0324-8267 Wydawnictwo Uniwersytetu Jagiellońskiego Oral microbiome dynamics in Postmortem Interval estimation: research standards and guidelines Zmienność mikrobiomu jamy ustnej w określaniu czasu zgonu: standardy i rekomendacje badawcze Haenel Aleksandra author Grzybowski Tomasz author Skonieczna Katarzyna author Katedra Medycyny Sądowej, Collegium Medicum im. Ludwika Rydygiera w Bydgoszczy, Uniwersytet Mikołaja Kopernika w Toruniu, Katedra Medycyny Sądowej, Zakład Genetyki Molekularnej i Sądowej, Wydział Lekarski, Collegium Medicum Uniwersytetu Mikołaja Kopernika Correspondence to: Aleksandra Haenel katarzyna.skonieczna@cm.umk.pl Correspondence to: Tomasz Grzybowski Correspondence to: Katarzyna Skonieczna katarzyna.skonieczna@cm.umk.pl 04 06 2025 Vol. 75 (1) 2025 48 55 Copyright © 2025 2025 This research was funded by NCU Research University Excellence Initiative, Poland, ForensOMICs and DEBUTS 6 grant Determination of the postmortem interval (PMI) is a crucial aspect of forensic investigations as it verifies an alibi or narrows down suspects. Nevertheless, PMI estimation remains one of the most challenging problems in forensic science. Currently used methods are influenced by various biotic and abiotic factors affecting decomposition. Thus, determining the time of death largely depends on the skills and experience of the forensic experts. Consequently, currently used procedures are prone to inaccuracies. Lately, gut microbiome analysis has proven useful in determining the time of death. Furthermore, emerging evidence indicates that profiling the oral microbiome may also provide valuable insights into PMI estimation. In this review, we examine published reports on oral microbiome and highlight the methodological limitations that diminish their scientific value. Therefore, we summarize the research standards and guidelines for oral microbiome studies to enhance the accuracy and reproducibility of PMI studies. Consequently, standardization of this type of research could lead to the development of innovative approaches that may be implemented into routine forensic genetics practice. 1 Henssge C. Death time estimation in case work I. The rectal temperature time of death nomogram. Forensic Sci Int. 1988; 38, 209-236 2 Madea B. Methods for determining time of death. Forensic Sci Med Pathol 2016, 12, 451-485 3 Kaliszan M., Hauser R., Kernbach-Wighton G. Estimation of the time of death based on the assessment of post mortem processes with emphasis on body cooling. Legal Medicine 2009, 11, 111-117 4 Dell’Aquila M., De Matteis A., Scatena A., Costantino A., Camporeale M.C., De Filippis A. Estimation of the time of death: where we are now? Clin Ter. 2021, 172: 109-112 5 Buchan M.J.; Anderson G.S. Time Since Death: A Review of the Current Status of Methods used in the Later Postmortem Interval. Canadian Society of Forensic Science Journal 2001, 34, 1-22 6 Cieśla J, Skrobisz J, Niciński B, Kloc M, Mazur K, Pałasz A, Javan GT, Tomsia M. The smell of death. State-of-the-art and future research directions. Front Microbiol. 2023, 14, 1260869 7 Amendt J.; Krettek R.; Zehner R. Forensic entomology. Naturwissenschaften 2004; 91, 51-65 8 Henssge, C. Decomposition and postmortem changes of the human body. Forensic Sci. Int. 1993, 61, 75-86 9 Payne J.A. A summer carrion study of the baby pig Sus Scrofa Linnaeus. Ecol 1965; 46, 592-602 10 Wójcik J, Tomsia M, Drzewiecki A, Skowronek R. Thanatomicrobiome – State Of The Art And Future Directions. Advancements of Microbiology 2021, 60: 21-29 11 Regueira-Iglesias A, Balsa-Castro C, Blanco-Pintos T, Tomás I. Critical review of 16S rRNA gene sequencing workflow in microbiome studies: From primer selection to advanced data analysis. Mol Oral Microbiol. 2023; 38: 347-399 12 Schmidt C. S.; Amend J. P. The microbiology of decomposition: a review. Nat. Rev. Microbiol., 2009; 7, 493-504 13 DeBruyn J.M.; Hauther K.A. Postmortem succession of gut microbial communities in deceased human subjects. PeerJ., 2017; 5, e3437 14 Hyde E.R.; Haarmann D.P.; Lynne A.M.; Bucheli S.R.; Petrosino J.F. The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition. PLoS One., 2013; 8, e77733 15 Guo J.; Fu X.; Liao H.; Hu Z.; Long L.; Yan W.; Ding Y.; Zha L.; Guo Y.; Yan J.; Chang Y.; Cai J. Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing. Sci Rep. 2016; 6, 24197 16 Carter DO, Yellowlees D, Tibbett M. Cadaver decomposition in terrestrial ecosystems. Naturwissenschaften. 2007; 94, 12-24 17 Johnson J.S.; Spakowicz D.J.; Hong B.Y.; Petersen L.M.; Demkowicz P.; Chen L.; Leopold S.R.; Hanson B.M.; Agresta H.O.; Gerstein M.; Sodergren 18 E.; Weinstock G.M. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat Commun. 2019; 10, 5029 19 Buetas E.; Jordán-López M.; López-Roldán A.; D’Auria G.; Martínez-Priego L.; De Marco G.; Carda-Diéguez M.; Mira A. Full-length 16S rRNA gene sequencing by PacBio improves taxonomic resolution in human microbiome samples. BMC Genomics. 2024; 25, 310 20 López-Aladid R.; Fernández-Barat L.; Alcaraz-Serrano V.; Bueno-Freire L.; Vázquez N.; Pastor-Ibáñez R.; Palomeque A.; Oscanoa P.; Torres A. Determining the most accurate 16S rRNA hypervariable region for taxonomic identification from respiratory samples. Sci Rep. 2023; 13, 3974 21 Ashe E.C.; Comeau A.M.; Zejdlik K.; O’Connell S.P. Characterization of Bacterial Community Dynamics of the Human Mouth Throughout Decomposition via Metagenomic, Metatranscriptomic, and Culturing Techniques. Front Microbiol. 2021; 12, 689493 22 Adserias-Garriga J.; Quijada N.M.; Hernandez M.; Rodríguez Lázaro D.; Steadman D.;.Garcia-Gil L.J. Dynamics of the oral microbiota as a tool to estimate time since death. Mol Oral Microbiol. 2017; 32, 511-516 23 Javan G.T.; Finley S.J.; Can I.; Wilkinson J.E.; Hanson J.D.; Tarone A.M. Human Thanatomicrobiome Succession and Time Since Death. Sci Rep. 2016; 6 ,29598 24 Ogbanga N.; Nelson A.; Gino S.; Wescott D.J.; Mickleburgh H.L.; Gocha T.P.; Procopio N. The impact of freezing on the post-mortem human microbiome. Front. Ecol. Evol. 2023; 11; 1151001 25 Pechal J.L.; Schmidt C.J.; Jordan H.R.; Benbow M.E. Frozen: Thawing and Its Effect on the Postmortem Microbiome in Two Pediatric Cases. J Forensic Sci. 2017; 62; 1399-1405 26 Pittner S.; Bugelli V.; Benbow M.E.; Ehrenfellner B.; Zissler A.; Campobasso C.P.; Oostra R.J.; Aalders M.C.G.; Zehner R.; Lutz L.; Monticelli F.C.; Staufer C.; Helm K.; Pinchi V.; Receveur J.P.; Geißenberger J.; Steinbacher P.; Amendt J. The applicability of forensic time since death estimation methods for buried bodies.in advanced decomposition stages. PLoS One. 2020; 15, e0243395 27 Hyde E.R.; Haarmann D.P.; Lynne A.M.; Bucheli S.R.; Petrosino J.F. The living dead: bacterial community structure of a cadaver at the onset and end of the bloat stage of decomposition. PLoS One. 2013; 8; e77733 28 Huang X.; Zeng J.; Li S.; Chen J.; Wang H.; Li C.; Zhang S. 16S rRNA, metagenomics and 2bRAD-M sequencing to decode human thanatomicrobiome. Sci Data. 2024; 11; 736 29 Gupta V.K.; Paul S.; Dutta C. Geography, Ethnicity or Subsistence-Specific Variations in Human Microbiome Composition and Diversity. Front Microbiol. 2017; 8; 1162 30 Cho H.W.; Eom Y.B. Forensic Analysis of Human Microbiome in Skin and Body Fluids Based on Geographic Location. Front Cell Infect Microbiol. 2021; 11; 695191 31 Govender P.; Ghai M. Population-specific differences in the human microbiome: Factors defining the diversity. Gene. 2025; 933; 148923 32 Mark Welch J.L.; Rossetti B.J.; Rieken C.W.; Dewhirst F.E.; Borisy G.G. Biogeography of a human oral microbiome at the micron scale. Proc Natl Acad Sci U S A. 2016; 113; E791-800 33 Mark Welch J.L.; Dewhirst F.E.; Borisy G.G. Biogeography of the Oral Microbiome: The Site-Specialist Hypothesis. Annu Rev Microbiol. 2019; 73; 335-358 34 Baker J.L.; Mark Welch J.L.; Kauffman K.M.; McLean J.S.; He X. The oral microbiome: diversity, biogeography and human health. Nat Rev Microbiol. 2024; 22; 89-104 35 Amos G.C.A.; Logan A.; Anwar S.; Fritzsche M.; Mate R.; Bleazard T., Rijpkema S. Developing standards for the microbiome field. Microbiome. 2020; 8; 98 36 Qian X.B.; Chen T.; Xu Y.P.; Chen L.; Sun F.X.; Lu M.P.; Liu Y.X. A guide to human microbiome research: study design, sample collection, and bioinformatics analysis. Chin Med J (Engl). 2020; 133; 1844-1855 37 Zaura E, Pappalardo VY, Buijs MJ, Volgenant CMC, Brandt BW. Optimizing the quality of clinical studies on oral microbiome: A practical guide for planning, performing, and reporting. Periodontol 2000. 2021; 85; 210-236 38 Mirzayi C.; Renson A.; Genomic Standards Consortium; Massive Analysis and Quality Control Society; Zohra F.; Elsafoury S.; Geistlinger L.; Kasselman L.J.; Eckenrode K.; van de Wijgert J.; Loughman A.; Marques F.Z.; MacIntyre D.A.; Arumugam M.; Azhar R.; Beghini F.; Bergstrom K.; Bhatt A.; Bisanz J.E.; Braun J.; Bravo H.C.; Buck G.A.; Bushman F.; Casero D.; Clarke G.; Collado M.C.; Cotter P.D.; Cryan J.F.; Demmer R.T.; Devkota S.; Elinav E.; Escobar J.S.; Fettweis J.; Finn R.D.; Fodor A.A.; Forslund S.; Franke A.; Furlanello C.; Gilbert J.; Grice E.; Haibe-Kains B.; Handley S.; Herd P.; Holmes S.; Jacobs J..P.; Karstens L.; Knight R.; Knights D.; Koren O.; Kwon D.S.; Langille M.; Lindsay B.; McGovern D.; McHardy A.C.; McWeeney S.; Mueller N.T.; Nezi L.; Olm M.; Palm N.; Pasolli E.; Raes J.; Redinbo MR.; Rühlemann M.; Balfour Sartor R.; Schloss PD.; Schriml L.; Segal E.; Shardell M.; Sharpton T.; Smirnova E.; Sokol H.; Sonnenburg J.L.; Srinivasan S.; Thingholm L.B.; Turnbaugh P.J.; Upadhyay V.; Walls R.L.; Wilmes P.; Yamada T.; Zeller G.; Zhang M.; Zhao N.; Zhao L.; Bao W.; Culhane A.; Devanarayan V.; Dopazo J.; Fan X.; Fischer M.; Jones W.; Kusko R.; Mason CE.; Mercer T.R.; Sansone SA.; Scherer A.; Shi L.; Thakkar S.; Tong W.; Wolfinger R.; Hunter C.; Segata N.; Huttenhower C.; Dowd J.B.; Jones H.E.; Waldron L. Reporting guidelines for human microbiome research: the STORMS checklist. Nat Med. 2021; 27; 1885-1892 39 Ferdous T.; Jiang L.; Dinu I.; Groizeleau J.; Kozyrskyj A.L.; Greenwood C.M.T.; Arrieta M.C. The rise to power of the microbiome: power and sample size calculation for microbiome studies. Mucosal Immunol. 2022; 15; 1060-1070 40 Stumptner C.; Stadlbauer V.; O’Neil D.; Gessner A.; Hiergeist A.; Zatloukal K.; Abuja P.M. The Pre-Analytical CEN/TS Standard for Microbiome Diagnostics-How Can Research and Development Benefit? Nutrients. 2022; 14; :1976 41 Bang E.; Oh S.; Ju U.; Chang H.E.; Hong J.S.; Baek H.J.; Kim K.S.; Lee H.J.; Park K.U. Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms. Sci Rep. 2023; 13; 10086