In the current context of forensic science, the term forensic microbiology is typically defined as the study of microorganisms, including their DNA and other biomolecules, to aid in the investigation of crimes or other legal cases. However, in the realm of bioterrorism, forensic microbiology encompasses the illegal deployment of biological agents capable of releasing toxins with dangerous effects on the population (Budowle et al., 2003).

An emerging branch of forensic microbiology, developed in recent years, involves the investigation of microbes associated with decomposition (Iancu et al., 2015, 2018; Javan et al., 2016; Metcalf et al., 2017; Singh et al., 2018), since it is well known that microbes are the driving factor of decomposition and pathogenic spread (Damann et al., 2015; Hauther et al., 2015; Johnson et al., 2016; Adserias-Garriga et al., 2017; DeBruyn and Hauther, 2017; Harrison et al., 2020). In this regard, studies used pig carcasses (Iancu et al., 2015; DeBruyn and Hauther, 2017; Forger et al., 2019), rat carcasses (Metcalf et al., 2013; Guo et al., 2016), and human cadavers (Humphreys et al., 2013; Hauther et al., 2015; Finley et al., 2016; Jeong et al., 2016) exposed in various geographical locations and seasons, to investigate the microbiome patterns during decomposition. The intended application of the forensic microbiome investigation is represented by the postmortem interval (PMI) estimation (Metcalf et al., 2013; Belk et al., 2018). Most of the studies related to the postmortem microbial diversity from human bodies were performed in the USA, primarily in Michigan (Pechal et al., 2018), Texas (Hyde et al., 2013), Alabama (Can et al., 2014), and Tennessee (Damann et al., 2015; Hauther et al., 2015; Johnson et al., 2016; Adserias-Garriga et al., 2017; DeBruyn and Hauther, 2017), while scarce studies were performed in Europe. To emphasize bacteria as “postmortem clocks,” several studies (Hyde et al., 2013; Bucheli and Lynne, 2016) addressed the species/community’s identification and their succession. As previously demonstrated, the postmortem microbiome presents predictable patterns associated with different decomposition stages (Bucheli and Lynne, 2016); however, the specificity of the microbiome related to each individual and antemortem conditions must be taken into consideration. In addition to the individual microbiome characteristics, the diversity and dynamics varies among body locations (Javan et al., 2019). Thus, in our study we chose to sample and investigate face and hand areas, as these are the most exposed to exogenous factors; intending to distinguish common trends among all of this diversity.

A few studies (Pechal et al., 2018; Kodama et al., 2019; Ahannach et al., 2021; Tozzo et al., 2022) aimed to investigate the postmortem microbiome of human skin, as a non-invasive collection method, but also in aiming to associate skin bacteria with personal objects (Gouello et al., 2021; Speruda et al., 2022). These studies revealed that the decomposition microbiome may be linked to antemortem conditions (Pechal et al., 2018). Although these previous studies stated that bacterial decomposition indicators may not be present in all cases and ubiquitous species are not particularly useful for specifying the manner and/or cause of death, the current study aims to propose the usage of ubiquitous microbes and their relative abundances as markers of the PMI, precisely given their cosmopolite presence that can be quantified and related to antemortem and/or postmortem conditions. Considering that each person hosts a different microbiota, composed of up to 1,000 different bacteria species, which in turn varies across body sites and time (Turnbaugh et al., 2007), and that the discriminatory power of the microbiome could be similar to that of a fingerprint (Costello et al., 2009; Grice et al., 2009; Fitz-Gibbon et al., 2013), investigating common taxa/colonization patterns could lead to a better understanding of these variations after death.

Since skin is the most extensive human organ, harboring unique niches of microbes varying from site to site (Costello et al., 2009), and taking into consideration a non-invasive collection technique (via sterile swabs) the current research aimed to investigate skin postmortem microbial signatures. Moreover, since scarce studies aimed to investigate the link between microbiome and cause and manner of death, our studied intended to provide critical information related to the human postmortem skin microbiome signatures along the first 12 h since morgue receiving, by investigating not only the diversity, but also the association with various antemortem conditions. To obtain an overview of the postmortem microbial patterns, skin samples associated with the face and hands of eight human bodies received at the morgue, in Iași (Romania), were investigated during April and May 2021. Due to the highly conserved regions, 16S rRNA is widely used as a genetic marker for the identification of microbial communities (Roy et al., 2021). Moreover, as previously observed (Bell et al., 2018), Next Generation Sequencing (NGS) is best suited for the identification of microbial signatures. Hence, in the current study, the collected skin samples were investigated via NGS Illumina MiSeq.

Decomposition starts from the gastrointestinal tract driven primarily by the lack of response from the immune system and by a low level of oxygen within the cells. While thanatomicrobiome profiles (Javan et al., 2019) are exhibiting correlations with different decomposition stages, skin postmortem microbiomes could reveal similar characteristics for the early postmortem interval. Although necrobiome studies expanded in recent years (Javan et al., 2016, 2019; Oliveira and Amorim, 2018) aiming to help in the PMI estimation, many more studies need to be developed, to better understand the skin postmortem microbiome; given the specificity of each individual’s microbiota. In this context, our research focused on examining the skin postmortem microbiome, with the aim of identifying similarities that would help understand microbial signatures throughout early decomposition.

Our research theory was founded on the following research questions aiming to provide data that could help in elucidating the role of microbes as PMI estimators: (a) are postmortem skin microbiome patterns similar in regard to the human cadavers arrived at the morgue within the first 12 h? (b) if variations are detected, are those triggered by the cause of death or by the antemortem medical conditions? (c) can shared microbial taxa be used as PMI indicators?

Of the eight cases, we successfully generated a total of 7,951,382 high-quality merged sequence reads [median (IQR): 49,578 (33,136-69,377)], which were then mapped to 16,555 amplicon sequence variants (ASVs).

We present here the outcomes regarding the skin postmortem microbial investigation of human bodies 12 h after morgue receiving. Most of the studies to date focused on investigating decomposition and microbiome in terrestrial environments (Tuomisto et al., 2013; Hauther et al., 2015), and over more extended periods of time, while the current study focused on the first 12 h upon morgue receiving of the bodies. This approach was used, since this is the typical timeframe for keeping cadavers at the morgue (at that facility), hence, trying to bring the current experimental research as close as possible to real cases. Sometimes, the collection of skin or tissue samples at the crime scene is not possible, therefore, only the morgue samples could be accessible. In a hypothetical case, limited-to-no information from the crime scene would be available to a forensic microbiologist, such as temperature and relative humidity records, living conditions, e.g., A better communication/collaboration between death investigators, forensic pathologists, and forensic microbiologists would help overcome difficulties and fill in the information gaps. Moreover, access to medical records and autopsy reports could prove to be crucial in understanding the microbial distribution after death.

As previously stated (Ahn and Hayes, 2021), an individual’s microbiome is influenced by both macroenvironmental factors (toxicological, location of discovery, socioeconomic, etc.) and microenvironmental factors (alcohol, diet, etc.); hence, this explains the specific necrobiome clustering based on medical backgrounds and cause of death, in the current experiment. Understanding the medical background would help tremendously in the identification of potential bacterial species as PMI estimators. Since the microbiome fulfills multiple functions within the living organism, like different metabolic functions, immune system development, and nutrient synthesis, researching the cause and manner of death, together with the dynamics of the decomposition process, will provide a clearer understanding of the microbiome distribution patterns throughout decomposition.

The built environment (location of body discovery; Gilbert and Stephens, 2018) is included within the macroenvironmental factors that can influence the microbial structure of each individual. In the present experiment, a distinct microbiome structure was observed between decedents found at an apartment building and other locations, such as hospital, stairway, and a house. Recognizing that the built environment has an impact on the individual’s microbiome (Sharma et al., 2019), the place of death must be considered when investigating skin postmortem microbial signatures. Among the microenvironmental factors, alcohol played an important role in understanding the microbiome distribution in the current experiment. Previous studies on animal models (Kantorski et al., 2007) showed an increase in Streptococcus mutans colonization correlated with 20% of alcohol intake. For the case with the highest alcohol concentration, M8, Streptococcus spp. was present, but did not represent a dominant taxon. Nevertheless, the results revealed a decrease in bacterial richness and diversity associated to an elevated blood alcohol concentration, similar to previous findings (Signoretto et al., 2010; Thomas et al., 2014). Liver cirrhosis has been associated with a unique increase in Fusobacteria (Chen et al., 2011), while in our experiment, an increase in abundance was observed in M8, representing the case associated with the highest alcohol concentration, 1.81 g%. It is of note that previous studies investigated the microbiome diversity linked with macro and microenvironmental factors in living individuals. Similarities between previous and current studies could be explained by the microbiome reflection of antemortem conditions during early PMI. Nevertheless, antemortem conditions (medical records, living environment, dietary factors) could play a key role in shaping the postmortem microbiome communities over a more extended postmortem period (Carter, 2020).

Previous data (Lutz et al., 2020) showed that the microbial beta diversity patterns could be of help in predicting the cause or manner of death. This is portrayed in the current research, as different microbial signatures have been associated with a specific cause of death, such as cardiovascular disease. Moreover, studies on heart thanatomicrobiome (Bell et al., 2018) revealed differences in bacterial diversity between males and females. Proteobacteria has been identified as predominant phylum for cardiovascular disease associated deaths (Metcalf et al., 2013; Shin et al., 2015). Kaszubinski et al. (2020) showed that death related to cardiovascular disease or natural causes present a higher beta diversity dispersion and a lower alfa diversity (Pechal et al., 2018; Pearson et al., 2019). However, age, other causes of death, lifestyle, must be considered when investigating beta and alfa diversities.

In one of these previous studies (Pechal et al., 2018) the living environment explained most of the microbial diversity. Actinobacteria and Bacteroidetes were observed to decrease during decomposition, while Proteobacteria presented an increasing trend. In our investigation, Actinobacteria and Firmicutes slightly decreased over the first 12 h, while Bacteroidetes and Proteobacteria increased. In previous studies Firmicutes was found to be a constant marker between different body areas (Roy et al., 2021), while this phylum and Proteobacteria were found to be the predominant taxa identified from buccal swabs collected from cadavers in different decomposition stages (Sguazzi et al., 2022; Ogbanga et al., 2023). The increase in Bacteroidetes is in contrast with previous findings (Pechal et al., 2018), however, the sampling areas must be taken into consideration (face and hands vs. eyes, nose, mouth). Staphylococcus and Streptococcus, common genera identified in living humans, prevailed in most of the analyzed skin samples, while being previously identified as dominant taxa associated with the mouth samples after more than 24 h after death (Pechal et al., 2018). As previously shown (Speruda et al., 2022) the presence of Bacilli, like Staphylococcus and Streptococcus species can also indicate an infection or a contamination. Furthermore, Staphylococcus and Streptococcus were identified (Hyde et al., 2013; Javan et al., 2016; Johnson et al., 2016; Adserias-Garriga et al., 2017; Pechal et al., 2018) as important PMI indicators by machine-learning algorithms, confirmed by our results, emphasizing the need for targeted studies on these two genera.

Prior studies (Sampson and Mazmanian, 2015; Sharon et al., 2016) revealed that the gut microbiome can play a role in the nervous system functions, thus, potentially influencing various nervous system disorders. In the current research, two cases were associated with medical records encompassing dementia, while one case out of the two presented additional medical conditions. Although the one case that had a clear medical record associated with dementia clustered separate of all samples, it is not clear if this is the sole factor, or if the sex could be another element to be considered. Nevertheless, in addition to the forensic implications, understanding the gut-brain microbiome-nervous system interactions could help in developing preventive therapies and treatments of various neurologic disorders. When Pechal et al. (2018) investigated the relationship postmortem microbiome—health status, a low microbial diversity was found to be associated with heart conditions, similar to our findings. A more complete set of medical conditions as reflected by the medical records could provide further insights into postmortem microbiome, as a chronic health condition or habit, like alcohol consumption, can impact the microbial biodiversity, as previously reported in living humans (Eckburg et al., 2005; Cabrera-Rubio et al., 2012).

Schmedes et al. (2017) identified Propionibacterium acnes as the species present in all body sites, while in the current study the Propionibacterium sp. was identified only in 86 samples, from the total of 150 analyzed. This difference can be explained by the sequencing approach, as Schmedes et al. (2017) used shotgun metagenomics. Moreover, the same authors observed that the hand samples yield accurate rates of classification, even though they contained fewer shared phylotypes between the two hands of the same individual. It is important to consider that the hands are the target of frequent recolonization by daily activities.

As Kodama et al. (2019) revealed that the postmortem skin microbiomes remain stable in the first 24 h, as in our study (in the first 12 h) with little to no variation. Moreover, these microbial communities show potential in establishing associations with personal objects, such as medical devices, mobile phones, keyboard, computer mice, and fabrics (Fierer et al., 2010; Meadow et al., 2014; Lax et al., 2015; Lee et al., 2016).

According to Oh et al. (2016), healthy individuals maintain their skin communities for up to 2 years (despite daily life perturbations). Pechal et al. (2018) suggested that the bacterial taxa in the first 24–48 h after death would represent antemortem bacterial communities.

It was previously discussed that two individuals are having in common only 13% of all identified phylotypes (Speruda et al., 2022), so focusing the research on shared taxa/patterns could lead to a better understanding of the role of microbes in decomposition. Among shared taxa, Clostridium spp. was observed during the first 72 h postmortem, up to the entire duration of decomposition (Plueckhahn, 1986; Javan et al., 2016, 2019), similar to our study, where Clostridium sensu stricto was predominant for the face samples. However, the representatives of this genus have been observed mostly in organ samples toward the end of the bloat stage (Hyde et al., 2013, 2015; Can et al., 2014; DeBruyn and Hauther, 2017).

Differences in the thanatomicrobiome communities were recorded in male vs. female cadavers (Javan et al., 2016); Pseudomonas species being representative for females, while Rothia was found to be prevalent in males. In the current research such comparison is not supported, given the 1:7 female to male ratio, however, differences could be observed. The human microbiome is not only dynamic or sex dependent (Turnbaugh et al., 2007; Sguazzi et al., 2022), but it is also concentrated in certain areas of the body, constituting specific niches, such as the intestines (Roy et al., 2021). Our alfa diversity results showed differences between body areas as well. Therefore, it is crucial to investigate these specific niches, such as the skin postmortem microbiome, to better understand what intrinsic and extrinsic factors, macro, and microenvironments, are creating these variations, to identify common putative bacteria as PMI estimators. Moreover, as previously emphasized (Kaszubinski et al., 2020) the microbiome specificity for certain body sites is stable in the first 48 h, reflecting the antemortem conditions. Age is another factor affecting the microbiome during, and after death. Aging is considered to have an impact on the intestinal microbiome (Xu et al., 2019; Brooks et al., 2023), the diversity decreasing with age (Leite et al., 2021), while, Proteobacteria being observed in higher abundances in older living subjects. During the current experiment, Firmicutes was more abundant in cases involving older adults, however, these observations are made postmortem. As our study revealed, the postmortem skin microbiome has potential in understanding the cause and manner of death, if corroborated with the medical records data. Future experiments including the corroboration between chronological age, the number of associated diseases, the number of medications used, and the microbiome diversity would be of great help to provide additional information. A standardized sampling area(s) would help in further comparisons of the postmortem skin microbiomes even between continents. Depending on the body’s state of decomposition, the selection of a precise sampling location would be helpful to be included in a standardized sampling protocol (Metcalf, 2019). A standardized collection protocol calls the need of standardized protocols for DNA extraction, amplification, sequencing, and data analysis. Additionally, the time elapsed between death and the collection of samples must be considered. This is important since microbiome changes are dynamic, the diversity being influenced by the external environment, and the internal microenvironment. Due to the limited number of analyzed cases, the current research must be continued and expanded, as studies from different continents/countries are paramount for understanding the role and application of the microbiome in forensic investigations worldwide.

Samples representing face and hands skin microbiome were investigated from eight cases within 12 h upon arrival at the morgue. The taxonomic characteristics of the skin microbiota varied across different body sites, with no significant differences in taxonomic profiles between the two collection times (T0 and T1). The investigation also showed that different microbial signatures could be associated with a specific cause of death, such as cardiovascular disease, while an elevated blood alcohol level could be associated with a decrease in bacterial richness and diversity. Moreover, the places where the bodies were discovered seemed to play an important role in explaining microbiome diversity.

Given the predictive patterns and ubiquitousness of microbes, these taxa have great potential to be used as PMI estimators. Human skin is personalized, however, some microbes were shared between cases, like Staphylococcus and Streptococcus. As many bodies are discovered in the first 48 h after death, knowledge of skin microbial diversity would constitute a powerful tool for the PMI estimation, in cases where additional methods cannot be used. The resulted outcomes provided an overview on the postmortem skin microbiome in the 12 h upon morgue receiving of the bodies; extensive future studies are required to complement the current data, including the investigation of a greater number of cases.

The original contributions presented in this study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/, PRJNA940997.

The studies involving human participants were reviewed and approved by Institute of Legal Medicine Iași (no. 26067/03.23.2021) and the Institutional Biosafety Committee (IBC) University of North Dakota (UND; no. IBC-202111-011). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

LI performed the experimental design, data interpretation, and wrote the manuscript. VJ performed sample and metadata collection. AM, SA, and LI analyzed and interpreted the raw data. All authors contributed to the article and approved the submitted version.

This research received financial support from the Start-up research funding (LI), College of Arts and Sciences, University of North Dakota.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.