Plant Debris) at the Burning Tree (A) mastodon site (Licking Co., Ohio) and at the Heisler (B) mastodon site (Calhoun Co., Mich.). The matrix and major constituents of each feature are labelled and Munsell soil color codes are given for all components except gravel. Sample numbers (cited also in the text and tables) are shown in representative locations.

FIG. 2

Photograph of a block of material cut at right angles and removed from the margin of one of the sand-filled intestinal features at the Heisler mastodon site (Calhoun Co., Mich.). The narrow, curved, dark zone evident on the strongly illuminated face of the block is comminuted plant debris inferred to be original intestinal contents. Such material was the source of samples 17 and 18 from the Heisler site. Granular material enclosed by the zone of plant debris is sand, inferred to have been introduced by human activity. Diameter of trowel handle (left background) is ca. 2.5 cm.