Microbial Witness: Unraveling Mysteries with Forensic Microbiomes

Abstract

Recent breakthroughs in forensic sciences, bioinformatics and next-generation sequencing technologies have broadened the application of microbiome analysis as an upcoming forensic tool. Studying the variation of the microbial flora and their profile, as well as the interactions among microorganisms, hosts, and the environment, are recent topics in microbiome research worldwide. Such novel microbiome applications have created a wide range of additional opportunities for the advancement of the forensic science. There are many modern forensic uses for the microbiome, such as postmortem identification, geolocation inference, and post-mortem interval prediction.

1. Introduction

In the context of forensics, the term “microbiome” refers to the collective community of microorganisms, including bacteria, fungi, viruses, and other microbes, that inhabit a specific environment or surface of interest, such as a crime scene, a victim’s body, or evidence recovered from a crime scene [1]. These microorganisms leave behind unique traces and patterns, which forensic scientists can analyze and utilize to gain valuable insights and aid investigations. More Simply, The microbiome consists of all microorganisms and their DNA that inhabit a particular environment. Soils and oceans have distinct microbiomes, as do humans, animals, and vegetation [2].

Microbiomes in forensics can be found in various locations, including soil, skin, hair, and even on objects associated with a crime. By studying the composition and diversity of these microbial communities, forensic experts can potentially determine vital data that might help them in their daily work. These contributions include the time of death, postmortem interval, geographical origin, and other crucial information about a crime or incident [3]. Using microbiomes in forensics is an emerging field, offering a novel and promising approach to complement traditional forensic techniques and contribute to more accurate and comprehensive investigations [4].

Every crime scene contains microbes used as tangible evidence for over a century. Low-cost, high-throughput technologies enable the rapid accumulation of molecular data and the application of sophisticated machine-learning algorithms and artificial intelligence to develop criminal justice-relevant, generalizable predictive models. Integrating microbiome and metabolomic data has the potential to advance microbial forensics significantly [1, 5].

2. The microbiome analysis pathway

The microbiome analysis pathway involves several steps, from sample collection to data analysis. Figure 1 is a basic outline of these critical stages. It’s important to note that this is a simplified schema. However, the process can be more complex and involve additional steps depending on the specific case and available resources. Additionally, specialized training and expertise in microbiome analysis and forensic science are required to accurately perform and interpret the results.

Figure 1.

A simplified schema summarizing the microbiome analysis pathway during forensic investigations.

3. Microbial forensics

Microbial Forensics is the term applied to the science of using microbiological techniques to analyze evidence for forensic attribution in situations spanning from bioterrorism to fraud, pathogen outbreaks, transmission, or the unintentional release of a biological agent or toxin. Biological and non-biological evidence are targeted for detection and characterization in microbial forensic investigations [6, 7].

Biological agents include microorganisms, protozoa, fungi, and toxins. Non-biological evidence, including additives, growth media, delivery devices, intelligence, etc. Biological and non-biological evidence can generate investigative leads and infer manufacturing and distribution processes [2, 8].

Forensic microbiomes in criminal investigations are expanding beyond biocrime, bioterrorism, and epidemiology. They are currently used to elucidate causes of death [such as drownings, toxicology, hospital-acquired infections, unanticipated child death, and shaken baby syndrome] and to aid in identifying mortals using microbiomes from the skin, hair, and body fluids. In addition, soil microbiomes assist in geolocation, while thanato-microbiome and epi-necrotic microbial communities are used to predict posthumous ages [9, 10]. Microbiomes are currently considered modern, reliable forensic investigation tools due to their current and prospective applications in various forensic investigations [3, 5].

The topic of microbiome forensics has been propelled by significant advancements in sequencing technology and computational pipelines, enabling the in-depth investigation of microbial communities with a high degree of species diversity. This methodology diverges from microbial forensics, which predominantly uses procedures tailored to detect and classify specific taxa of interest [11, 12].

4. The potential for microbial DNA as a source of trace evidence

Microbial forensics is a field that primarily concerns itself with the identification of certain strains of microorganisms linked to acts of terrorism, disease outbreaks, and instances of contamination. This discipline has extensively examined and investigated several strains for decades, employing a detailed analysis of genetic variations within individual genomes [13, 14, 15]. In contrast, the postmortem alterations and trace evidence signatures associated with microorganisms exhibit distinct variations in microbial communities, which can be most effectively analyzed using microbiome methodologies [16]. Numerous study groups have provided evidence of the potential of using microbiome data to estimate the postmortem interval (PMI), identify concealed burial sites, and establish connections between individuals and things or environments based on their skin microbiota [17, 18, 19]. The characteristics of microbial communities by employing advanced sequencing techniques were used to analyze gene markers that provide information about the evolutionary relationships or taxonomic classification (16S rRNA, 18S rRNA, and ITS). Additionally, statistical and computational methods, including machine learning, were utilized to analyze the data [1, 20].

5. Examples of microbiome forensic applications

Forensic microbiomes offer a growing capacity for microbial community identification and characterization, opening the door for microbial evidence in cases involving human identification, geolocation, and postmortem interval estimation, among other applications, as seen in Figure 2.

Figure 2.

Microbiome applications in forensic sciences.

5.1 Postmortem interval estimation

Postmortem interval (PMI) estimation has always been an essential tool in forensic science. PMI has traditionally been estimated using physical, histomorphological, and biochemical techniques. However, preserving the forensic samples used in PMI estimation and surpassing the time limit for endogenous substance degradation largely compromise these traditional methods [21, 22, 23]. The organism undergoes irreversible physical and chemical changes after death. As time progresses, the accuracy of the current methodologies for estimating PMI diminishes [24]. For decades, forensic entomology has been used to provide valuable estimates of time elapsed since death, but only if the death occurred between days and weeks ago. After this time limit, accuracy declines dramatically [25]. Here come the benefits of Metagenomics offering an alternative solution to this issue. Cadaver decomposes progressively after death due to the action of microorganisms. In the interim, tissue degradation products also progressively accumulate. As the duration of death increases, the quantity of spoilage microorganisms and products varies according to specific patterns. Therefore, the law of microbial community succession can be utilized to estimate PMI. Recently, the importance of metagenomic analysis in PMI estimation has grown [3, 8].

Different decay stages indicate the elapsed time since death, which can be calculated using changes in the variability and quantity of the microbiome in human corpses after death. However, it is essential to note that the bacterial succession that occurs at different stages of decomposition is influenced by physiological changes, fresh, bloated, active, and advanced decay, dry remains, and gender type. Each has diverse bacterial populations, which suggests the potential of microbiomes in criminal investigations to estimate the postmortem interval. In the future, it may be possible to investigate different microbiome communities in terms of decomposition to determine the actual time since death in addition to gender-specific microbiomes [7, 26].

Similar and repeatable microbiomes influence mammalian decomposition in various hosts and environments. This ecological hypothesis supports the PMI estimation method based on the microbiome. Most experiments have been conducted on animal models by obtaining postmortem samples from the abdomen, skin, scalp, and soil of experimental animals’ cadaver decomposition systems. These studies yielded PMI estimates with a small mean absolute error. Scalp data yielded the lowest level of error [27].

The lack of a predictive model based on many human cadaver samples primarily restricts the method’s application in forensic investigations. However, the potential for developing cadaveric microbiomes as a “clock” for estimating human PMI is immense [5].

5.2 Identification or individualization of unknown bodies

Individualization through skin microbiomes as a distinct and unique trait is a dream coming to reality through microbiome studies. A person’s microbiome can be stable over time, making microbiome characterization potentially pertinent for forensic human identification. The human body’s largest organ, the skin, is a complex living ecosystem supporting diverse microbial communities [14]. This helps establish the connection between human contact and evidence and that certain minor species are unique to particular individuals and have the capacity for personal recognition. Various substrates may facilitate the transmission of skin microbiome through direct or indirect skin-to-skin contact. The influence of individual microbial communities in public and private spaces revealed that microbiome variation is more significant between individuals than within the same individual [3].

5.3 Microbiomes in body fluid characterization

In contrast to body fluids like blood and semen, which are generally present in large quantities, traces of other fluids, such as vaginal fluid, urine, and sweat, have an essential role in DNA evidence. Identification is very obvious in forensic investigation cases of mixed body fluid, and it becomes a challenge to identify them separately [6, 9]. Microbial markers have been proposed as an alternative method of distinguishing between mixed body fluids. Each type of body fluid has a unique microbial makeup that can be used as bio-indicators, with the microbial composition inferring the kind of body fluid present. Different body fluids carry different types of bacteria that can be identified in this way. For instance, vaginal discharge often contains Lactobacillus crispatus, Lactobacillus jensenii, and Atopobium vaginae. Saliva often contains Streptococcus salivarius and Streptococcus mutans [28].

Particularly in the cases of an alleged sexual assault, microbial communities’ identification will undoubtedly be beneficial, whereby the spatial placement of such biological samples can fix sexual relationships and support the testimony of victims or suspects [11].

5.4 Stain deposition time estimation

The time estimation since the deposition of a stain at a crime scene can be a valuable tool for law enforcement to evaluate the alibis of identified suspects and witnesses and generate investigative leads to determine the appropriate perpetrator [14]. Until recently, the forensic application of studying microbial composition in a microbiome setting has not been thoroughly explored. However, new research has demonstrated that DNA and RNA analysis can identify time-dependent changes in the microbial composition of human biological traces [12, 17]. Estimating the time elapsed since an individual’s salivary stain deposition, for example, can be primarily achieved using DNA profiling of commensal bacteria. This method exhibits an average error rate of 5 days when applied within the initial 30-day period following the deposition of the stain. Nevertheless, additional investigation is required to gain a more comprehensive understanding and provide a more accurate representation of the microbial alterations pertaining to this application [27].

5.5 Geolocation during crime scene investigations

The human microbiome can be influenced by various circumstances, some of which may serve as a means to deduce the geographical origin of a host through the examination of microbiome samples obtained from human evidence found at crime scenes. The criteria encompassed in this analysis comprise geographical latitude, industrialization in the country of residency, and cultural and societal components [25].

Microbial community composition of things belonging to individuals residing within the same geographical places has more similarity to those of the individuals living in different areas. Human microbiome analysis has the potential to determine the city of origin for human biological samples accurately. Identifying the geographic sources of unidentified cadavers was facilitated by the appearance of Helicobacter pylori strains in the context of forensic casework. In the event of a satisfactory outcome, integrating microbiome-based geolocation analysis and bio-geographic ancestry [BGA] research utilizing human ancestry informative DNA markers will yield supplementary geographic data about the individual being examined [18].

5.6 Other forensic science applications of microbiomes

Microbiome analysis in forensic medicine encompasses other various applications, including ethnicity and possible living conditions, determining the cause of death, such as drowning, cardiovascular-related fatalities, and deaths associated with drug usage. In addition, the genotyping of microorganisms related to sexually transmitted illnesses can serve as a valuable tool for identifying the primary source of infection, a factor of particular significance within the legal and law enforcement domains [29]. In some instances, establishing a connection between the infectious microbe present in the suspect, the source of infection, and the victim, the infected individual, has been found beneficial. This approach has been valuable in identifying the perpetrator of child abuse [15].

Metagenomic research of microbial communities can yield insights about an individual’s racial and ethnic background. For example, individuals residing in economically disadvantaged rural regions who have not been exposed to commercial antibiotics exhibit more bacterial diversity and enhanced functionality in their skin than those utilizing alternative therapeutic antibiotics [7]. Nonetheless, variations in the composition of the human microbiome among different ethnic groups are influenced to some extent by factors such as dietary patterns, lifestyle choices, and geographical surroundings [12].

6. The challenges and ethical perspectives surrounding forensic microbiome analysis

The utilization of metagenomics in the field of forensics is now in its nascent stages. The utilization of microbiomes as a shred of exclusive evidence for individual identity, geolocation inference, and postmortem interval [PMI] estimates has not yet received official approval. There is a lack of established operational principles and requirements for microbiological evidence extraction, packaging, transportation, and preservation. Furthermore, there is a pressing need to enhance the trustworthiness of microbiome tools utilized in forensics. Further validation is necessary to establish the specificity and stability of individual microorganisms in comparison to human DNA markers [30].

Further investigation is needed to enhance our understanding of the microbiome’s trustworthiness in the context of forensic applications and to establish error rates that can be deemed trustworthy. To effectively tackle the issue at hand, it is imperative to employ a sample size of considerable magnitude and utilize quantitative machine learning methodologies [18]. Various well-established methods, such as K-nearest neighbor models, random forest models, and neural networks, have been extensively used in forensic applications involving the microbiome, namely in the domains of classification and regression [4]. Machine learning techniques have demonstrated a clear benefit in effectively handling multidimensional datasets, particularly in the context of microbiome data. However, it is necessary to do the quantitative computation of pertinent forensic factors. There exist notable disparities between the existing evaluation techniques employed in elucidating microbiome evidence and the conventional likelihood calculation utilized for human DNA [21]. Additional study is required for the evaluation criteria to gain acceptance within the forensic scientific community [8].

However, it is imperative to have a sample size that is large enough for machine learning techniques to achieve satisfactory performance. Hence, it is essential to create microbiome databases to employ their proper use in forensic science effectively. Implementing forensic DNA databases has facilitated law enforcement agencies in identifying or eliminating individuals connected to criminal activities [24]. Furthermore, this technological advancement enabled the identification of serial criminals through the correlation of several cases, enhancing forensic evidence’s evidentiary significance [19]. The application of microbiomes as a forensic tool has been hindered by the fragmented condition of publically available databases despite the sequential creation of several microbiome databases, such as the Human Microbiome Project [HMP] and Earth Microbiome Project [EMP]. Using 16S rRNA gene sequencing data obtained from publically accessible sources can facilitate the identification and characterization of the Forensic Microbiome Database [FMD], enabling the derivation of insights about its original location of discovery. There is a growing demand for more databases to cater to diverse forensic applications [31]. Ultimately, the establishment of awareness is a crucial component in rendering forensic science admissible. The cost of training and acquiring equipment for sequencing microbiomes is substantial. The reduction in prices will be facilitated by advancements in sequencing technology and increased computational capabilities. The aforementioned concerns hold significant importance in forensic microbiomes and are expected to be resolved with the advancement of related scientific research. In addition to its previous uses, metagenomics has the potential to yield significant improvements in forensic pathology, toxicology, and drug addiction testing. In summary, integrating metagenomics into forensics can offer novel insights and resolutions for forensic identification [32].