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

First report of isolate & culture of a bacterium from ancient human samples & dental pulp in particular.

Highlights

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This original article is the first report of isolate and culture of a bacterium from ancient human samples and dental pulp in particular.

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The dental pulp is a mirror of the individual's infectious state at the time of death

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Ancient dental pulp culture yielded to the identification and isolation of the bacterium Clostridium tertium responsible for septicemia during World War I,

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

paleomicrobiology

dental pulp

culturomics

soldier

WWI

The soil inhabitant Clostridium tertium (C. tertium) has been described as an opportunistic pathogen associated with gangrenous wounds in the context of World War I (Henry, 1918). In 1917, Captain Herbert Henry from the British Royal Army Medical Corps reported that a quarter of the 100 wounds he observed in British and French soldiers were infected with C. tertium (King et al., 1963). C. tertium, a microorganism further detected in the oral cavity and intestines, has been further associated with necrotising gangrene and bacteraemia (Henry, 1918; Ray et al., 2003; Vanderhofstadt et al., 2010). In this situation, its deadly potential was confirmed after cumulative reports of deadly C. tertium bacteraemia, notably in neutropenic patients (Miller et al., 2001; Wazir et al., 2019).

Using an original culture protocol, we diagnosed a C. tertium bacteraemia in a French soldier who died in 1914, at the beginning of World War I.

1. MATERIALS AND METHODS

1.1. Archaeological investigations

After war declaration between France and Germany on August 3rd 1914, the first weeks of conflict involved territories close to the village of Spincourt, Great-East France (latitude: 49.3333; longitude: 5.6667), regarded as the heart of the « Border battle ». After German troops defeated at Mangiennes, a frontal battle against French troops retreating towards Verdun, took place in Spincourt between August 20th and August 25th. Fights raged and losses were heavy (27,000 deaths only for August 22nd). Fights ended-up with Spincourt firing and its occupation by German troops, until October 1918 (Verna et al., 2020). Few days after August fights, German troops assured the funeral gestion of deads including the French ones into a provisional cemetery. In the aftermath of war during the 1920’s, numerous bodies were unearthed from the provisional cemetery to be returned to their families and be reinsulated in military necropolis. Starting February 2017, the provisional military cemetery was investigated by Institut National Recherche Archéologique Préventive, disclosing testifying of an organised gestion of corpses (graves rigorously aligned, coffin inhumation) and 31 remaining skeletons by October 2018 (Verna et al., 2021). Examination of these skeletons testified of the violence of fightings (multiple traumas on skeleton, supernumerary anatomical part in coffin). Skeleton were examined using the Diagnose Sexuelle Probabiliste v2 (DSP2) software method for sex estimation and Lovejoy's quotation for age (Lovejoy et al., 1985; Brůžek et al., 2017). A total of 32 teeth were collected from the 31 skeletons for palaeomicrobiological investigations.

1.2. Paleomicrobiological investigations

Thirty-two teeth were investigated for palaeomicrobiology, one tooth for each one of the 31 soldiers and two teeth (45 and 34) for individual SQ517. The external surface of each tooth was cleaned with sterile gauze soaked with pure ethanol and bleach for 30 seconds (Figure 1). Pre-opening, fracture and pulp extirpation were all carried out under anaerobiosis to avoid any direct exposure of the dental pulp to atmospheric oxygen. Accordingly, all instruments necessary for opening the teeth and extracting the pulp, including a small circular diamond saw and its motor or excavator, were placed under an anaerobic hood (Don Whitley, Bingley, UK) before the manipulation. Dental pulps were extracted with specific tools (an excavator) following a previously described protocol (Drancourt et al., 1998). Pulps were rehydrated for one minute with 10 µL sterile phosphate buffered saline (PBS) and 0.5 µL of rehydrated dental pulp were inoculated onto a 5% sheep blood agar Petri plate (Becton Dickinson GmbH, Heidelberg, Germany) (Figure 1). A negative control culture medium was opened in the anaerobiosis hood at the beginning of the manipulation to assess the hood's sterility. A piece of sterile gauze soaked with sterile water was placed under each culture plate to keep humidity, and plates were placed into a microaerophilic bag (BD GasPak, EZ Pouch Systems, Becton-Dickinson, Franklin Lakes, NJ, US) incubated at 37°C under a 5% CO2 atmosphere (Figure 1). Each microaerophilic bag also contained one negative control inoculate with 10 µL sterile PBS. Sediments in direct contacts with individual SQ517’s tooth 45, tooth 34 and left tibia were put in culture before decontamination, following the same protocol described above.

1.3. C. tertium identification

Colonies observed by daily naked eye inspection were stained by Gram-staining (bioMérieux, Craponne, France) and further identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) (Seng et al., 2009).

The minimum inhibitory concentration (MIC) of amoxicillin–clavulanic acid (30 mg/L), erythromycin (15 mg/L), metronidazole (4 mg/L), and clindamycin (2 mg/L) (Vanderhofstadt et al., 2010; Salvador et al., 2013) was determined for each C. tertium isolate by Scan 1200® (Interscience, Saint-Nom-la-Bretèche, France).

The C. tertium isolate Q5690 was further investigated by whole genome sequencing (WGS) as previously described (Muñoz et al., 2019). Briefly, DNA was extracted using the standard EZ1 protocol from isolate Q5690, from strain SP2622 initially isolated in 1923 (Johnson and Francis, 1975) (DSMZ 2485, Leibniz Institute, Braunschweig, Germany), and from a previous C. tertium (Q6181) cultivated in our laboratory. DNAs were sequenced on Illumina Miseq and sequencing reads were assembled using Spades software (Bankevich et al., 2012). In silico DNA–DNA hybridization (DDH) was used to delineate bacterial species (Rossello-Mora, 2006) and C. tertium Q5690 genomic sequence was compared to those retrieved in public databases including C. tertium MGYG HGUT 01328 reference genome. Mugsy software was used for genome alignments (Angiuoli and Salzberg, 2011) and a phylogenetic tree was done using Raxml software (Stamatakis, 2014) incorporating the maximum likelihood method (ML) algorithm, with 1,00 bootstrap replicates. Genes related to pathogenicity and virulence factors were searched in the literature and sequences available in the Virulence Factor Database (VFDB) (Chen, 2004). Antibiotic resistance profiling was achieved by using Abricate pipeline comparaison with CARD (McArthur et al., 2013), ARG-ANNOT (Gupta et al., 2014), Restfinder (Zankari et al., 2012) and Bacterial Antimicrobial Resistance Reference Gene Database. SNPs analyses were performed with SNP-sites on (Angiuoli and Salzberg, 2011; Page et al., 2016).

1.4. Fluorescence in situ hybridization (FISH)

C. tertium was specifically detected in the dental pulp specimen by fluorescent in situ hybridisation (FISH), incorporating probe 3′-CTCCAACCCTAGTAAACCCCT-5′ labelled with Alexa fluor-488 and targeting the specific C. tertium gene fur coding for a transcriptional regulator following the MGYG HGUT 01328 reference genome annotation on Dfast software. The gene sequence was blasted on NCBI against the Clostridium genus (taxid 1485) database yielded 100% identity and 87% coverage with C. tertium and 99.76% identity and 87% coverage with Clostridium perfringens. Then, a specific probe was designed from this gene using NCBI primers and yielded 100% identity and coverage only with C. tertium on NCBI blast against the Clostridium genus (taxid 1485) database. Briefly, the dental pulp of individual SQ517 was fixed for three hours in Sandison's rehydration solution (aqueous formaldehyde 1%, 96% ethanol and 5% aqueous sodium carbonate) (Collini et al., 2014) and smeared on microscopic slade using Cytospin (ThermoFisher, Illkirch, France). In situ hybridization was performed with a hybridiser (Dako, Les Ulis, France) at 65°C for 10 minutes and then at 37° overnight, following a previously reported protocol (Millogo et al., 2020). Dental pulp of the individual SQ536, which remained negative in culture for C. tertium was used as a negative control to assess the probe specificity.

2. RESULTS

2.1. Archaeological investigations

After meticulous inspection and bacterial identification of the 32 dental pulps, only teeth from the individual SQ517 yielded C. tertium as reported below. He was a man aged 30-35 years at the time of his death. The anthropological examination of the skeleton of individual SQ517 revealed two notches in the left tibia, evocative of the impact of a cold steel weapon (knife or bayonet), surrounded by a periosteal reaction indicating that the wounds occurred before death; a fracture to the right zygomatic branch and the right condyle of the mandible; left and right rib fractures which may have occurred at the time of death or post-mortem. A bullet impact was also observed with an entry point in the anterior part of the ilium and the exit point in the posterior part with significant bone loss and no sign of healing (Figure 2).

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https://www.sciencedirect.com/science/article/pii/S2666517421000699

Figure 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.

Figure 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.