Forensic Science International: Genetics 59 (2022) 102686

Contents lists available at ScienceDirect

Forensic Science International: Genetics

journal homepage: www.elsevier.com/locate/fsigen

Research Paper

Microbial DNA in human nucleic acid extracts: Recoverability of the

microbiome in DNA extracts stored frozen long-term and its potential and
ethical implications for forensic investigation
Giulia Sguazzi a, b, 1, Hayley L. Mickleburgh c, d, 1, Stefano Ghignone e, Samuele Voyron e, f,
Filippo Renò a, Mario Migliario g, Federica Sellitto h, Flavia Lovisolo a, Giulia Camurani a,
Nengi Ogbanga h, Sarah Gino a, 2, Noemi Procopio d, h, *, 2
a
Department of Health Science, University of Piemonte Orientale, via Solaroli 17, 28100 Novara, Italy
b
CRIMEDIM – Center for Research and Training in Disaster Medicine, Humanitarian Aid and Global Health, Università del Piemonte Orientale, Via Lanino, 1-28100
Novara, Italy
c
Department of Cultural Sciences, Linnaeus University, Växjö, Sweden
d
Forensic Anthropology Center, Texas State University, San Marcos, TX, USA
e
Institute for Sustainable Plant Protection (IPSP) - Turin Unit - National Research Council (CNR), 1-10125 Turin, Italy
f
Department of Life Sciences and Systems Biology, University of Torino, V.le P.A. Mattioli 25, 10125 Turin, Italy
g
Department of Translational Medicine, University of Piemonte Orientale, via Solaroli 17, 28100 Novara, Italy
h
Forensic Science Research Group, Faculty of Health and Life Sciences, Applied Sciences, Northumbria University, NE1 8ST, Newcastle Upon Tyne, UK

A R T I C L E I N F O A B S T R A C T

Keywords: Human DNA samples can remain unaltered for years and preserve important genetic information for forensic
Microbiome investigations. In fact, besides human genetic information, these extracts potentially contain additional valuable
Forensic genetics information: microbiome signatures. Forensic microbiology is rapidly becoming a significant tool for estimating
Cold cases
post-mortem interval (PMI), and establishing cause of death and personal identity. To date, the possibility to
Metabarcoding
Cadaveric decomposition
recover unaltered microbiome signatures from human DNA extracts has not been proven. This study examines
Storage guidelines the microbiome signatures within human DNA extracts obtained from six cadavers with different PMIs, which
were stored frozen for 5–16 years. Results demonstrated that the microbiome can be co-extracted with human
DNA using forensic kits designed to extract the human host’s DNA from different tissues and fluids during
decomposition. We compared the microbial communities identified in these samples with microbial DNA
recovered from two human cadavers donated to the Forensic Anthropology Center at Texas State University
(FACTS) during multiple decomposition stages, to examine whether the microbial signatures recovered from
“old” (up to 16 years) extracts are consistent with those identified in recently extracted microbial DNA samples.
The V4 region of 16 S rRNA gene was amplified and sequenced using Illumina MiSeq for all DNA extracts. The
results obtained from the human DNA extracts were compared with each other and with the microbial DNA from
the FACTS samples. Overall, we found that the presence of specific microbial taxa depends on the decomposition
stage, the type of tissue, and the depositional environment. We found no indications of contamination in the
microbial signatures, or any alterations attributable to the long-term frozen storage of the extracts, demon­
strating that older human DNA extracts are a reliable source of such microbial signatures. No shared Core
Microbiome (CM) was identified amongst the total 18 samples, but we identified certain species in association
with the different decomposition stages, offering potential for the use of microbial signatures co-extracted with
human DNA samples for PMI estimation in future. Unveiling the new significance of older human DNA extracts
brings with it important ethical-legal considerations. Currently, there are no shared legal frameworks governing
the long-term storage and use of human DNA extracts obtained from crime scene evidence for additional research
purposes. It is therefore important to create common protocols on the storage of biological material collected at

* Correspondence to: Northumbria University, Room A107, Ellison Building, Newcastle upon Tyne NE1 8ST, UK.
E-mail address: [email protected] (N. Procopio).
1
Giulia Sguazzi and Hayley Mickleburgh contributed equally to first-authorship.
2
Sarah Gino and Noemi Procopio considered co-last authors and contributed equally to this work.

https://doi.org/10.1016/j.fsigen.2022.102686
Received 18 November 2021; Received in revised form 8 March 2022; Accepted 9 March 2022
Available online 23 March 2022
1872-4973/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

crime scenes. We review existing legislation and guidelines, and identify some important limitations for the
further development and application of forensic microbiomics.

1. Introduction 1.1. The human postmortem microbiome

Human DNA can remain unchanged for years and extracts stored at The human postmortem microbiome develops from the microbial
− 20 ◦ C or − 80 ◦ C can preserve important genetic information for long communities already present in the body before death, and from com­
periods of time, which can be submitted as evidence in court [1]. This munities that colonize the body after death [8]. Human cadavers host
makes such extracts particularly valuable in (older) cases in which other large numbers of microorganisms and provide an attractive habitat for
evidence has been destroyed and further analyses are required. Forensic several microbial communities. These include the thanatomicrobiome
genetics uses various techniques for human DNA extraction, including (microbial communities and fungi located in human internal organs and
traditional methods such as organic extraction (phenol-chloroform) [2] cavities, that succeed antemortem microbes within 48 h postmortem
and extraction with Chelex 100 Resin [3], depending on the type of [12]) and the epinecrotic microbial and fungal communities which
biological material. Over time and with the possibility of automating reside on the external surfaces of the decomposing body [8,13–15]. The
multiple steps in DNA analysis, many laboratories have switched to solid signature of the thanatomicrobiome and epinecrotic microbial com­
phase extraction (e.g., ion exchange columns, magnetic beads) in which munities is affected by the microbes present in the antemortem micro­
DNA is selectively bound to a substrate such as silica particles. In this biome of the deceased and individual biological and lifestyle factors
way, the DNA is retained while the proteins and other cellular compo­ [16]. The antemortem microbiome is known to vary depending on
nents are washed away, releasing the DNA in a purified form. The anatomical location on the body (body site) [17], sex [18,19], age [20,
extracted human DNA is typically stored at − 20 ◦ C, or even at − 80 ◦ C in 21], geographical origin of the individual [22], health condition [23,
order to prevent the activity of nucleases and preserve DNA for genetic 24], body size [18,19,25] and lifestyle including diet [19,26,27],
profile typing [4]. alcohol consumption [27,28], physical activity [28] and smoking habits
Previous research has not clarified whether human DNA extracts also [29–31]. By far the largest and most diverse of the antemortem micro­
preserve the microbiome – which comprises “all of the genetic material bial communities resides in the gut [31]. Bacterial decomposition of the
within a microbiota (the entire collection of microorganisms in a specific body commences in the intestines and spreads from there to the other
niche, such as the human gut)” [5]. While the field of forensic micro­ organs, generally following a certain order: the brain, stomach, bowel,
biomics is still in development, the huge potential of microbiome liver, and pancreas are affected before muscles, tendons and bones [8].
analysis to contribute to forensic investigations has already been The rate of decomposition is determined by several abiotic and biotic
demonstrated, and includes potential applications for postmortem in­ variables, including temperature (the higher the temperature, the
terval (PMI) estimation [6], human identification [7], biological sex quicker the decomposition, within limits), cause of death (in septic
determination [8], and even manner and cause of death [9]. The po­ conditions, putrefaction occurs more rapidly), or the presence of
tential to recover microbiome signatures from human DNA extracts held clothing or blankets. Other factors that influence the rate of decompo­
in storage opens up new possibilities for the development and validation sition include submersion in water, drying of fluids due to ventilation of
of forensic microbiomics methods [10], and ultimately, new opportu­ the environment, body size, and presence of external trauma with
nities for the investigation of older criminal cases for example by consequent loss of blood [32].
providing new estimation of the PMI, or new information on the identity The natural fluctuations in the number and types of microorganisms
of an individual or the cause of death. associated with a cadaver have great potential to be harnessed for the
This study examines microbial signatures recovered from human development of precise and accurate methods for estimation of the PMI
DNA extracts obtained from six cadavers which were stored frozen for [33–36]. Currently, PMI is generally estimated based on changes to the
between 5 and 16 years. The type and number of microbial communities body, including cooling, lividity, and rigor mortis, followed by gross
identified in these samples are compared with those from human ca­ morphological changes. These are generally divided into five stages of
davers donated to the Forensic Anthropology Center at Texas State decomposition: fresh, bloat, active decay, advanced decay, and partially
University (FACTS), retrieved using a commercial DNA extraction kit. or completely skeletonized remains (e.g., Galloway et al. and Megyesi
The study aims to understand: (1) whether the microbiome recovered et al. [37,38]). These methods are widely used in forensic anthropology,
from human DNA extracts is consistent with the human postmortem even though they tend to offer broad estimates only, and they suffer
microbiome, or whether the presence of microbial communities in these from problems with accuracy and precision [32]. During the early stages
extracts could result from contamination, (2) the effects of prolonged of decomposition, the cadaveric ecosystem consists mostly of the bac­
periods of storage at − 20 ◦ C on the survival of the microbiome in human teria that live in and on the human body before death. After death, the
DNA extractions, (3) whether the microbial differences that are known physical and chemical barriers of the immune system that limit bacterial
to characterise the different stages of decomposition can be observed in migration break down, facilitating movement of bacteria into nearby
these old human DNA extractions, and (4) whether the burial environ­ tissues [39]. Internal organs are normally considered to be sterile in
ment, the geographical location where the body was found, the tissue healthy adults, but within 24 h postmortem, microbes start to proliferate
type, and/or the cause of death influence the recovered microbiome. In [40]. Due to their persistent sterility for up to five days postmortem, the
addition, this study examines some important legal frameworks and liver and pericardial fluids are therefore optimal sampling sites to
guidelines for the storage and re-use of DNA extracts. Re-analysis of such evaluate the degree of postmortem microbial migration [41]. Endoge­
samples and broad comparative studies offer important ways to further nous microbial populations succeed in a predictable way over the course
develop the field of forensic microbiomics and to generate new infor­ of the decomposition [42,43]. The thanatomicrobiome has been found
mation for old forensic cases, yet legislation on how long samples and to be less influenced by external factors than the epinecrotic microbial
DNA extractions can be retained differs considerably by country [10,11] community [15], leading researchers to target shifts in the thanatomi­
and often depends on the outcome or conclusion of criminal crobiome in developing a microbiome-based PMI estimation method [6,
proceedings. 34,44–47].
Several studies on human and animal decomposition have identified
common trends in microbiome shifts during decomposition, including a
drop in microbial diversity associated with the initial stages of

2
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

decomposition [16,34,35,48,49] and the presence of four predominant body donation program complies with all legal and ethical standards
phyla during decomposition: Bacteroidetes, Acidobacteria, Actino­ associated with the use of human remains for scientific research in the
bacteria and Firmicutes [34,35,49,50]. Postmortem microbial succes­ USA.
sion patterns are strongly influenced by oxygen availability, which Finally, we examined the legal framework for processing personal
causes a shift from aerobic to anaerobic taxa during the bloat stage [51]. data (biological sex and age) collected during sampling for forensic
Microbes proliferate in blood, liver, spleen, heart and brain in a investigative purposes, as well as for data that may derive from subse­
time-dependent manner and their relative abundances vary in different quent laboratory activity (the DNA profile). For such additional con­
body organs and at specific postmortem intervals [8]. For example, siderations, see Suppl. Material.
reproductive organs start to decay later than other internal organs
during decomposition [52]. The composition of the postmortem 2.2. Chemicals and materials
microbiome also differs by biological sex, with Pseudomonas sp. only
having been identified in female cadavers, and Rothia sp. only in male Nucleospin FLB and Nucleomag 96 Blood kit was purchased from
cadavers [8]. Organ thanatomicrobiome analyses suggest that faculta­ Macherey-Nagel (Düren - Nordrhein-Westfalen, Germany). Platinum
tive anaerobes, such as Lactobacillus, predominate in the “short PMI” Hot Start PCR Master Mix 2x was purchased from ThermoFisher Scien­
timeframe, while in a “long PMI” timeframe a predominance of obligate tific (Waltham, MA USA). 1x Accuprime Pfx Supermix and SequalPrep™
anaerobes like Clostridium is observed [53]. Notably, Firmicutes (e.g., Normalisation kit was purchased from Invitrogen™ (ThermoFisher
Clostridium, Peptoniphilus, and Bacillus) represent a stable and constant Scientific, Waltham, MA USA). QIAamp PowerFecal Pro DNA Kit was
biomarker in microbial communities derived from different body loca­ purchased from QIAGEN (Hilden, Germany). 16S rRNA primers and
tions [33]. Even the manner of death (natural, accidental, suicide, ho­ agarose were purchased from Sigma-Aldrich (UK). PhiX Control v3 and
micide, undetermined) has been shown to influence the MiSeq Reagent Kits v2 were purchased from Illumina (Illumina Inc.,
thanatomicrobiome in various organs [33,39,52]. Cambridge, UK).

2. Materials and methods 2.3. Samples

2.1. Ethical approval A total of eighteen samples were collected from 8 human cadavers (5
males and 3 females), derived from Italian court cases (n = 6) and
This study was submitted and approved in Italy by the Novara donated to the FACTS (n = 2). The six cadavers (five males, one female)
Intercompany Ethics Committee (CE 24/21) and in the United Kingdom from Italian court cases were discovered in different locations in North-
by the Northumbria University Ethics Committee (submission ref. 24514 west Italy (Piedmont and Liguria regions). The cadavers were found in
and 29218). The study included samples collected from (1) deceased different stages of decomposition, including one in active decomposi­
individuals who have been subjected to forensic genetic analysis by one tion, two in advanced decomposition, and one partially skeletonised.
of the authors at the request of the Judicial Authority, and whose genetic Two were found in charred condition (Table 1). The time intervals re­
material is stored at the Medical Forensic Laboratory of the Department ported for these cases in Table 1 indicates either the PMI or the time
of Health Sciences at the University of Eastern Piedmont in Novara, and between the discovery and sampling of the subject, as well as the human
(2) deceased individuals whose body was donated to the Forensic An­ DNA extraction from the tissue/biological fluid, and ranged between
thropology Center at Texas State University (FACTS) for forensic taph­ four days and seven years. Several tissues/body fluids, including mus­
onomic research. Before submitting the study to the two ethics cles, blood and organs, were sampled at the scene for the purpose of
committees (Novara Intercompany Ethics Committee and Northumbria obtaining human DNA for forensic genotyping. Storage duration of the
University Ethics Committee), we considered the existing ethical DNA extracts at − 20 ◦ C prior to the start of the present study is reported
frameworks for using biological samples (or their derivatives) taken in Table 1.
from deceased subjects. In particular, we examined the issue of consent The two cadavers donated to the FACTS were placed to decompose
to participate in the study. Two main frameworks are relevant in this outdoors at the FARF. The cadavers were placed unclothed in flexed
context: a framework for samples belonging to juridical cases and a supine body positions to decompose naturally in shallow oval-shaped
framework for samples collected from donated cadavers. pits, one of which remained open during decomposition and one of
For biological material (and/or samples derived from it through which was covered immediately with soil. A metal cage was placed over
laboratory analysis) taken for judicial purposes from deceased subjects the open pit experiment to protect the remains from large scavengers.
and stored at the Forensic Medical Laboratory of the Department of Other than this, the pits and cadavers were exposed to the natural ele­
Health Sciences of the University of Eastern Piedmont, the "Provision ments, including weather, insects and small scavengers. Oral and tooth
relating to the processing of particular categories of data, pursuant to swabs were collected at different decomposition stages (Table 1). Swabs
art. 21, paragraph 1 of Legislative Decree 10 August 2018, n. 101" were stored frozen at − 20 ◦ C. DNA extractions were made for the pur­
(Annex I, point 5.3) of the Italian Guarantor for the protection of per­ pose of the present study and the extracts were processed immediately.
sonal data, applies. This provision stipulates that obtaining the informed Therefore, no storage period at − 20 ◦ C is reported for these extractions.
consent of the deceased is not possible and therefore not required.
Permission from the legal next-of-kin is also not required, although in 2.4. DNA extracts isolated for human identification purposes in Italian
practical implementation of the provision the legal next-of-kin will be court cases
informed, and in the case that they indicate that sampling and/or
analysis is against the wishes of the deceased, the research will not be Eleven tissue samples (SG01-02-04-05-08-10-14-15-18-21-22) were
further pursued. collected during forensic investigations from six cadavers found in
The sampling and analysis of tissues from deceased persons whose various locations in North-West Italy (Piedmont and Liguria regions)
bodies were donated to FACTS, was conducted in accordance with the (Table 1). The tissues were collected either at the scene, from the burial
Texas Uniform Anatomical Gift Act (Health and Safety Code Chapter environment or during autopsy using sterile scalpels. The blood sample
692A) [54]. Whole body donations studied during decomposition out­ (SG02) was collected using a sterile swab. DNA extraction was carried
doors at the Forensic Anthropology Research Facility (FARF; the human out using Nucleomag 96 Blood kit combined with KingFisher mL
taphonomy facility managed by FACTS located in San Marcos, Texas) (ThermoFisher Scientific, Waltham, MA USA). Quantitation was con­
are acquired for scientific research purposes, through the expressed and ducted in different ways depending on sample type (tissues or blood).
documented willing of the donors and/or their legal next of kin. The Details on the methods used to quantify the material and to obtain full

3
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

Table 1
Samples and associated biological and sampling information: age, sex, decomposition stage, type of tissue or body fluid, deposition type, sampling dates, time elapsed
between the death/disappearance/discovery of the subject and the sampling of tissue/biological fluid for each forensic sample and DNA storage length at − 20 ◦ C.
Sample Age Sex and Decomposition Tissue/body Deposition type Disappearance/death/ Time elapsed Sampling Storage at
code individual stage fluids discovery between death/ date − 20 ◦ C
number discovery and
sampling

Cadavers from North-west Italy

SG01 38 Male 1 Charred Heart Car accident near Turin Death: 11/12/2004 4 days post- 15/12/2004 16 years
SG02 Blood (TO) mortem
SG04 40 Male 2 Partial Spleen Mountain near Turin Disappearance: 6 days post- 05/04/2005 15 years
SG05 skeletonization Quadriceps (TO) November discovery (March
2004Discovery: March 2005)
2005
SG08 – Male 3 Active decay Liver River bank, near Cuneo Discovery: March 2007 Not available March 2007 13 years
(CN)
SG10 – Male 4 Advanced decay Quadriceps Found exposed near the Discovery: 03/07/ 7 years post-death 10/05/2010 11 years
seaside in Savona (SV), 2003
then buried in mountain
near SV
SG14 37 Female 1 Advanced decay Quadriceps Little river bank, near Disappearance: 23/01/ 6 days post- 24/10/2014 6 years
SG15 Asti (AT) 2014Discovery: 18/ discovery
SG18 10/2014
SG21 66 Male 5 Charred Quadriceps Homicide near Cuneo Discovery: 14/11/ 4 days post- 18/11/2015 5 years
SG22 Heart (CN), found in a car 2015 discovery
Body donors studied at the Forensic Anthropology Research Facility (FARF)
SG103 91 Female A Fresh Buccal swab Open pit Date of death: 18/04/ 10 days post- 28/04/2015 –
2015 mortem
SG104 Active Decay Buccal swab 16 days post- 04/05/2015 –
mortem
SG105 Fresh Tooth 3.2 5 years, 2 months, 28/04/ –
12 days post- 2015a30/
mortem 06/2020b
SG106 Full Tooth 4.3 5 years, 2 months, 03/12/ –
skeletonization 12 days post- 2015a 30/
mortem 06/2020b
SG107 67 Female B Fresh Buccal swab Buried Date of death: 05/05/ 2 days post- 07/05/2015 –
2015 mortem
SG108 Fresh Tooth 3.1 5 years, 1 month, 07/05/ –
25 days post- 2015a30/
mortem 06/2020b
SG109 Full Tooth 4.1 5 years, 1 month, 17/8/2017a –
skeletonization 25 days post- 30/06/
mortem 2020b
a
date of tooth extraction;
b
date of tooth swabbing.

genetic profiles can be found in the Suppl. Material. we excluded the use of amplification-based quantification methods to
overcome any potential limitation. Subsequently, microbial commu­
2.5. Samples collected from donated human cadavers nities were targeted by amplifying the 16 S rRNA locus [56] using for­
ward 515FB (GTGYCAGCMGCCGCGGTAA) and reverse 806RB
A total of seven tooth and buccal swabs (SG103-104-105-106-107- (GGACTACNVGGGTWTCTAAT) primers [49,57,58] in order to test if
108-109) were collected from the two cadavers at different stages during any microbial DNA was present prior to any sequencing approach. PCR
decomposition (Table 1). Microbial DNA was extracted using the negative controls were run in each analysis to perform a quality check of
QIAamp PowerFecal Pro DNA Kit following the protocol reported in the amplifications and in all cases these controls gave negative results.
Procopio et al. [55]. Biological material from the buccal mucosa of both PCR reaction mixtures were set up following Procopio et al. [49] as
body donors studied at FARF was collected using a sterile swab which follows: 12.5 μL master mix (Platinum Hot Start PCR Master Mix 2x),
was rubbed along the inside of the left and right buccal mucosa (10 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM) and
strokes each side), during the fresh and active stages of decomposition. 0.5–1.5 μL template DNA in a final reaction volume of 25 μL. The
In addition, biological material was collected from the surface of thermocycler conditions were set up as follows: denaturation at 94 ◦ C for
extracted single-rooted teeth during fresh and skeletonized decomposi­ 2 min; 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 52 ◦ C or
tion stages, by gently rubbing all surfaces of the extracted tooth for a 40 s and extension at 68 ◦ C for 30 s; final extension at 68 ◦ C for 10 min
duration of 30 s using sterile swabs. and maintenance of the samples at 4 ◦ C. The PCR products obtained
were checked on 1.5% (w/v) agarose gels (run in TAE buffer at 100–120
2.6. Microbiome analysis V).
After confirming that microbial DNA was present in all 18 samples,
All 18 samples were quantified (or re-quantified, in the case of the extracts were sent to the NUOmics DNA Sequencing Research Facility
samples from the Italian court cases) immediately prior to the execution (Northumbria University, Newcastle, UK) for the amplification and
of the microbiome analyses using NanoDrop One Microvolume UV-Vis sequencing of the hypervariable region V4 of the 16 S ribosomal RNA
Spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) gene using the Illumina Miseq Next Generation Sequencer (Illumina Inc.,
(Table S1). NanoDrop was the method chosen for quantification as we Cambridge, UK), following the gold standards suggested by the Earth
expected the presence of PCR inhibitors in the DNA extracts; therefore, Microbiome Project to target and sequence the highly variable V4 region

4
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

of the 16 S rRNA gene for microbial identification. We followed the signal they provided on the agarose gel (Fig. 1A and B). The raw data
method proposed by Kozich et al. [59] and adopted and described in generated for this study have been deposited at the NCBI Sequence Read
detail in Procopio et al. [55]. Archive (SRA-NCBI; https://www.ncbi.nlm.nih.gov/sra) under Project
Paired-end reads from each sample were sequenced with forward accession number PRJNA773228 and BioSample accession numbers
and reverse reads in separate files and processed by means of the SAMN22451803-SAMN22451820.
microbiome bioinformatics platform QIIME2 (Quantitative Insights Into From the 18 samples, the sequencing provided a total of 1453,775
Microbial Ecology 2), v.2021.2 [60,61]. Denoising and quality control, useful reads, after DADA2 denoising step, with a variable distribution in
including removal of chimeras, were achieved by means of the DADA2 the samples, from a minimum of 500 to a maximum of 202,701 (Suppl.
[61] plugin and to avoid low quality sequences reads were truncated Fig. S1). The numbers of identified ASVs ranged between 11 and 516 per
(250 bp for forward, 240 bp for reverse reads). The classifier adopted for sample. An initial number of 1864 ASVs was identified, subsequently
the taxonomic assignment was Silva v.138 (99% OTUs full-length se­ reduced to 1822 after removal of contaminants, and to 1388, after ASVs
quences) [62]. with fewer than five reads were removed. Among the 1388 ASVs, 22
were Archaea, four were unassigned, and 1362 were Bacteria.
First, an MDS/PCoA on unweighted-UniFrac distance was drawn in
2.7. Statistical analysis order to identify possible groupings among different decomposition
stages or body sites (Fig. 2). Samples taken at similar decomposition
Statistical analyses were performed within the computing environ­ stages cluster closer together than samples collected at different stages
ment R (https://www.R-project.org/). All the taxon abundances were of decomposition. In particular, samples in advanced decomposition and
calculated and graphically plotted with the aid of the PHYLOSEQ fully skeletonised were clearly separated from the other decomposition
v.1.22.3 package [63,64]. Prior to performing formal analyses and stages on the first principal component. Charred, fresh and active decay
creating the figures, pre-processing steps were applied to the Amplicon samples clustered on the same side of the PCoA but were separated on
Sequence Variants (ASV) counts: ASVs with fewer than 5 reads were their second principal component. Samples were not clustered in a clear
filtered out and abundances were standardized to the median way according to the anatomical location on the body (e.g., quadriceps
sequencing depth according to McMurdie and Holmes [64]; ASVs samples belonging to different individuals at different decomposition
recognized as mitochondrial or chloroplast sequences were also stages did not cluster together).
excluded. The Indicator Species Analysis (ISA), (a classification-based Phylum abundances were then evaluated to identify the most prev­
method to access the statistical significance of the relationship be­ alent phyla on the whole sample (Suppl. Fig. 2). Firmicutes species were
tween species occurrence/abundance and groups of sites) was per­ the most abundant, and together with Proteobacteria they represented
formed using the multipatt function from the indicspecies v.1.7.9 R 79.8% of the overall population. Actinobacteriota and Bacteroidota
package, with 999 permutations [65] in order to assess whether ASVs shared similar abundances (~8%), followed by Chloroflexi (~1%) and
were significantly associated with a particular stage of decomposition. Acidobacteriota (~1%).
Phylum abundances examined according to the decomposition stage
3. Results showed the presence of specific phyla at specific post-mortem stages, i.
e., Actinobacteriota were only found in fresh and in fully skeletonised
Quantification and optical density results obtained with NanoDrop at remains, Cyanobacteria only in fully skeletonised remains, and Asgar­
the time of the study are reported in Suppl. Table S1. The samples darchaeota only in charred cadavers). Others (i.e., Firmicutes and Pro­
extracted with the kit specifically designed for the microbiome (SG103- teobacteria) were found in all samples (Fig. 3). Evaluation based on the
SG109) resulted in lower DNA abundances than the others. The 16 s two different modes of deposition (i.e., open pit versus shallow burial)
rRNA gene was successfully amplified in all 18 samples, as shown by the

Fig. 1. Agarose gel electrophoresis of the PCR product of the 16s rRNA gene for DNA extracts. A) Lanes L: Promega 1 kb DNA ladder; lane P: positive control (E. coli,
BL21(DE3)); lane N: negative control; lanes 1–22: showing amplified 16s rRNA genes from samples SG01 - SG22. B) Lanes L: Promega 1 kb DNA ladder; lane P:
positive control (E. coli, BL21(DE3)); lane N: negative control; lanes 103–109: showing amplified 16 s rRNA genes from samples SG103 - SG109.

5
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

Fig. 2. PCoA (Principal Coordinates Analysis) on unweighted-UniFrac distance. Different shapes represent different body sites used for the DNA extraction, whereas
different colours represent different decomposition stages.

revealed an increased abundance of taxa belonging to the Bacillales 4. Discussion

order in “Female B” (buried) in comparison with in “Female A” (open
pit). No other major differences were observed (Suppl. Fig. S3). 4.1. Laboratory analyses
This study did not reveal a Core Microbiome (CM) shared amongst all
18 samples. One ASV (“d46e2205”, see Suppl. Table S2 for the full An important aim of this study was to investigate the presence and
identifiers) belonging to the genus of Escherichia-Shigella was identified survival rate of microbial signatures in old human DNA extracts that
in all but one sample (SG107). The same ASV was shared across all were stored frozen for extended periods of time, obtained from bodies in
samples derived from Italian forensic casework. Among the fresh DNA different stages of decomposition. DNA quantification results, extracted
extracts from body donors, the CM was instead characterised by the using Nucleomag 96 Blood kit, unsurprisingly returned higher DNA
Enterococcus identified by the ASV “9908fffa” (see Suppl. Table S2 for concentrations in the Italian samples than for the more recent extracts,
the full identifiers). which were processed with the QIAamp PowerFecal Pro DNA Kit.
The indicator species analysis (ISA), run with a significance level of Whereas the Italian extracts contained both human and microbial DNA,
0.05, identified several ASVs were closely associated with specific the PowerFecal Pro DNA kit specifically targets microbial DNA, resulting
groups of interests (Suppl. Data S1). By grouping the samples according in notably lower concentrations of total DNA. Almost all the ratios
to the time in which DNA was extracted (e.g., fresh vs. frozen extracts) calculated for the optical density are in the range of what is considered
we found 50 ASVs (p-value <0.05) characterising specifically the group to be normal for forensic samples and this reflect the nature of the
of fresh extracts, including six ASVs with a p-value < 0.01 (“9908fffa”, samples included in the study.
“68a9a8d8”, “e93b3133”, “0ed3a683”, “ff9d93d7” and “581a5501”). All samples included in the study yielded successful amplification of
When considering the older frozen DNA extracts, we instead found only the 16s rRNA gene, despite the fact that some differences in the intensity
two ASVs (“43fddf15” and “1a4f8fdc”, p-value <0.05). When samples of the amplicons were visible on the agarose gel (i.e., SG02-18-21 bands
were grouped based on the stage of decomposition, ISA showed 25 were quite faint and SG05 band was the most intense one). The samples
species associated with the “fresh” stage of decomposition with a p-value with the lightest bands include two extracts from two charred in­
< 0.05 (including 21 with a p-value <0.01), 21 species associated with dividuals (SG02 from blood and SG21 from quadriceps), and one from a
the “advanced decay” stage (of which 10 had a p-value <0.01), nine body in advanced stage of decomposition (SG18). It is possible that the
species associated with “partial skeletonization” (p-value <0.05), 99 weak signal of the SG18 band was caused by technical issues associated
ASVs associated with “full skeletonization” (p-value <0.05) and two with the gel, because two biological replicates extracted from the same
associated with the “charred” remains (one with p-value <0.05, one tissue type from the same individual (SG14-15) both gave a stronger
with p-value <0.01). signal. Other charred samples from the heart muscle (in particular SG01)
also resulted in a relatively weak amplification in comparison with the
overall sample, likely as a result of the charred condition of the remains.
In order to test if the microbiome recovered from old human DNA
extracts is consistent with the expected human postmortem microbiome,

6
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

Fig. 3. Phylum abundances arranged by decay stage, built on data from 18 samples divided as follows: fresh (n = 4), active decay (n = 2), advanced decay (n = 4),
skeletonised partial (n = 2), skeletonised full (n = 2), charred (n = 4). Taxonomic classification based on Silva v.138.

or whether the microbial communities found in these samples could where their abundance was notably lower. Firmicutes are a large group
have resulted from contamination during sample processing or during of bacteria which typically characterise the human bacterial flora [66]
frozen storage, we compared Italian casework extracts with extractions and are commonly found in association with soil and aquatic environ­
from samples collected during human decomposition experiments at ments [67,68]. High abundances of Firmicutes have frequently been
FARF. The separation of the samples in the PCoA along the first axis reported in decomposition studies [44,49,50,69–71], particularly in
shows that newly extracted tooth swabs from skeletonised samples cadavers with large PMIs. The majority of the recovered Firmicutes
(FARF) share a similar microbiome profile with extracts taken from consisted of Clostridia, followed by Bacilli. Clostridia are well-known
quadriceps from two individuals in an advanced stage of decomposition obligate anaerobes that are abundant in soil but are also symbiotic
(Italian judicial cases). Samples are further separated along the second bacteria in the human gut. They are able to colonise human tissues
dimension, in particular those taken from fresh cadavers and those from rapidly after death [72] and are highly abundant in decomposing re­
charred individuals. The fact that all samples but one (SG107) shared mains with both short and long PMIs [73]. Bacilli are primarily aerobic,
one specific taxa further shows that there are some recurring taxa both although several taxa in this phylum are anaerobic but aerotolerant
in “fresh” and in “old” microbial DNA samples, and suggests that the [74]. They are commonly found in the gastrointestinal tract and have a
microbiome sequenced from “old” extracts did not originate from recent proteolytic effect in the cadaver [75]. Bacilli have been reported as one
contamination. of the most abundant classes in decomposition studies that examined the
Based on our findings, the prolonged storage of DNA extracts at different stages of decomposition [71,74,76].
− 20 ◦ C does not impede the successful sequencing of microbial ASVs. The second most prevalent phyla are Proteobacteria, which are
According to the number of reads obtained in the library, “fresh” and facultative or obligate anaerobes and decomposers that are reported to
“old” extracts are similar. The samples which generated the lowest be one of the most abundant taxa in and on decaying carcasses and
number of reads and ASVs obtained (SG01-02; stored frozen for 16 cadavers [33,36,44,50,77]. The Proteobacteria identified in this study
years), and two other samples that yielded only a small number of reads were found to be equally represented by Alphaproteobacteria and
(SG21-22; stored frozen for 5 years), consisted of charred samples. In Gammaproteobacteria. Alphaproteobacteria are usually abundant,
these cases, the low yield appears to be associated with the condition of particularly during active decomposition in grave soil samples but also
the cadaver rather than the duration of frozen storage, as other non- during advanced decomposition in both soil and skin samples [34].
charred samples which were stored for extended durations (SG04-05) Gammaproteobacteria have been identified in several studies of bodies
resulted in a relatively high number of reads (e.g., SG04 yielded the with large PMIs, and have been found in different anatomical locations
highest number). and various depositional environments [8,42,44]. Interestingly, most of
Among the most prevalent phyla in the study, Firmicutes are the the Proteobacteria identified in this study were found in the charred
most abundant in all samples, with the exception of charred samples, samples.

7
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

Amongst the less abundant phyla, we identified nearly equal abun­ While our results demonstrate that certain taxa were indeed associated
dances of Bacteroidota and Actinobacteriota. Bacteroidota have been predominantly with specific decomposition stages, the potential in­
found in high concentrations in soils during cadaveric and carcass fluences of the burial environment in this case are as yet unclear. For
decomposition [34,49,78] but also in and on cadavers [12,44,79]. In instance, phylum Sumerlaeota was found almost uniquely in the samples
this study, they were most abundant in samples collected during from two skeletonised cadavers. Sumerlaeota bacteria are not yet fully
advanced decomposition. Actinobacteriota have previously been found understood, due to a paucity of studies so far, but there is evidence to
in fresh cadavers in oral samples [47,80] but also in partially and fully suggest they are associated with several different soil types and marine
skeletonised remains [36] and in grave soils [81]. This concurs with our sediments [95]. They are characterised by the presence of multiple
study, in which Actinobacteriota were found to be most abundant in peptidases which allow them to degrade organic matter and adapt to
fresh oral swabs and in oral swabs taken from skeletonised remains. both aerobic and anaerobic conditions [95]. So far, they have not been
While some of these phyla were associated with most of the stages of reported to be involved in the process of cadaveric decomposition, and
decomposition represented in our sample, certain phyla were predom­ considering the type of samples (tooth swabs) and the environment from
inantly associated with specific stages. Fusobacteriota were found which the skeletonised samples were collected (FARF, i.e., the same
mostly in fresh samples, Sumerlaeota mostly in fully skeletonised sam­ environment and soil) it is possible that the recovered Sumerlaeota
ples, and Acidobacteriota mostly in charred samples. The ISA results result from the local burial environment rather than the stage of cadaver
highlighted that several taxa (identified at genus level) were statistically decomposition. Similarly, Fusobacteriota were identified only in fresh
significantly associated with certain individuals or decomposition samples (oral and tooth swabs). These bacteria are known to be involved
stages. Interestingly, the group of charred samples are differentiated with dental pathologies and infections [96] and therefore their presence
from the other samples by the presence of the taxa Pyrinomonas (p-value in these fresh samples is unsurprising and may be associated with the
0.0017) and Cupriavidus (p-value 0.0472) genera which were identified type of tissue, rather than the stage of decomposition of the remains.
in all four charred samples. Pyrinomonas (from the Greek “pyrino”, born Sample SG08, which generated amongst the lowest number of reads
of fire, and “monas”, unit) are thermophilic bacteria that have been and had a very low variety of ASVs, was obtained from the liver of “Male
found in geothermally heated savanna soils from volcanic fumaroles 3”, a cadaver which was recovered in active decomposition stage in the
[82,83]. To our knowledge, no studies have analysed the microbiome of proximity of a river bank in March in northern Italy. In this case, the cool
burnt remains, and Pyrinomonas has not previously been observed in river bank, particularly during the winter months in temperate climates
association with cadaveric remains. Cupriavidus is a genus that includes (such as in Piedmont) may have reduced or hindered microbial growth,
bacteria that are predators of other soil bacteria and fungi. These are affecting the number of taxa identified in this extract. Microorganisms
highly resistant to metals and their growth is stimulated by the presence and microbial activity are strongly influenced by temperature (microbial
of copper [84]. Cupriavidus bacteria have been found both in soil and in activity increases as temperature increases and slows with decreasing
clinical samples [85], but they have not been reported in fire-related temperatures), and it is known that temperature affects the microbial
contexts before. Besides these two indicator species, we observed decomposition of cadavers in soil [97]. By comparison, samples
additional taxa that are highly abundant in the burnt samples, including SG14-15-18 show a notably higher variety of bacteria than SG08. These
Vulcaniibacterium (Gammaproteobacteria, Xanthomonadaceae), Escher­ samples were also collected from a cadaver (“Female 1”) found in the
ichia-Shigella (Gammaproteobacteria, Enterobacteriaceae), Pseudomonas proximity of a river bank during winter, however both the environment
(Gammaproteobacteria, Pseudomonadaceae), Methylobacter­ and the type of tissue sampled differed considerably from those collected
ium-Methylorubrum (Alphaproteobacteria, Rhizobiales), Aeribacillus from “Male 3”. In fact, the cadaver of “Female 1” was found in an
(Bacilli, Bacillaceae), Romboutsia (Clostridia, Peptostreptococcaceae), advanced stage of decomposition after a prolonged period of time from
Paraclostridium (Clostridia, Peptostreptococcaceae) and Clos­ when this individual was reported missing (nine months), and was
tridium_sensu_stricto_1 (Clostridia, Clostridiaceae). Some of these taxa almost fully submerged in a wet slime/mud characteristic of the river
have been frequently reported to be associated with decomposing re­ bank environment. Because the internal organs had liquefied and were
mains and carcasses (i.e., Pseudomonadaceae, Xanthomonadaceae, not distinguishable, only muscle tissue was sampled. The collected
Enterobacteriaceae [1,44,49,86–88]). Vulcaniibacterium, similarly to muscle tissue sample was contaminated with the surrounding mud and
Pyrinomonas, has been isolated from a geothermally heated soil samples decomposition liquids, in contrast to the liver sample collected from
[88], and their presence in charred samples is therefore not completely “Male 3”. The difference in the variety of bacteria identified in both
unexpected. Pseudomonas are able to produce heat resistant enzymes, cases is likely due to the combined effects of temperature/seasonality,
including lipases and peptidases [89,90]. Methylobacterium-Methyloru­ humidity, PMI/decomposition stage and the type of tissue sampled.
brum spp. and Aeribacillus spp. are reported to be resistant to exposure to SG10 also comprised a muscle sample, collected from “Male 4”, a
high temperatures [91,92]. Bacteria of the Clostridia class are obligate cadaver in advanced stage of decomposition. In this case the cadaver
anaerobes which are able to produce resilient endospores; Clostridium was recovered from a coffin buried in a cemetery in the Ligurian
genus spores in particular are able to survive extreme and extended Apennines, an area which is characterised by low temperatures. SG10
heating conditions [93]. Clostridium species have also been frequently revealed a lower number of identified microbial species and read counts
reported in decomposition studies, and they are considered to be ubiq­ than SG14-15-18, which may have been related to the cold environment
uitous post-mortem communities both in early and in advanced which is likely to have led to slower microbial growth than in the river
decomposition stages [73]. In sum, the taxa represented in the charred bank environment. Furthermore, “Male 4” was sampled seven years
samples consist of bacterial species that are capable of surviving extreme after burial, so the expected microbial presence, variety and activity was
conditions including low oxygen levels and high temperatures (e.g., a lower than the one expected for “Female 1”.
burning car). These results suggest that burnt cadavers contain a distinct We compared the two donated cadavers to assess the potential effect
microbiome, and they warrant further study of the microbiome in burnt of exposure of the body versus burial of the body. “Female A” was placed
remains as well as investigation of the potential for developing specific in a pit that remained open, and “Female B” was buried in a shallow
microbiomic PMI estimation methods for burnt remains. grave. No major differences in the microbial composition of the tooth
Due to the limited size of the sample and the number of replicates in swabs of these individuals were identified, although Bacillales order
each category, as well as the multiple variables potentially affecting the were more abundant in “Female B” than in “Female A” (Suppl. Data S1).
results (i.e., diverse body recovery environments, cause of death, type of Bacillales belong to Firmicutes phylum and include Bacillus, Listeria and
tissue sampled), it is not possible to assess whether the identified mi­ Staphylococcus genera. Several bacteria belonging to Bacillus spp. were
crobial signatures are consistent with signatures described in the liter­ found to be notably more present in the buried donor. Bacillus are
ature as associated with specific stages of decomposition [34,51,69,94]. ubiquitous in soil and represent a large percentage of bacterial residents

8
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

in the soil habitat [98]. While the lack of replicates impedes attaching Recommendations document which states that “the cell material of
significant conclusions to this observation, the presence of a high crime scene stains from which a DNA profile has been generated is
abundance of Bacillus spp. in the swab taken from the buried individual, usually stored” [103]. In this case again, the definition seems to be quite
is therefore not unexpected. We did not observe any other major dif­ generic and it leaves room for interpretation; specifically, there is no
ferences between the microbiome of these two samples. These results clear guidance on the duration of this storage, nor if DNA extracts should
generally concur with the findings of other analyses conducted on the or should not be considered as “cell material”. The document provides
same cadavers (including an isotope study [99,100] and a bone prote­ guidelines on how to deal with “reference samples”, their preservation
omics study [101]), which showed that the difference in mode of and storage, but lacks information on how to deal with casework sam­
placement (shallow open pit vs. shallow burial) did not play a significant ples. ENFSI suggests considering the possibility of performing additional
role in biomolecule preservation and changes in the isotopic signatures DNA testing when there are doubts about the identity of the donor of the
of different tissues. trace, implying a need to preserve DNA material obtained from case­
Our findings emphasise the fact that the presence and recovery of work samples (e.g., in cases where the original biological sample was
specific microbial taxa is dependent on a number of important variables, exhausted during previous attempts of DNA extraction).
including the stage of decomposition of the cadaver, modification of the In Italy the question is still open. The law n. 85 of 30 June 2009, (Law
remains (e.g., burning), the type of tissue sampled, and the environment n. 85, 2009), that introduced the national DNA database, and the Decree
in which decomposition took place. It is essential to understand the ef­ of the President of the Republic 7 April 2016, n. 87 (Regulation con­
fects of these variables in order to evaluate recovered microbiomes and taining provisions for the implementation of law no. 85) highlight
to further develop and validate microbiomics approaches for forensic technical and procedural criteria, and procedures for the sampling and
purposes. storage of biological samples (“reference samples”). However, there is
no mention at all of the preservation of the biological material obtained
4.2. Legislative frameworks for the storage of DNA extracts from forensic from the evidence (“casework samples”). The only reference in the
casework Italian legislation to preservation is given in art. 10 paragraph 2 of Law
85/2009, which considers the possibility of reopening unsolved cases
The results obtained from this work underscore the previously noted without, however, any mention of methods and duration. In this regard,
importance [4,102] of the storage and preservation of what was ob­ art. 13 (Law n. 85, 2009) regulates the deletion of profiles and the
tained from the biological traces found at the crime scene (i.e., extracts destruction of biological samples following acquittal with a final sen­
of DNA, RNA, proteins). The potential value of such samples for both the tence or if the crime does not exist. In all other cases, as provided for in
methodological and technical advancement of microbiomics, and paragraph 4 art. 13, the profile remains in the database no more than 40
application of microbiomics in forensic practice are significant. A lack of years, while the biological sample is stored no more than 20 years. With
consistency in the long-term preservation of such samples, as well as regards to DNA extracts, section II of the DPR, chapter IV art. 24, states
variation in legislative frameworks for their use for additional research that they must be destroyed after complete typing, therefore precluding
purposes, risks undermining this largely unexplored source of the possibility of obtaining a new typing in the future. “Casework
information. samples”, which in Italy are instead known as “biological evidence”, are
On a global level there is no shared vision regarding storage of such only mentioned in art. 6 (Law n. 85, 2009) but again, there is no mention
extracts: a shared framework or legislation on this topic is lacking and of measures for preservation and future usage modalities of DNA ex­
guidelines differ considerably between countries. The National Institute tracts derived from them.
of Standards and Technology (NIST), through the Technical Working Recently in the United Kingdom the National Police Chiefs’ Council
Group on Biological Evidence Preservation, suggested best practice (NPCC) has agreed to the guidelines adopted by Police Forces in England
guidelines for evidence handlers in order to ensure the integrity, to and Wales, in which this question has been covered in more detail. In the
prevent the loss, and to reduce the premature destruction of biological NPCC Forensic Retention Guidance v. 1.0 the preservation of the
evidence after collection [10]. They recommend retaining indefinitely generated material, which is the material created during the examina­
any biological evidence collected during homicide investigations for tion of an evidence, is discussed. This encompasses different kinds of
open and/or charges filed cases. For other types of crime and case sta­ samples such as slide mounted fibres, scanning electron microscope
tuses they advise different retention lengths (at a minimum, for the stubs, but also DNA extracts. According to this document, generated
duration of the statute of limitations) on a case-by-case basis. According material is retained or returned by the Forensic Unit, the existence and
to this document (page IV), biological evidence consists of any “sample location of this material is documented on the case file and the retention
of biological material – hair, tissue, bones, teeth, blood, semen, or other period depends on the offence (case category)3 and can range from a
bodily fluids – or evidence items containing biological material”. It is not minimum of 1 year to a maximum of 30 years [104].
clear if this definition encompasses DNA extracts, and therefore it is not In sum, there is a distinct lack of consistency in legislation and
clear whether the listed recommendations apply to these samples or not. guidelines internationally, which significantly impacts the capability to
However, in the paragraph referring to the packaging and storing of perform additional research on old cases using new technologies and
biological evidence, specific recommendations on the storage of therefore also limits the potential to solve them.
extracted DNA are provided, with the indication that the best way for
long-term storage of DNA extracts (e.g., > 72 h) is the frozen preserva­ 5. Conclusions
tion (e.g., ≤ − 10 ◦ C).
The European Forensic Genetics Network of Excellence (EURO­ This study shows that it is possible to recover microbial signatures
FORGEN-NoE) provided an audit of legislative frameworks within the from “old” human DNA extracts comparable to those from recently
European Union for the collection, retention and use of forensic DNA extracted microbial DNA samples from decomposing cadavers. The
profiles, however the document refers to either biological samples microbial communities we identified from DNA extracts from casework
(intended as the biometric material obtained from a suspect, also known samples collected during different stages of decomposition are consis­
as “reference sample”) or to genetic profiles obtained from those. No tent with those found in other studies with similar post-mortem intervals
specific guidelines on the preservation of DNA extracts derived from and with the recent FACTS samples we included in this study.
“casework samples” (e.g., evidence obtained at the crime scene) are
provided [11], although such samples are mentioned in the document.
The European Network of Forensic Science Institutes (ENFSI) DNA 3
Major crime (MoPI group 1 offences), Serious crime (MoPI group 2 of­
Working Group created a DNA Database Management Review and fences), and Non-serious/volume crime (MoPI group 3 offences).

9
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

We can conclude that long-term storage of DNA extracts at − 20 ◦ C Migliario: Funding acquisition; Federica Sellitto: Investigation; Flavia
does not affect the survival of the microbial signature during a time­ Lovisolo: Investigation; Giulia Camurani: Investigation; Roles/Writing
frame of between 5 and 16 years. By contrast to the decomposition stage, - original draft; Nengi Ogbanga: Investigation; Roles/Writing - original
the type of tissue, and the environmental conditions in which the draft; Sarah Gino: Conceptualization; Funding acquisition; Methodol­
decomposition took place, the duration of frozen storage does not seem ogy; Project administration; Supervision;Roles/Writing - original draft;
to affect microbial DNA yield and successful sequencing. The microbial Writing - review & editing; Noemi Procopio: Conceptualization;
signatures contained in material related to older casework therefore Funding acquisition; Methodology; Project administration; Supervision;
represent an important source of medico-legal and scientific information Roles/Writing - original draft; Writing - review & editing.
pertaining to long time frames and PMI estimation.
We found a high abundance of Firmicutes and Proteobacteria in our Acknowledgements
study, which is consistent with other decomposition studies conducted
on various PMIs and different anatomical locations and depositional NUOmics Facility is acknowledged for conducting the sequencing of
environments. We also identified phyla that are predominantly associ­ the samples. The authors also gratefully acknowledge the donors and
ated with specific stages of body decomposition, such as Fusobacteriota their next of kin for allowing the use of donated bodies to perform this
taxa in the fresh stage and Sumerlaeota taxa in the skeletonised stage. research, as well as the Forensic Anthropology Research Facility (FARF),
Furthermore, this study identified for the first time the presence of the outdoor human decomposition facility associated with the Forensic
Pyrinomonas and Cupriavidus genera in charred samples. These taxa have Anthropology Center at Texas State University (FACTS).
previously been found in association with volcanic soils and in clinical
samples, but they have hitherto never been reported in cadaveric sam­ Declaration of Interests
ples. This observation expands on the very limited current knowledge on
burnt and charred remains, and opens important new avenues for The authors declare no conflict of interest.
research.
Considering the potential future use of older human DNA extracts to
Appendix A. Supporting information
advance microbiomics approaches, such as for PMI estimation, we argue
that there is a need for the scientific community and legislators to define
Supplementary data associated with this article can be found in the
a common standard and protocol for retaining case materials for the
online version at doi:10.1016/j.fsigen.2022.102686.
purposes of new analyses and future investigation, prosecution or ap­
peals. The distinct differences between countries at present impede the
References
comparative studies that are needed in order to further develop the
forensic applications of microbiomics. Amongst the inconsistencies [1] J.M. Robinson, Z. Pasternak, C.E. Mason, E. Elhaik, Forensic applications of
found, the use of different terminologies to indicate reference and microbiomics: a review, Front. Microbiol. 11 (2021), 608101.
casework samples, limits the potential for comparative studies and [2] S. Köchl, H. Niederstätter, W. Parson, DNA extraction and quantitation of forensic
samples using the phenol-chloroform method and real-time PCR, in: Forensic
complicates correct interpretation of the few existing guidelines. DNA Typing Protocols, Springer, 2005, pp. 13–29.
For this reason, it is essential to clarify and standardise the termi­ [3] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple
nology that has to be used in forensic genetics laboratories when extraction of DNA for PCR-based typing from forensic material, Biotechniques 10
(1991) 506–513.
referring to different types of evidence, samples and DNA extracts. [4] G. Sguazzi, F. Lovisolo, S. Gino, Is Genomic DNA extracted and stored at-20◦ C for
Within individual national frameworks, the lack of clear guidelines on long time useful in forensic field? Forensic Sci. Int. Genet. 7 (Supplement Series)
the precise legal status of and appropriate procedures for retaining and (2019) 629–631.
[5] Nature.com, Microbiome, (n.d.). 〈https://www.nature.com/subjects/mic
storing extracts, means that the potential for their use to yield new case
robiome〉. (Accessed 27 August 2021).
evidence and advance forensic microbiomics, currently remains largely [6] A. Belk, Z.Z. Xu, D.O. Carter, A. Lynne, S. Bucheli, R. Knight, J.L. Metcalf,
unused. To maximise the chances of obtaining new relevant information Microbiome data accurately predicts the postmortem interval using random
forest regression models, Genes 9 (2018) 104.
from old samples for the resolution of cold cases it is highly important
[7] A.E. Woerner, N.M.M. Novroski, F.R. Wendt, A. Ambers, R. Wiley, S.E. Schmedes,
that appropriate scientific bioethics and jurisprudence frameworks are B. Budowle, Forensic human identification with targeted microbiome markers
developed in tandem with rapidly progressing technological using nearest neighbor classification, Forensic Sci. Int. Genet. 38 (2019) 130–139.
innovations. [8] G.T. Javan, S.J. Finley, I. Can, J.E. Wilkinson, J.D. Hanson, A.M. Tarone, Human
thanatomicrobiome succession and time since death, Sci. Rep. 6 (2016) 29598.
[9] S.F. Kaszubinski, J.L. Pechal, K. Smiles, C.J. Schmidt, H.R. Jordan, M.H. Meek, M.
Funding E. Benbow, Dysbiosis in the dead: human postmortem microbiome beta-
dispersion as an indicator of manner and cause of death, Front. Microbiol. 11
(2020) 2212.
This work was supported by UKRI through a Future Leaders [10] S.M. Ballou, M.C. Kline, M.D. Stolorow, M.K. Taylor, S.R. Williams, P.S.
Fellowship [MR/S032878/1] awarded to Noemi Procopio; and partially Bamberger, B. Yvette, L.Brown, C.E. Jones, R. Keaton, The Biological Evidence
by the Università del Piemonte Orientale [FAR 2017] to Filippo Renò, Preservation Handbook: Best Practices for Evidence Handlers, 2013.
[11] K. Reed, D. Syndercombe Court, A Comparative Audit of Legislative Frameworks
Mario Migliario and Sarah Gino. The taphonomic experiments at FARF within the European Union for the Collection, Retention and Use of Forensic DNA
were supported by the European Research Council under the European Profiles, London, 2016.
Union’s Seventh Framework Program (FP7/2007-2013)/ERC Synergy [12] J.L. Pechal, C.J. Schmidt, H.R. Jordan, M.E. Benbow, A large-scale survey of the
postmortem human microbiome, and its potential to provide insight into the
grant agreement no. 319209, the Leiden University Fund Byvanck grant living health condition, Sci. Rep. 8 (2018) 1–15.
number 5604/30-4-2015/Byvanck, and the 2017 Royal Netherlands [13] E. Ventura Spagnolo, C. Stassi, C. Mondello, S. Zerbo, L. Milone, A. Argo, Forensic
Academy of Arts and Sciences (KNAW) “National Postdoc Prize” awar­ microbiology applications: a systematic review, Leg. Med. 36 (2019) 73–80.
[14] W. Zhou, Y. Bian, Thanatomicrobiome composition profiling as a tool for forensic
ded to Hayley Mickleburgh.
investigation, Forensic Sci. Res. 3 (2018) 105–110.
[15] G. Javan, S. Finley, Z. Abidin, J. Mulle, The thanatomicrobiome: a missing piece
Author contributions of the microbial puzzle of death, Front. Microbiol. 7 (2016).
[16] D.O. Carter, The importance of microbial communities in the estimation of the
time since death, in: J. Hayman, M. Oxenham (Eds.), Estimation of the Time since
Giulia Sguazzi: Investigation; Roles/Writing - original draft; Hayley Death: Current Research and Future Trends, Academic Press, London, UK, 2020,
L. Mickleburgh: Conceptualization; Methodology; Roles/Writing - pp. 109–139.
original draft; Writing - review & editing; Stefano Ghignone: Data [17] G.I. Perez, Z. Gao, R. Jourdain, J. Ramirez, F. Gany, C. Clavaud, J. Demaude,
L. Breton, M.J. Blaser, Body site is a more determinant factor than human
curation; Formal analysis; Software; Samuele Voyron: Data curation; population diversity in the healthy skin microbiome, PLoS One 11 (2016),
Formal analysis; Software; Filippo Renò: Funding acquisition; Mario e0151990.

10
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

[18] C. Haro, O.A. Rangel-Zúñiga, J.F. Alcalá-Díaz, F. Gómez-Delgado, P. Pérez- [49] N. Procopio, S. Ghignone, A. Williams, A. Chamberlain, A. Mello, M. Buckley,
Martínez, J. Delgado-Lista, G.M. Quintana-Navarro, B.B. Landa, J.A. Navas- Metabarcoding to investigate changes in soil microbial communities within
Cortés, M. Tena-Sempere, J.C. Clemente, J. López-Miranda, F. Pérez-Jiménez, forensic burial contexts, Forensic Sci. Int. Genet. 39 (2019).
A. Camargo, Intestinal microbiota is influenced by gender and body mass index, [50] S.J. Finley, J.L. Pechal, M.E. Benbow, B.K. Robertson, G.T. Javan, Microbial
PLoS One 11 (2016), e0154090. signatures of cadaver gravesoil during decomposition, Microb. Ecol. 71 (2016)
[19] C. Dominianni, R. Sinha, J.J. Goedert, Z. Pei, L. Yang, R.B. Hayes, J. Ahn, Sex, 524–529.
body mass index, and dietary fiber intake influence the human gut microbiome, [51] E.R. Hyde, D.P. Haarmann, J.F. Petrosino, A.M. Lynne, S.R. Bucheli, Initial
PLoS One 10 (2015). insights into bacterial succession during human decomposition, Int. J. Leg. Med.
[20] P.W. O’Toole, I.B. Jeffery, Gut microbiota and aging, Science 350 (2015) 129 (2015) 661–671.
1214–1215. [52] H. Lutz, A. Vangelatos, N. Gottel, A. Osculati, S. Visona, S.J. Finley, J.A. Gilbert,
[21] M. Lu, Z. Wang, Microbiota and aging, Adv. Exp. Med. Biol. (2018) 141–156. G.T. Javan, Effects of extended postmortem interval on microbial communities in
[22] J.H. Shin, M. Sim, J.Y. Lee, D.M. Shin, Lifestyle and geographic insights into the organs of the human cadaver, Front. Microbiol. 11 (2020) 3127.
distinct gut microbiota in elderly women from two different geographic locations, [53] I. Can, G.T. Javan, A.E. Pozhitkov, P.A. Noble, Distinctive thanatomicrobiome
J. Physiol. Anthropol. 35 (2016) 1–9. signatures found in the blood and internal organs of humans, J. Microbiol.
[23] M.S.H. Akash, F. Fiayyaz, K. Rehman, S. Sabir, M.H. Rasool, Gut microbiota and Methods 106 (2014) 1–7.
metabolic disorders: advances in therapeutic interventions, Crit. Rev. Immunol. [54] Texas Revised Uniform Anatomical Gift Act, Health and Safety Code Chapter
39 (2019). 692A, (n.d.). 〈https://statutes.capitol.texas.gov/Docs/HS/htm/HS.692A.htm〉.
[24] M. Egert, R. Simmering, C.U. Riedel, The association of the skin microbiota with (Accessed 3 March 2022).
health, immunity, and disease, Clin. Pharmacol. Ther. 102 (2017) 62–69. [55] N. Procopio, F. Lovisolo, G. Sguazzi, S. Ghignone, S. Voyron, M. Migliario,
[25] Y. Yun, H.N. Kim, S.E. Kim, S.G. Heo, Y. Chang, S. Ryu, H. Shin, H.L. Kim, F. Renò, F. Sellitto, G. D’Angiolella, P. Tozzo, L. Caenazzo, S. Gino, “Touch
Comparative analysis of gut microbiota associated with body mass index in a microbiome” as a potential tool for forensic investigation: a pilot study,
large Korean cohort, BMC Microbiol. 17 (2017) 1–9. J. Forensic Leg. Med. (2021), 102223.
[26] M. Sánchez-Tapia, A.R. Tovar, N. Torres, Diet as regulator of gut microbiota and [56] P.C.Y. Woo, S.K.P. Lau, J.L.L. Teng, H. Tse, K.-Y. Yuen, Then and now: use of 16S
its role in health and disease, Arch. Med. Res. 50 (2019) 259–268. rDNA gene sequencing for bacterial identification and discovery of novel bacteria
[27] J.S. Bajaj, Alcohol, liver disease and the gut microbiota, Nat. Rev. Gastroenterol. in clinical microbiology laboratories, Clin. Microbiol. Infect. 14 (2008) 908–934.
Hepatol. 16 (2019) 235–246. [57] A.E. Parada, D.M. Needham, J.A. Fuhrman, Every base matters: assessing small
[28] F. Gallè, F. Valeriani, M.S. Cattaruzza, F. Ubaldi, V. Romano Spica, G. Liguori, subunit rRNA primers for marine microbiomes with mock communities, time
S. Calimeri, R. Bono, G. Privitera, L. Fabiani, F. D’Aloisio, G. Baccari, E. Leoni, series and global field samples, Environ. Microbiol. 18 (2016) 1403–1414.
S. Tafuri, G. Liguori, G. Brandi, G. Gervasi, A. Dell’Eva, A. Gradilone, [58] A. Apprill, S. McNally, R. Parsons, L. Weber, Minor revision to V4 region SSU
C. Frangella, A. la Torre, Exploring the association between physical activity and rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton,
gut microbiota composition: a review of current evidence, Ann. Di Igiene 31 Aquat. Microb. Ecol. 75 (2015) 129–137.
(2019) 582–589. [59] J.J. Kozich, S.L. Westcott, N.T. Baxter, S.K. Highlander, P.D. Schloss,
[29] G. Yu, S. Phillips, M.H. Gail, J.J. Goedert, M.S. Humphrys, J. Ravel, Y. Ren, N. Development of a dual-index sequencing strategy and curation pipeline for
E. Caporaso, The effect of cigarette smoking on the oral and nasal microbiota, analyzing amplicon sequence data on the miseq illumina sequencing platform,
Microbiome 5 (2017) 1–6. Appl. Environ. Microbiol. 79 (2013) 5112–5120.
[30] Z. Savin, S. Kivity, H. Yonath, S. Yehuda, Smoking and the intestinal microbiome, [60] E. Bolyen, J.R. Rideout, M.R. Dillon, N.A. Bokulich, C.C. Abnet, G.A. Al-Ghalith,
Arch. Microbiol. 200 (2018) 677–684. H. Alexander, E.J. Alm, M. Arumugam, F. Asnicar, Y. Bai, J.E. Bisanz, K. Bittinger,
[31] R. Sender, S. Fuchs, R. Milo, Revised estimates for the number of human and A. Brejnrod, C.J. Brislawn, C.T. Brown, B.J. Callahan, A.M. Caraballo-Rodríguez,
bacteria cells in the body, PLoS Biol. 14 (2016), e1002533. J. Chase, E.K. Cope, R. da Silva, C. Diener, P.C. Dorrestein, G.M. Douglas, D.
[32] D.J. Wescott, Recent advances in forensic anthropology: decomposition research, M. Durall, C. Duvallet, C.F. Edwardson, M. Ernst, M. Estaki, J. Fouquier, J.
Forensic Sci. Res. 3 (2018) 278–293. M. Gauglitz, S.M. Gibbons, D.L. Gibson, A. Gonzalez, K. Gorlick, J. Guo,
[33] D. Roy, S. Tomo, P. Purohit, P. Setia, Microbiome in death and beyond: current B. Hillmann, S. Holmes, H. Holste, C. Huttenhower, G.A. Huttley, S. Janssen, A.
vistas and future trends, Front. Ecol. Evol. 9 (2021) 75. K. Jarmusch, L. Jiang, B.D. Kaehler, K. bin Kang, C.R. Keefe, P. Keim, S.T. Kelley,
[34] J.L. Metcalf, L.W. Parfrey, A. Gonzalez, C.L. Lauber, D. Knights, G. Ackermann, G. D. Knights, I. Koester, T. Kosciolek, J. Kreps, M.G.I. Langille, J. Lee, R. Ley, Y.
C. Humphrey, M.J. Gebert, W. van Treuren, D. Berg-Lyons, A microbial clock X. Liu, E. Loftfield, C. Lozupone, M. Maher, C. Marotz, B.D. Caraballo-Rodríguez,
provides an accurate estimate of the postmortem interval in a mouse model D. McDonald, L.J. McIver, A. v Melnik, J.L. Metcalf, S.C. Morgan, J.T. Morton, A.
system, Elife 2 (2013), e01104. T. Naimey, J.A. Navas-Molina, L.F. Nothias, S.B. Orchanian, T. Pearson, S.
[35] J.L. Pechal, T.L. Crippen, M.E. Benbow, A.M. Tarone, S. Dowd, J.K. Tomberlin, L. Peoples, D. Petras, M.L. Diener, E. Pruesse, L.B. Rasmussen, A. Rivers, M.
The potential use of bacterial community succession in forensics as described by S. Robeson, P. Rosenthal, N. Segata, M. Shaffer, A. Shiffer, R. Sinha, S.J. Song, J.
high throughput metagenomic sequencing, Int. J. Leg. Med. 128 (2014) 193–205. R. Spear, A.D. Swafford, L.R. Thompson, P.J. Torres, P. Trinh, A. Tripathi, P.
[36] F.E. Damann, D.E. Williams, A.C. Layton, Potential use of bacterial community J. Turnbaugh, S. Ul-Hasan, J.J.J. van der Hooft, F. Vargas, Y. Vázquez-Baeza,
succession in decaying human bone for estimating postmortem interval, E. Vogtmann, M. von Hippel, W. Walters, Y. Wan, M. Wang, J. Warren, K.
J. Forensic Sci. 60 (2015) 844–850. C. Weber, C.H.D. Williamson, A.D. Willis, Z.Z. Xu, J.R. Zaneveld, Y. Zhang,
[37] A. Galloway, W.H. Birkby, A.M. Jones, T.E. Henry, B.O. Parks, Decay rates of Q. Zhu, R. Knight, J.G. Caporaso, Reproducible,interactive, scalable and
human remains in an arid environment, J. Forensic Sci. 34 (1989) 607–616. extensible microbiome data science using QIIME 2, Nat. Biotechnol. 37 (2019)
[38] M.S. Megyesi, S.P. Nawrocki, N.H. Haskell, Using accumulated degree-days to 852–857.
estimate the postmortem interval from decomposed human remains, J. Forensic [61] B.J. Callahan, P.J. McMurdie, M.J. Rosen, A.W. Han, A.J.A. Johnson, S.P. Holmes,
Sci. 50 (2005) 1–9. DADA2: high-resolution sample inference from Illumina amplicon data, Nat.
[39] G.T. Javan, S.J. Finley, S. Tuomisto, A. Hall, M.E. Benbow, D.E. Mills, An Methods 13 (2016) 581–583.
interdisciplinary review of the thanatomicrobiome in human decomposition, [62] C. Quast, E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, F.
Forensic Sci. Med. Pathol. 15 (2019) 75–83. O. Glöckner, The SILVA ribosomal RNA gene database project: improved data
[40] W. Gevers, Biochemical aspects of cell death, Forensic Sci. 6 (1975) 25–29. processing and web-based tools, Nucleic Acids Res. 41 (2012) D590–D596.
[41] S. Tuomisto, P. Karhunen, R. Vuento, J. Aittoniemi, T. Pessi, Evaluation of [63] P.J. McMurdie, S. Holmes, phyloseq: an R package for reproducible interactive
postmortem bacterial migration using culturing and real-time quantitative PCR, analysis and graphics of microbiome census data, PloS One 8 (2013), e61217.
J. Forensic Sci. 58 (2013) 910–916. [64] P.J. McMurdie, S. Holmes, Waste not, want not: why rarefying microbiome data is
[42] J.M. DeBruyn, K.A. Hauther, Postmortem succession of gut microbial inadmissible, PLoS Comput. Biol. 10 (2014), e1003531.
communities in deceased human subjects, PeerJ 5 (2017), e3437. [65] M. de Cáceres, P. Legendre, Associations between species and groups of sites:
[43] K.A. Hauther, K.L. Cobaugh, L.M. Jantz, T.E. Sparer, J.M. DeBruyn, Estimating indices and statistical inference, Ecology 90 (2009) 3566–3574.
time since death from postmortem human gut microbial communities, J. Forensic [66] J.F. Petrosino, S. Highlander, R.A. Luna, R.A. Gibbs, J. Versalovic, Metagenomic
Sci. 60 (2015) 1234–1240. pyrosequencing and microbial identification, Clin. Chem. 55 (2009) 856–866.
[44] J. Guo, X. Fu, H. Liao, Z. Hu, L. Long, W. Yan, Y. Ding, L. Zha, Y. Guo, J. Yan, [67] A.S. Wieczorek, O. Schmidt, A. Chatzinotas, M. von Bergen, A. Gorissen, S. Kolb,
Y. Chang, J. Cai, Potential use of bacterial community succession for estimating Ecological functions of agricultural soil bacteria and microeukaryotes in chitin
post-mortem interval as revealed by high-throughput sequencing, Sci. Rep. 6 degradation: a case study, Front. Microbiol. 10 (2019) 1293.
(2016) 24197. [68] D. Zhao, X. Cao, R. Huang, J. Zeng, Q.L. Wu, Variation of bacterial communities
[45] S.J. Finley, M.E. Benbow, G.T. Javan, Microbial communities associated with in water and sediments during the decomposition of Microcystis biomass, PLoS
human decomposition and their potential use as postmortem clocks, Int. J. Leg. One 12 (2017), e0176397.
Med. 129 (2015) 623–632. [69] E.R. Hyde, J.L. Metcalf, S.R. Bucheli, A.M. Lynne, R. Knight, Microbial
[46] J.L. Metcalf, Estimating the postmortem interval using microbes: Knowledge gaps communities associated with decomposing corpses. Forensic Microbiology, Wiley
and a path to technology adoption, Forensic Sci. Int. Genet. 38 (2019) 211–218. Online Books, John Wiley & Sons Ltd,, The Atrium, Southern Gate, Chichester,
[47] H.R. Dash, S. Das, Thanatomicrobiome and epinecrotic community signatures for West Sussex, UK, 2017, pp. 245–273.
estimation of post-mortem time interval in human cadaver, Appl. Microbiol. [70] J.L. Metcalf, Z.Z. Xu, S. Weiss, S. Lax, W. van Treuren, E.R. Hyde, S.J. Song,
Biotechnol. (2020) 1–16. A. Amir, P. Larsen, N. Sangwan, Microbial community assembly and metabolic
[48] N. Procopio, S. Ghignone, S. Voyron, M. Chiapello, A. Williams, A. Chamberlain, function during mammalian corpse decomposition, Science 351 (2016) 158–162.
A. Mello, M. Buckley, Soil fungal communities investigated by metabarcoding [71] E.R. Hyde, D.P. Haarmann, A.M. Lynne, S.R. Bucheli, J.F. Petrosino, The living
within simulated forensic burial contexts, Front. Microbiol. (2020) 1686. dead: bacterial community structure of a cadaver at the onset and end of the bloat
stage of decomposition, PloS One 8 (2013), e77733.

11
G. Sguazzi et al. Forensic Science International: Genetics 59 (2022) 102686

[72] J.A. Morris, L.M. Harrison, S.M. Partridge, Postmortem bacteriology: a re- geothermally heated soil sample, and reclassification of Lysobacter thermophilus
evaluation, J. Clin. Pathol. 59 (2006) 1–9. Wei et al. 2012 as Vulcaniibacterium thermophilum comb. nov, Antonie Van
[73] G.T. Javan, S.J. Finley, T. Smith, J. Miller, J.E. Wilkinson, Cadaver Leeuwenhoek 104 (2013) 369–376.
thanatomicrobiome signatures: the ubiquitous nature of Clostridium species in [89] C. Baur, M. Krewinkel, I. Kutzli, B. Kranz, M. von Neubeck, C. Huptas,
human decomposition, Front. Microbiol. 8 (2017) 2096. M. Wenning, S. Scherer, M. Stoeckel, J. Hinrichs, Isolation and characterisation of
[74] J.W. Castle, D.M. Butzbach, G.S. Walker, C.E. Lenehan, F. Reith, K.P. Kirkbride, a heat-resistant peptidase from Pseudomonas panacis withstanding general UHT
Microbial impacts in postmortem toxicology, Forensic Microbiol. (2017) processes, Int. Dairy J. 49 (2015) 46–55.
212–244. [90] D.M. Adams, T.G. Brawley, Factors influencing the heat resistance of a heat-
[75] R.C. Janaway, S.L. Percival, A.S. Wilson, Decomposition of human remains, in: resistant lipase of Pseudomonas, J. Food Sci. 46 (1981) 673–676.
Microbiology and Aging, Springer, 2009, pp. 313–334. [91] K.J. Szwetkowski, J.O. Falkinham, Methylobacterium spp. as emerging
[76] L.P. Chun, M.J. Miguel, E.N. Junkins, S.L. Forbes, D.O. Carter, An initial opportunistic premise plumbing pathogens, Pathogens 9 (2020) 149.
investigation into the ecology of culturable aerobic postmortem bacteria, Sci. [92] S. Harirchi, Z. Etemadifar, F. Yazdian, M.J. Taherzadeh, Efficacy of
Justice 55 (2015) 394–401. polyextremophilic Aeribacillus pallidus on bioprocessing of beet vinasse derived
[77] K. Dong, Y. Xin, F. Cao, Z. Huang, J. Sun, M. Peng, W. Liu, P. Shi, Succession of from ethanol industries, Bioresour. Technol. 313 (2020), 123662.
oral microbiota community as a tool to estimate postmortem interval, Sci. Rep. 9 [93] A. Rodriguez-Palacios, J.T. LeJeune, Moist-heat resistance, spore aging, and
(2019) 1–9. superdormancy in Clostridium difficile, Appl. Environ. Microbiol. 77 (2011)
[78] K.L. Cobaugh, S.M. Schaeffer, J.M. DeBruyn, Functional and structural succession 3085–3091.
of soil microbial communities below decomposing human cadavers, PLoS One 10 [94] J.L. Metcalf, D.O. Carter, R. Knight, Microbiology of death, Curr. Biol. Mag. 26
(2015), e0130201. (2016) R543–R576.
[79] S. Tuomisto, P.J. Karhunen, T. Pessi, Time-dependent post mortem changes in the [95] Y. Fang, Y. Yuan, J. Liu, G. Wu, J. Yang, Z. Hua, J. Han, X. Zhang, W. Li, H. Jiang,
composition of intestinal bacteria using real-time quantitative PCR, Gut Pathog. 5 Casting light on the adaptation mechanisms and evolutionary history of the
(2013) 1–5. widespread Sumerlaeota, Mbio 12 (2021) e00350–21.
[80] J. Adserias-Garriga, N.M. Quijada, M. Hernandez, D. Rodríguez Lázaro, [96] T. Thurnheer, L. Karygianni, M. Flury, G.N. Belibasakis, Fusobacterium species
D. Steadman, L.J. Garcia-Gil, Dynamics of the oral microbiota as a tool to and subspecies differentially affect the composition and architecture of supra-and
estimate time since death, Mol. Oral Microbiol. 32 (2017) 511–516. subgingival biofilms models, Front. Microbiol. 10 (2019) 1716.
[81] J. Adserias-Garriga, M. Hernández, N.M. Quijada, D.R. Lázaro, D. Steadman, [97] D. Carter, D. Yellowlees, M. Tibbett, Temperature affects microbial
J. Garcia-Gil, Daily thanatomicrobiome changes in soil as an approach of decomposition of Cadavers (Rattus rattus) in Contrasting soils, Appl. Soil Ecol. 40
postmortem interval estimation: an ecological perspective, Forensic Sci. Int. 278 (2008) 129–137.
(2017) 388–395. [98] G. Santoyo, M. del, C. Orozco-Mosqueda, M. Govindappa, Mechanisms of
[82] P.K. Wüst, B.U. Foesel, A. Geppert, K.J. Huber, M. Luckner, G. Wanner, biocontrol and plant growth-promoting activity in soil bacterial species of
J. Overmann, Brevitalea aridisoli, B. deliciosa and Arenimicrobium luteum, three Bacillus and Pseudomonas: a review, Biocontrol Sci. Technol. 22 (2012) 855–872.
novel species of Acidobacteria subdivision 4 (class Blastocatellia) isolated from [99] L.M. Kootker, I.C.C. von Holstein, J. Broeders, D.J. Wescott, G.R. Davies, H.
savanna soil and description of the novel family Pyrinomonadaceae, Int. J. Syst. L. Mickleburgh, The effects of decomposition and environment on antemortem H-
Evolut. Microbiol. 66 (2016) 3355–3366. Pb-Sr isotope compositions and degradation of human scalp hair: Actualistic
[83] M.A. Crowe, J.F. Power, X.C. Morgan, P.F. Dunfield, K. Lagutin, W.I.C. Rijpstra, taphonomic observations, Forensic Sci. Int. 312 (2020), 110336.
M. Vyssotski, J.S.S. Damste, K.M. Houghton, J.L.J. Ryan, Pyrinomonas [100] L.M. Kootker, S.T.M. Ammer, D.J. Wescott, G.R. Davies, H.L. Mickleburgh,
methylaliphatogenes gen. nov., sp. nov., a novel group 4 thermophilic member of Diagenesis of human bone, tooth enamel, dentine in short time frames:
the phylum Acidobacteria from geothermal soils, Int. J. Syst. Evolut. Microbiol. implications for Isotope Forensics., In Preparation, (n.d.).
64 (2014) 220–227. [101] H.L. Mickleburgh, E.C. Schwalbe, A. Bonicelli, H. Mizukami, F. Sellitto, S. Starace,
[84] N.S. Makkar, L.E. Casida Jr., Cupriavidus necator gen. nov., sp. nov.; a D.J. Wescott, D.O. Carter, N. Procopio, Human bone proteomes before and after
nonobligate bacterial predator of bacteria in soil, Int. J. Syst. Evolut. Microbiol. decomposition: investigating the effects of biological variation and taphonomic
37 (1987) 323–326. alteration on bone protein profiles and the implications for forensic proteomics,
[85] P. Vandamme, T. Coenye, Taxonomy of the genus Cupriavidus: a tale of lost and J. Proteome Res. 20 (2021) 2533–2546.
found, Int. J. Syst. Evolut. Microbiol. 54 (2004) 2285–2289. [102] S. Gino, C. Robino, C. Torre, DNA typing of liquid blood samples stored at 4◦ C for
[86] J. Handke, N. Procopio, M. Buckley, D. van der Meer, G. Williams, M. Carr, 15years, in: Proceedings of the Eighteenth International ISFH Congress, Elsevier,
A. Williams, Successive bacterial colonisation of pork and its implications for 2000, 476–478.
forensic investigations, Forensic Sci. Int. 281 (2017) 1–8. [103] ENFSI DNA Working Group, DNA-Database Management Review and
[87] F. Tuccia, E. Zurgani, S. Bortolini, S. Vanin, Experimental evaluation on the Recommendations, 2016.
applicability of necrobiome analysis in forensic veterinary science, [104] S. Marshall, Retention, Storage and Destruction of Materials and Records relating
MicrobiologyOpen 8 (2019), e00828. to Forensic Examination, (2021).
[88] T.-T. Yu, E.-M. Zhou, Y.-R. Yin, J.-C. Yao, H. Ming, L. Dong, S. Li, G.-X. Nie, W.-
J. Li, Vulcaniibacterium tengchongense gen. nov., sp. nov. isolated from a

12