International Journal of Molecular Sciences Article The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing Valeria D’Argenio 1,2, Marielva Torino 3, Vincenza Precone 1, Giorgio Casaburi 1,†, Maria Valeria Esposito 1, Laura Iaffaldano 1, Umberto Malapelle 4, Giancarlo Troncone 4, Iolanda Coto 1,2, Paolina Cavalcanti 5, Gaetano De Rosa 6, Francesco Salvatore 1,7,* and Lucia Sacchetti 1,* 1 CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; [email protected] (V.D.); [email protected] (V.P.); [email protected] (G.C.); [email protected] (M.V.E.); [email protected] (L.I.); [email protected] (I.C.) 2 Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy 3 Department of Paleopathology, University of Naples Suor Orsola Benincasa, via Suor Orsola 10, 80125 Naples, Italy; [email protected] 4 Department of Public Health, University of Naples Federico II, Naples, via Pansini 5, 80131 Naples, Italy; [email protected] (U.M.); [email protected] (G.T.) 5 Microbiology Unit, Hospital of Cosenza, via San Martino, 87100 Cosenza, Italy; [email protected] 6 Department of Advanced Biomedical Sciences, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy; [email protected] 7 IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico)-Fondazione SDN, via Gianturco 113, 80143 Naples, Italy * Correspondence: [email protected] (F.S.); [email protected] (L.S.); Tel.: +39-081-746-3648 (F.S.); +39-081-373-7827 (L.S.); Fax+39-081-746-3133 (F.S.); +39-081-373-7808 (L.S.) † Present address: Life Sciences Lab, University of Florida, Kennedy Space Center, Merritt Island, FL 32953, USA. Academic Editor: William Chi-shing Cho Received: 28 October 2016; Accepted: 3 January 2017; Published: 6 January 2017 Abstract: The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative “infection” at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most “informative” area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary’s report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes, and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations. Keywords: metagenomics; human microbiome; cold case; next generation sequencing Int. J. Mol. Sci. 2017, 18, 109; doi:10.3390/ijms18010109 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 109 2 of 9 1. Introduction Int. J. Mol. Sci. 2017, 18, 109 2 of 10 Past cases of death from unknown causes, particularly those of famous personalities, are commonly1. Introduction referred to as “mysterious deaths”. In many instances, such cases can now be solved thanks toPast advances cases inof researchdeath from and unknown to the advent causes, of modernparticularly technologies those of famous [1,2]. Notably, personalities, identification are of causescommonly of death referred in ancientto as “mysterious “cold cases” deaths”. may In also many shed instances, light on such the cases evolution can now of be a diseasesolved [3]. Thisthanks article concernsto advances a young in research boy who and died to at the the ad endvent of theof 18thmodern century technolo fromgies a putative [1,2]. Notably, infection of a boneidentification wound (possibly of causes osteomyelitis). of death in ancient His parents“cold cases” feltthat may their also sonshed was light not on receivingthe evolution appropriate of a treatmentdisease because [3]. This his article condition concerns was a young not correctly boy who diagnosed. died at the Consequently,end of the 18th century they took from the a unusualputative step infection of a bone wound (possibly osteomyelitis). His parents felt that their son was not receiving of preserving a fragment of the affected bone for future analysis. Thus, the fragment was sealed by a appropriate treatment because his condition was not correctly diagnosed. Consequently, they took public notary in a small envelope (see the documentation in the Supplementary File 1 and Figure S1) the unusual step of preserving a fragment of the affected bone for future analysis. Thus, the untilfragment the study was described sealed inby thisa public paper. notary in a small envelope (see the documentation in the SupplementaryNext-generation File technology1 and Supplementary for high-throughput Figure S1) until sequencingthe study described of nucleic in this acidspaper. has been a breakthroughNext-generation in the field technology of metagenomics, for high-throughpu and hast producedsequencing aof wealth nucleic of acids data has about been various a environmentalbreakthrough niches in the in medicalfield of metagenomics, research, particularly and has in produced the field ofa wealth infectious of data diseases about [4 ,various5]. The aim of ourenvironmental study was toniches try to in identifymedical theresearch, cause particularly of death of in this the boyfield usingof infectious contemporary diseases [4,5]. technologies The and methods.aim of our study was to try to identify the cause of death of this boy using contemporary technologies and methods. 2. Results 2. Results Hematoxylin and eosin (H and E) stained sections of the specimen showed the features typical of Hematoxylin and eosin (H and E) stained sections of the specimen showed the features typical bone tissue and aggregates of granulocytes, which is indicative of an inflammatory reaction (Figure1A). of bone tissue and aggregates of granulocytes, which is indicative of an inflammatory reaction To minimize the risk of external contamination and obtain a sufficient quantity of DNA-rich material, (Figure 1A). To minimize the risk of external contamination and obtain a sufficient quantity of we isolatedDNA-rich a piecematerial, of the we infectedisolated a bone piece by of laser the infected microdissection bone by laser under microdissection sterile conditions. under sterile Figure 1B showsconditions. the pre-microdissected Figure 1B shows area,the pre-microdissect and Figure1Ced the area, post-microdissected and Figure 1C the area.post-microdissected Sex-determining regionarea. Y (SRY)Sex-determining analysis confirmed region Y (SRY) that analysis the DNA confirmed was from that a malethe DNA (Figure was1 fromD). a male (Figure 1D). Figure 1. Bone sample characterization. (A) Microscopy image (hematoxylin and eosin staining) Figure 1. Bone sample characterization. (A) Microscopy image (hematoxylin and eosin staining) showing the features typical of bone tissue and inflammatory infiltration; example of a laser showing the features typical of bone tissue and inflammatory infiltration; example of a laser captured captured microdissected area of the bone sample: (B) shows the pre-microdissected area × microdissected(40× resolution), area of(C the) the bone green sample: line encircles (B) shows the microdissected the pre-microdissected area; (D) areasex-determining (40 resolution); region( CY ) the greenamplification line encircles revealed the microdissected the sex of the area;patient: (D )lane sex-determining 1: ladder (100–1200 region bp); Y amplificationlane 2: sample; revealed lane 3: the sex ofnegative the patient: control; lane lanes 1: ladder4 and 5: (100–1200male controls; bp); lanes lane 6–8: 2: sample; uncharged lane slots. 3: negative control; lanes 4 and 5: male controls; lanes 6–8: uncharged slots. Int. J. Mol. Sci. 2017, 18, 109 3 of 9 Int. J. Mol. Sci. 2017, 18, 109 3 of 10 WeWe analyzed analyzed the the entire entire bacterial bacterial microbiome microbiome of the of bone the sample bone bysample a next-generation by a next-generation sequencing approach,sequencing as describedapproach, as under described Methods under (see Methods the Section (see the 4.5 ).Section A total 4.5). of A 7,357,569 total of 7,357,569 base pairs base (bp) correspondingpairs (bp) corresponding to 14,172 16S sequencesto 14,172 16S (mean sequence sequences (mean length, sequence 528.7 bp length, and average 528.7 qualitybp and scoreaverage 34.18) werequality generated. score 34.18) After were pre-quality generated. filtering After (see pre-quality Section 4.6filtering for details), (see Section 12,829 4.6 high for qualitydetails), sequences 12,829 werehigh considered quality sequences for subsequent were considered analysis. In for detail, subseq 243uent representative analysis. In operationaldetail, 243 taxonomicrepresentative units (OTUs)operational