Abstract
Microbial forensics represents a promising tool to strengthen traditional forensic investigative methods and fill related knowledge gaps. Large-scale microbiome studies indicate that microbial fingerprinting can assist forensics in areas such as trace evidence, source tracking, geolocation, and circumstances of death. Nevertheless, the majority of forensic microbiome studies focus on soil and internal organ samples, whereas the microbiome of skin, mouth, and especially vaginal samples that are routinely collected in sexual assault and femicide cases remain underexplored. This review discusses the current and emerging insights into vaginal, skin, and salivary microbiome-modulating factors during life (e.g., lifestyle and health status) and after death (e.g., environmental influences and post-mortem interval) based on next-generation sequencing. We specifically highlight the key aspects of female reproductive tract, skin, and mouth microbiome samples relevant in forensics. To fill the current knowledge gaps, future research should focus on the degree to which the post-mortem succession rate and profiles of vaginal, skin, and saliva microbiota are sensitive to abiotic and biotic factors, presence or absence of oxygen and other gases, and the nutrient richness of the environment. Application of this microbiome-related knowledge could provide valuable complementary data to strengthen forensic cases, for example, to shed light on the circumstances surrounding death with (post-mortem) microbial fingerprinting. Overall, this review synthesizes the present knowledge and aims to provide a framework to adequately comprehend the hurdles and potential application of vaginal, skin, and salivary post-mortem microbiomes in forensic investigations.
Introduction
Violence against women is an urgent global problem, as more than one-third of women worldwide has been victim of physical and/or sexual violence in their lifetime (; World Health Organization, 2013). Annually, 66,000 women and girls are victims of femicide, that is, intentional murder of women and girls because they are female (; World Health Organization and Pan American Health Organization, 2012). Remarkably, only 25% of reported rape cases in Europe lead to a conviction, often due to the difficulty of providing evidence (). Moreover, forensic experts are convinced that a large number of undetected homicides are misclassified in annual death statistics as natural deaths, suicides, or accidents (; ). The major obstacle is the difficulty of elucidating the circumstances surrounding death, including cause and manner (; ). Traditional forensic techniques, such as human DNA profiling, can provide critical evidence by linking biological traces to crime scenes and individuals or through victim identification (). However, they occasionally fall short because of human DNA degradation (Sijen, 2015; ) and need to be complemented with alternative techniques.
Recent advances in microbial profiling have uncovered that each individual is home to complex microbial communities (; ). These communities inhabit all surfaces of the human body (for example, orogastrointestinal tract, respiratory tract, urogenital tract, skin) and collectively represent the human microbiota, with their microbial DNA signatures forming the microbiome (Ursell et al., 2012; ). Recent research suggests that the microbiome could greatly aid forensic casework (; ; ; ; ). For example, the microbiome can serve as a personal microbial fingerprint that not only can associate individuals to objects () and geographical locations that they came in contact with (), but also can provide identifiable characteristics (). Moreover, many microbial cells contain robust cell walls that leave them better protected against degradation compared to human cells (Sijen, 2015). Nevertheless, the focus of microbial forensics to date has been predominantly on gut and soil samples (, ; ; ; ; ), whereas research on forensic implementation of the (post-mortem) microbiome of the female reproductive tract, skin, and oral cavity is lagging behind. Therefore, in this review, we provide a critical assessment of current research on the vaginal, skin, and oral/salivary microbiome in relation to their potential application in forensics, especially sexual assault and femicide cases.
Relevance of Vaginal, Skin, and Salivary Microbiome During Life for Forensic Casework
Vaginal, skin, and saliva samples represent some of the most commonly collected samples in forensic casework, including sexual assault cases (World Health Organization, 2003; ) and cases involving touch evidence (; ). These mucocutaneous niches are shaped by several microbiome-influencing factors (e.g., pH and oxygen) (; ). However, which of these factors have the largest effect is still unknown.
Vaginal samples are routinely collected in sexual assault cases (; ). While their microbiome is generally neglected in forensics, the less diverse composition of vaginal microbiota, its high microbial biomass, and protected anatomical location translate into its unique potential for microbial fingerprinting (Younes et al., 2018). Depending on the women’s ethnicity, the vaginal microbiome is generally dominated by Gram-positive Lactobacillus genera covered by a thick cell wall (i.e., Lactobacillus crispatus, Lactobacillus iners, Lactobacillus gasseri, and Lactobacillus jensenii) or a diverse microbiota dominated by non-lactobacilli such as Bifidobacterium, Gardnerella, Atopobium, and Prevotella (). Also fungal taxa mostly represented by Candida are detected, but generally in low abundances in healthy women (). Ongoing research suggests that the vaginal microbiome composition can be correlated to individual characteristics valuable in forensics, such as health status (), ethnicity (; ), sexual habits (), contraceptive use (Song et al., 2020), and pregnancy (Serrano et al., 2019), with various effect sizes that are not yet well mapped (Figure 1). For example, a longitudinal study found that sexual activity within 24 h of sampling has a significant negative impact on vaginal microbiome constancy as measured via the log Jensen–Shannon divergence rate (i.e., vaginal community deviation from constancy), independent of time in the menstrual cycle (). The vaginal microbiome could thus represent trace evidence in sexual assault cases indicating sexual intercourse in the last 24 h, in addition to providing links with other identifiable individual characteristics. However, whether this conclusion can be drawn from single, non-longitudinal samples after sexual intercourse needs to be investigated.
FIGURE 1
While vaginal samples are especially useful in sexual assault cases, the skin is probably the most commonly used source of forensic trace evidence, including skin under the victim’s fingernails (; ). However, the unique microbial trail left behind by skin shedding (; ) is often overlooked. The skin microbiome is dominated by Gram-positive Staphylococcus, Corynebacterium, Cutibacterium, Streptococcus, or Micrococcus, although Gram-negative Acinetobacter are also frequently isolated (). The skin microbiome composition varies depending on the body location (; ), host characteristics (e.g., age, lifestyle, and cohabitation) (Ross et al., 2017), and skin care (). Importantly, the skin microbiome is at an interface between the outside world and the body that undergoes most interactions with the environment. In fact, detectable amounts of skin microbiota can be transferred to objects such as a computer keyboard and mouse (), shoes and phones (), door handle, toilet seat, etc. (). Remarkably, this is not only limited to touched surfaces, but also extends to inhabited spaces through microbial clouds which are detectable within just a few hours (; ). However, whether built environment microbiota can be used as trace evidence remains to be substantiated (). An important knowledge gap is to what extent the skin microbiome can be detected on touched objects after a certain amount of time, potentially even after death. Establishing this link is complicated, as it depends on environmental parameters (e.g., temperature, moisture, and UV radiation) (), individual shedder status (i.e., amount of epithelium deposited on a substrate) (; ), and surface characteristics of the object ().
Saliva, as the primary oral cavity sample, is another widely used trace in forensic casework, especially for skin bite marks in sexual assault and child abuse cases (; ). The salivary microbiome is mostly dominated by the Gram-negative Neisseria, Prevotella, or Veillonella but also contains large proportions of Gram-positive Streptococcus taxa (e.g., Streptococcus salivarius and Streptococcus oralis) (Willis et al., 2018). Oral Gram-positive bacteria have recently been described as robust markers for highly degraded saliva samples, because of their higher resistance to degradation treatment (e.g., heat denaturation, microbial decomposition, and ultraviolet irradiation) compared to Gram-negative salivary bacteria, salivary α-amylase, and human DNA (). Individual characteristics that can influence the salivary microbiome composition include smoking (; Wu et al., 2016), dental hygiene (; ), general and oral health (Zhou et al., 2016; ), and socioeconomic status (; Figure 1). Importantly, shared environment at household level appears to more significantly determine the salivary microbiome than individual genetics (Shaw et al., 2017). Also intimate contact relevant in forensics, such as kissing (i.e., mixing of saliva), has been proposed to impact salivary microbial composition. Specifically, a transfer of approximately 80 million marker bacteria per intimate kiss of 10 s is observed, and partners have a more similar microbial community compared to unrelated individuals (). Further research into factors influencing the vaginal, skin, and oral/salivary microbiomes during life will allow their more targeted implementation in forensic casework.
The Detrimental Effects of Death on the Human Microbiome
While the human microbiome during life is widely studied, we are just beginning to understand the post-mortem microbial community dynamics and how it can be influenced by ante-mortem microbial communities and modulating factors. During different stages after death, many anatomical and immunological barriers break, causing fluids, chemicals, and microorganisms that normally would not interact, to come into contact with each other (). The post-mortem process facilitates the proliferation and relocation of microorganisms throughout the body and opens a gateway of cross-kingdom ecological interactions (; ).
The decomposition of a human body is a continuous process caused by enzymatic reactions, (bio)chemical metabolic pathways and the activity of vertebrates and invertebrates (). This process is divided into a series of observable stages: fresh, active decay (including bloating and leakage of effusion), advanced decay, and dry remains/skeletonized (; ). The course of these stages is partially determined by the diverse microbial communities occupying various internal and external body sites (; ). Most studies on the post-mortem microbiome focus on estimating the minimum period of time since death [i.e., post-mortem interval (PMI)] (). These studies aim to predict changes in the microbial composition of internal organs (e.g., gut, brain, liver, spleen, heart, etc.), also referred to as the post-mortem microbial clock (; ).
Interestingly, studies on internal organs of mice (; ), swine (), and human bodies (; ; ) have observed a shift in microbial communities from predominant aerobic microorganisms such as Staphylococcus and Enterobacteriaceae to more facultative anaerobic bacteria such as Proteobacteria, Firmicutes, and Bacteroidetes to obligate anaerobic organisms such as Clostridium, and finally spore-forming microorganisms such as Clostridium and Bacillus (Figure 1; ; , ; ). According to the “post-mortem Clostridium effect,” Clostridium species can be considered important drivers of this microbial shift due to their lipolytic enzymes (), proteolytic functions, and rapid generation time (). However, whether the findings from studies focusing on internal organs can be extended to mucocutaneous niches entails a different narrative.
Surprisingly, to date, no post-mortem microbiome studies have examined the vaginal microbiome succession after death (Table 1). This can be explained by the limited population of decomposing human bodies (mostly white males; >65 years old) studied at anthropological research facilities (). Nevertheless, found that reproductive organs (i.e., uterus and prostate) were the last internal organs to decay. Particularly, for the nulligravid uterus (i.e., never been pregnant), the post-mortem Clostridium effect was not observed in contrast to the prostate and other internal organs. Of note, during life, the uterine microbiome is distinct from the vagina with a significantly lower microbial biomass and colonization by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria (), and it is not clear to what degree bacterial transfer from the vagina to the uterus occurs after death. This highlights the underexplored potential of the female reproductive tract in post-mortem research.
TABLE 1
| Niches | Study aim | Sequencing | Population and sample size | Main outcome | Main pitfall | References |
| Brain, heart, liver, spleen, prostate, and uterus | Estimating minimum PMI and cause of death | 16S rRNA gene amplicon sequencing | 158 samples | Reproductive organs (uterus and prostate) were the last internal organs to decay during human decomposition | Larger population size is needed to further account for variation due to (a)biotic factors | |
| 40 human bodies (14 female, 26 male) | ||||||
| 6 body sites | ||||||
| Skin: nose and ear | Estimating minimum PMI | 16S rRNA gene amplicon sequencing | 144 samples | Machine learning model predicted the PMI with an average error of 2 days | Model was based on only four human bodies that were sampled longitudinally | |
| 21 human bodies | ||||||
| 2 body sites | ||||||
| Skin: left hip, right hip, left bicep, right bicep, left upper hip, right upper hip, left knee, groin, head | Understanding microbially mediated processes during decomposition on different soil substrates | 16S rRNA gene amplicon sequencing 18S rRNA gene amplicon sequencing ITS amplicon sequencing | 2 human bodies during winter | Soil type was not a dominant factor driving community development in the process of decomposition | Limited population size with no information on sex | |
| 3 skin sites | ||||||
| 143 days | ||||||
| 2 human bodies during spring | ||||||
| 8 skin sites | ||||||
| 82 days | ||||||
| Eyes, ears, mouth, nose, rectum, thigh skin | Estimating minimum PMI for buried bodies | 16S rRNA gene amplicon sequencing | 2 male bodies | Multidisciplinary methodology identified temporal changes in morphology, skeletal muscle protein decomposition, entomology, and microbiome for buried bodies | Model was based on only two human bodies of which multiple samples were taken | |
| 10 timepoints | ||||||
| Skin: right hand palm | Linking objects at the death scene to deceased individuals | 16S rRNA gene amplicon sequencing | 11 male bodies | Objects could be traced to deceased individual 75% of the time | Ante-mortem population was not always a demographic representation of the deceased study population | |
| 5 female bodies | ||||||
| 30 living individuals | ||||||
| 79 skin samples | ||||||
| 98 object samples | ||||||
| Eyes, nose,ears, mouth, umbilicus rectum | 1. Predicting the ante-mortem health condition of the deceased 2. Comparing three machine learning methods to predict PMI, location of death, and manner of death 3. Predicting cause and manner of death | 16S rRNA gene amplicon sequencing | 47 male bodies 141 female bodies 6 body sites 1 timepoint | 1. Microbial biodiversity from the mouth could predict ante-mortem host health condition (e.g., heart disease) 2. Analysis of post-mortem microbiota from more than thee anatomic areas had limited additional value 3. Beta-dispersion, and case demographic data reflected forensic death determination | Only one timepoint (majority of cases with estimated PMI of <72 h) which does not account for variability within a body | 1. 2. Zhang et al., 2019 3. |
| Mouth: palate, tongue, inner cheek mucosa and tooth surfaces | Estimating minimum PMI | 16S rRNA gene amplicon sequencing | 1 male body | Post-mortem microbial succession in the oral cavity changed in a temporal way according to oxygen availability | Limited population size with large variability | |
| 2 female bodies | ||||||
| 8 timepoints | ||||||
| 5 body sites | ||||||
| External auditory canal, eyes, nares, mouth, umbilicus, and rectum | Studying the impact of coexisting conditions such as frozen affect the human microbiome at the time of discovery | 16S rRNA gene amplicon sequencing | 1 male body 1 female body 3 timepoints | The microbial diversity increased throughout the thawing process | Association with time since death or cause of death | |
| Blood, brain, buccal cavity, heart, liver, and spleen | Estimating minimum PMI | 16S rRNA gene amplicon sequencing | 66 samples | Microbial communities demonstrated time-, organ-, and sex-dependent changes | Niche sampling was not equal for all deceased individuals | |
| 27 human bodies (12 female, 15 male) | ||||||
| 6 body sites | ||||||
| Mouth, external left/right cheeks external left/right bicep region, torso, and rectum | Studying outdoor decomposition under natural conditions | 16S rRNA gene amplicon sequencing and 454 pyro- sequencing | 1 male body | Shifts in community structure were recorded and associated with major decomposition and related events | Limited population size with large variability | |
| 1 female body | ||||||
| 10 timepoints | ||||||
List of human post-mortem microbiome studies which include female reproductive tract, skin and/or oral cavity samples in the last 5 years.
The bold terms refer to the most relevant niches.
While current research has focused on the potential of the skin microbiome as trace evidence (Tozzo et al., 2020), to the best of our knowledge, only have investigated whether actual objects from real death scenes (e.g., smoking pipes, medical devices, and phones) could be linked to the hand palm of the deceased through microbiome identification. The skin microbiome on the palm of the deceased remained stable up to 60 h after death, opening a window for individual microbiome identification even after death. It is noteworthy that this persistence of the skin microbiome into the early post-mortem period opens the possibility of also applying the post-mortem skin microbiome in PMI estimation. This is especially advantageous in cases where an autopsy is not requested, and a non-invasive microbiome sampling approach is best, because the most useful body sites for PMI estimation are external sites (e.g., skin).
Another body site easily accessible for microbiome and other sampling is the oral cavity. While its application for PMI estimation is yet to be studied in large populations, an increase of Firmicutes and Actinobacteria as the PMI increased was demonstrated (; ). Interestingly, mouth samples pre-bloating resembled the oral microbiome during life, whereas the mouth samples post-bloating contained gut bacteria such as Tenericutes that possibly migrated from the large intestine (). Overall, studies that include more body sites, like and others discussed in Table 1, could improve estimations.
The rate and pattern of decomposition are a mosaic system associated with biotic factors (e.g., individuality of the body, intrinsic and extrinsic bacteria, other microbes, and arthropods) and abiotic factors (e.g., weather, climate, humidity, and edaphic conditions) (; ; ; Figure 1). It is yet to be elucidated how the contact of skin and natural body openings (mouth and vagina) with the outside environment (clothing, soil, aquatic ecosystems, etc.) can differentially influence the post-mortem body site-specific microbiome. For the latter, the application of epinecrotic communities such as aquatic microbes on the post-mortem submersion interval estimation could be highly relevant in aquatic death investigations (; ; ). While the exact effect sizes are rarely reported (), abiotic factors, such as insects and soils beneath a decomposing body (; ; ; ), seasonal variation, and distinct climates (), but also exposure and clothing (), are some of the driving determinants of the microbial succession after death. Specifically, because of lack of thermoregulation, ambient temperatures () greatly affect the shift in nutrient availability and can thereby affect microbial community dynamics. Thus, while most studies have been performed in the United States (; ; ) with a few in Australia, Japan, and the United Kingdom (), post-mortem microbiome research in a wider range of climates should be encouraged.
Potential Hurdles and Considerations
Juries in the court of law have come to rely on physical evidence to corroborate a testimony (Shelton et al., 2007). However, before microbiome research can be reliably introduced into investigative and legislative casework, it has to be peer-reviewed, standardized, and accepted by the scientific community ().
Microbiome sequencing methods can be divided into those targeting specific parts of microbial DNA, such as the widely used 16S rRNA gene amplicon sequencing, or untargeted approaches, such as metagenomic shotgun sequencing. In-depth shotgun metagenomics is relatively new and currently more expensive than 16s rRNA gene amplicon sequencing, but it offers the advantage of sequencing the whole genetic content (microbial and human) of a sample with a higher taxonomic and functional resolution (; Schmedes et al., 2017; ; Walker and Datta, 2019). However, currently, amplicon sequencing is most widely implemented and thus relies on larger available datasets with metadata on microbiome-modulating factors necessary for increasing method accuracy of machine learning–based tools (; ; Zhang et al., 2019), allowing for larger meta-analyses (; Wang et al., 2018). Although both methods have specific limitations regarding taxonomic resolution, limit of specificity, and artificial bias are important when analyzing different types of samples. An integrative approach using both techniques could be implemented to rapidly advance the field, although this requires higher experimental costs (; ).
Importantly, results of microbiome studies vary due to differences in sampling, storage, processing, and data analysis (e.g., machine learning classification models) (; ). Thus, while the field expands, forensically oriented studies should contain standardized protocols with validated techniques. These methods should ensure reproducibility, sensitivity, and quantitative accuracy while defining and delineating the limitations (e.g., expected error rates, limit of detection, and limit of specificity) (; ). Minimizing the distinct impact of these variables on microbial profiling to reduce bias and skewing of the detected microbial composition is crucial in forensic evidence.
Future Perspectives and Concluding Remarks
Microbial forensics holds much potential; however, to integrate highly dimensional microbial data into routine investigative casework, several aspects need to be clarified. A key question is to what extent and for how long various individual factors shaping the vaginal, skin, and oral/salivary microbiome during life also play a role after death. These body sites are often inhabited by Gram-positive bacteria that are potentially more resistant to environmental and temporal degradation compared to Gram-negative bacteria and human DNA. In addition, vaginal, skin, and oral/saliva samples are routinely collected as critical components of sexual assault and femicide cases. Importantly, many sensitive individual characteristics can be associated with microbiome composition; however, the magnitude of these effects requires comprehensive investigation. A better understanding of the complex human body ecosystem during life and after death is necessary with the establishment of anthropological research facilities over different continents studying diverse populations and body sites. Hereby, we can facilitate discoveries especially related to female health and safety by comprehending how the post-mortem disturbance in the body homeostasis and its microbial communities make it more susceptible to the influences of the surrounding environment. While studies and regulations are complex specifically for the forensic field, the current and potential future possibilities of microbial forensics in phenotyping, identifying individuals, minimum PMI estimation, and the source of origin of a sample are highly important to consider and develop.
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Statements
Author contributions
SA, IS, and SL conceived and designed the manuscript. SA wrote the manuscript. IS made the figure. SA, IS, RD, EJ, and SL critically reviewed the manuscript and contributed with special attention towards their specific expertise. All authors contributed to the article and approved the submitted version.
Acknowledgments
We want to thank the entire research group ENdEMIC of the University of Antwerp, and the Forensic DNA Laboratories of the Antwerp University Hospital and University Hospitals of Leuven. We also want to thank the following funding agencies. SA was supported by the University Research Fund (BOF-DOCPRO 37054) of the University of Antwerp. IS was supported by the IOF-POC University of Antwerp funding ReLACT (FFI190115). SL was supported by the European Research Council (ERC) grant (Lacto-be 852600).
Conflict of interest
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.
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Summary
Keywords
post-mortem microbiome, trace evidence, microbial forensics, sexual assault, femicide, next-generation sequencing, thanatomicrobiome, epinecrotic communities
Citation
Ahannach S, Spacova I, Decorte R, Jehaes E and Lebeer S (2021) At the Interface of Life and Death: Post-mortem and Other Applications of Vaginal, Skin, and Salivary Microbiome Analysis in Forensics. Front. Microbiol. 12:694447. doi: 10.3389/fmicb.2021.694447
Received
13 April 2021
Accepted
14 June 2021
Published
28 July 2021
Volume
12 - 2021
Edited by
M. Eric Benbow, Michigan State University, United States
Reviewed by
Zachary M. Burcham, Colorado State University, United States; Baneshwar Singh, Virginia Commonwealth University, United States
Updates
Copyright
© 2021 Ahannach, Spacova, Decorte, Jehaes and Lebeer.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Sarah Lebeer, sarah.lebeer@uantwerpen.be
This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology
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