Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 4:3:100089.
doi: 10.1016/j.crmicr.2021.100089. eCollection 2022.

Whole genome sequence of bacteremic Clostridium tertium in a World War I soldier, 1914

Meucci M  1   2 , Costedoat C  3 , Verna E  3 , Adam F  3 , Signoli M  3 , Drancourt M  1   2 , Beye M  2 , Aboudharam G  1   4 , Barbieri R  1   2
Affiliations

Whole genome sequence of bacteremic Clostridium tertium in a World War I soldier, 1914

Meucci M et al. Curr Res Microb Sci. .

Abstract

Background: Dental pulp, encapsulating a blood drop, could be used to diagnose pathogen bacteraemia in archaeological materials using DNA-based techniques. We questioned the viability of such ancient pathogens preserved in ancient dental pulp.

Methods: After meticulous decontamination of 32 teeth collected from 31 World War I soldiers exhumed in Spincourt, France, dental pulps were extracted and cultured under strict anaerobiosis. Colonies were identified by mass spectrometry and whole genome sequencing. Fluorescent in situ hybridisation (FISH) was used for the direct microscopic detection of pathogens of interest in the dental pulp. All the experimental procedures included negative controls, notably sediments in contact with individual SQ517 to ensure that results did not arise from contamination.

Findings: Clostridium tertium was detected by FISH in two dental pulp specimens taken from a 1914 soldier. After a two-day incubation period, both dental pulp samples grew colonies identified by mass spectrometry and genome sequencing as C. tertium; whereas negative controls remained free of C. tertium in all the observations, and no C. tertium was founded in sediments. Skeletal remains of this soldier exhibited two notches in the left tibia evocative of a cold steel wound, and a probably fatal unhealed bullet impact in the hip bone.

Interpretation: Data indicated the presence of C. tertium in the dental pulp at the time of the death of one World War I soldier, in 1914. This observation diagnosed C. tertium bacteraemia, with war wounds as the probable portal of entry for C. tertium. Our C. tertium strains ante-dated by three years, the princeps description of this deadly opportunistic pathogen.

Keywords: Clostridium tertium; Culturomics; Dental pulp; Paleomicrobiology; Soldier; WWI.

PubMed Disclaimer

Conflict of interest statement

All authors declare no competing interests.

Figures

Fig. 1:
Fig. 1
Workflow summarizing culture of ancient dental pulps: Step 1: Teeth 45 and 34 were disinfected with pure ethanol and bleach. After the teeth were fractured, the dental pulp was extirpated under anaerobiosis; Step 1a: Sediment from the non-disinfected teeth of individual SQ517 and surrounding the tibia were used; Step 2: Dental pulp was mixed with 10uL of PBS; Step 2a: Sediment from the outer teeth and tibia were respectively mixed with 10μl and 150μl of PBS; Step 3: The rehydrated pulp was placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 3a: Rehydrated sediments were placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 4: Agar plates were incubated at 37° under a 5% CO2 atmosphere in a microaerophilic bag and were inspected daily.
Fig. 2:
Fig. 2
Osteological traumas on individual SQ517; A (I), (II) and (III): Marks on the left tibia resulting from a cold steel weapon; B: Bullet exit hole on the ilium of the left hip bone; C: Bullet entry hole on the ilium of the left hip bone; D and E: Perimortem fracture to the right zygomatic branch of the skull and to the mandibular condyle.
Fig. 3:
Fig. 3
C. tertium colonies yield from SQ517 individual after three days of incubation.
Fig. 4:
Fig. 4
Gram staining from Q5690 C. tertium strain under 100x optical microscopic lenses.
Fig. 5:
Fig. 5
Antibiogram: amoxicillin-clavulanic acid (AMC) 30 mg/L, erythromycin (E) 15 mg/L, metronidazole (MET) 4 mg/L and clindamycin (DA) 2 mg/L on Q5690. - Scan 1200®.
Fig. 6:
Fig. 6
Phylogenic tree of 20 Clostridium species: The tree underlines the proximity between 22 genomes from different Clostridium species. Q5690 appears closer to the reference genome MGYG-HGUT-01,328 than from Q6181 (modern diagnostic strain). The closest relative to C. tertium is Clostridium sartagoforme. All genomes can be found on NCBI.
Fig. 7:
Fig. 7
Phylogenic tree including all C. tertium genomes available on NCBI database: The tree underlines the proximity between several genomes from different C. tertium strain. Q5690 appears closer to the reference strain from 1923 (SP2622) than from the reference genome MGYG-HGUT-01,328. The closest relative to C. tertium Q5690 is strain BSD2780120874_150,323_E10. All genomes can be found on NCBI.
Fig. 8:
Fig. 8
FISH on SQ517 dental pulp: 1: filter AF555; 2: filter AF640; 3: Filter AF488; 4: DAPI; 5: MERCH.
See this image and copyright information in PMC

References

    1. Afouda P., Dubourg G., Cadoret F., Fournier P.E., Raoult D. Bacillus massiliglaciei’, a new bacterial species isolated from Siberian permafrost. New Microbes New Infect. 2016;15:92–93. - PMC - PubMed
    1. Angiuoli S.V., Salzberg S.L. Mugsy: fast multiple alignment of closely related whole genomes. Bioinformatics. 2011;27:334–342. - PMC - PubMed
    1. Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prijibelski A.V., Sirotkin A.V., Vyahhi N., Tesler G., Alekseyev M.A., Pevzner P.A. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012;19(5):455–477. - PMC - PubMed
    1. Barbieri R., Mai B.H.A., Chenal T., Bassi M.L., Gandia D., Camoin-Jau L., Drancourt M. A 2,000–year-old specimen with intraerythrocytic Bartonella quintana. Sci. Rep. 2020;10(1):1–8. - PMC - PubMed
    1. Brůžek J., Santos F., Dutailly B., Murail P., Cunha E. Validation and reliability of the sex estimation of the human os coxae using freely available DSP2 software for bioarchaeology and forensic anthropology. Am. J. Phys. Anthropol. 2017;164(2):440–449. - PubMed

LinkOut - more resources