A Review of Forensic Microbes in Crime Investigations

Volume 9, Issue 6, June – 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24JUN963
A Review of Forensic Microbes in

Crime Investigations
Vataparthi Pravallika1; Naga Jogayya.K2*; Satheesh Ampolu3 ; Sheerin Bashar4
1.
Assistant Professor, Department of Forensic Science, Centurion University of Technology and Management, Vizianagaram,
Andhra Pradesh.
2.
Associate Professor, School of Forensic Science, Centurion University of Technology and Management, Bhubaneswar.
3.
Associate Professor, Department of Chemistry, Centurion University of Technology and Management, Vizianagaram, Andhra
Pradesh.
4.
Research Scholar, School of Forensic Science, Centurion University of Technology and Management, Bhubaneswar.
* Corresponding Author:
Dr. Naga Jogayya.K
Associate Professor and HoD
School of Forensic Science
Centurion University of Technology and Management, Andhra Pradesh.
Abstract:- Advances in genetic sequencing technology, I. INTRODUCTION

enhanced microbiological sample methods, and rapidly
developing approaches in bioinformatics have all Over the past century and a half, microbiology's
contributed to the meteoric emergence of microbiomics contribution to forensic science has been minimal at best (1).
and metagenomics. Humans harbour vast microbial Sequencing of virally amplified DNA was implemented to
communities, both internally and outside, that are lend credence to allegations that a dentist at Florida in 1990,
constantly exchanging information with and shaping their United States had infected a number of his patients with the
environs. These interactions may be crucial to forensics Human Immunodeficiency Virus (2). These allegations were
because information about them can be gleaned from made in support of a lawsuit that was filed against the dentist.
human and environmental microbial profiles. Over the PCR-based bacterial genotyping was projected to be a helpful
reports of hundred studies, as metagenome markers in tool in future forensics due to the rapid growth of technology;
forensic science is an emerging field of forensic science, for instance, it was speculated during the time, forensic
microbiomes has a greater chance of becoming a specific science will be a emerge are for application of PCR-based
tool kit for forensic science to provide answers for genotyping (3). Substrate items might be linked to specific
personal identification, cause and manner of death, sites after the development of fungal and pollen spore
interpretation of PMI and geolocation of a suspect or examinations in the mid-1990s (4,5). However, the "scientific
accused. Although the application of microbiomics to discipline dedicated to analysing evidence from a
forensics has been extensively studied, most of its bioterrorism act, biocrime, or inadvertent
potential remains untapped because of insufficient sample microorganism/toxin release for attribution purposes" of
numbers, inaccurate models, and unrepresentative microbial forensics did not emerge until the early 2000s in
experimental conditions. Law enforcement agencies are reaction to the recently emerging bioterrorism threat (6,7).
unlikely to benefit from the limited microbiomics data
that is currently available. However, efforts are being Historically, a lack of accessible and affordable
made to find solutions to these problems, and evidence sequencing technology has hampered microbiological
gleaned from the microbiome may one day aid in forensic forensics (8,9) limiting many forensic applications to single-
investigations. Until more is learned about microbiomics, taxonomic investigations. This strategy has shifted
it is unlikely that this information will be useful to law significantly over the past decade because to developments in
enforcement. However, efforts are being made to find genomic sequencing technology and new approaches for
solutions to these problems, and it is possible that analysing complicated community datasets (and sometimes
evidence gleaned from the microbiome can one day aid in low biomass samples). The advancement of the microbial
forensic investigations. forensic toolbox (10,11) has been aided by the study of the
microbiome (12, 13) and metagenomics.
Keywords:- Microbiomics, Forensic Science, Law
enforcement, Microbiology.
IJISRT24JUN963 www.ijisrt.com 3088

The homicide conviction rate in England and Wales demonstrated that the larger distance between samples, larger
[United Kingdom] was 79% as of March 2019 (14), which is the variation in microbial community composition was,
just slightly higher than the homicide conviction rate in the indicating that the key determinants in shaping microbial
United States (70%) as of March 2019 (15). Conviction of communities are soil type and geographic location. Indeed, it
criminals is often difficult due to insufficient evidences (16, has been shown that patches can be discriminated using the
17, 18, 19), and false convictions are common worldwide. So soil microbiome (36, 37) successfully attributed 95.4% of soil
far, 375 people in the United States have been exonerated by bacterial profiles were able to be traced back to their
DNA testing, including 21 who spent time on death row, respective geographic origins by utilizing analysis of
according to the Innocence Project, falsely convicted similarity, non-metric multidimensional, abundance charts,
individuals were exonerated by national legal and public and k-nearest neighbor. Yet, because this was only credibility
policy organization (20). As a result, there is a considerable study-the total number of participants in the sample was
push from public, Judicial systems, and lawmakers to rather small (n = 19). Additional research is needed with
incorporate molecular techniques into the forensic arsenal. larger sample sizes and additional replications in order to get
There are many types of microorganisms in and around the a complete understanding of the possibilities of this strategy.
human body, in the environment, and on objects linked to a Analyzing the microbial communities found in a variety of
crime (21, 22, 23). It’s expected that the number of microbial soil samples [including those taken at crime scene, an pretext
cells to be more than or equal to the overall number of human site and any intermediary locations] may be beneficial to a
somatic cells (22,24). Conventional investigation techniques forensics inquiry. Using Ribosomal Intergenic Spacer
is been suggested which include the potential use of Analysis and 16S rRNA gene sequencing, researchers were
forensically relevant microbial profiles. (25, 26, 27, 28) able to accurately classify samples from a variety of soil
Computational tools, processing tools, and improved substrates (38). There is new data to suggest that at fine
methodologies are now being developed in the field of scales, 18S rRNA gene sequencing can be even more
bioinformatics. It’s unclear how useful microbiomic selective than conventional Mid-Infrared spectroscopy for
techniques could be in forensics at this time due to the field's eukaryotic organisms (39). In addition, the soil samples
early stage of development (29,30) and its lack of funding in retrieved from the shoes can help to determine the site of
the past (31). In this article, we discuss the use of origin by comparing the denaturing gradient gel
microbiomics in forensics thus far and its possible future electrophoresis-created soil bacterial 16S rDNA profiles (40).
uses.
Despite the promising future of this sector, there are still
Over thorough research, the topics of interest include obstacles to be overcome. For instance, the results of
personal identification, determination of biological sex, microbiomic investigations should be interpreted with
analysis of trace evidence, investigation of the method and caution due to a number of potential limiting factors
cause of death, PMI and exploration of alternative described by Pasternak (41). Microbiomes display a high
applications such as localization using animal microbiomes level of biological, physical and chemical variability in space
are just some of the areas that could benefit from geolocation. and time, and soil samples are heterogenous and immensely
complex, presenting a huge challenge to employing these in
II. CONCEPTS FROM FORENSIC BIOLOGY a forensic setting. Actinobacterial fingerprints differ greatly
between summer and winter at the same locations, which
 Geolocation suggests that there may be concerns that are time-related. This
In recent years, researchers have worked tirelessly to is proved by the fact that there is a correlation between time
better define the microbiome of different ecosystems, with a and actinobacterial fingerprints (42). According to Keet (43),
focus on urban areas and public transport. These studies have the fact that the composition of the soil microbiome is
shown that there may be different types of communities in susceptible to brought change on by abiotic soil
different parts of a city, as well as "molecular echoes" of circumstances and the patterns of plant communities offers a
environmental events and even a forensic capacity for substantial obstacle to the dependability of the results.
geospatial microbiomic data (32, 33, 34). Two primary facets
of geolocalization will be discussed here. Gravesoil and soil collected near the human and animal
cadavers can be used for the examination of metagenomics in
 Analysing the Substrate forensic investigations. Studied seasonal changes in
The forensic microbiology community is beginning to microbial community composition in soils near swine
see the value of analyzing soil microbial profiles. carcasses (44). They showed that microbial communities
Microbiomes in the rhizosphere and the bulk soil vary greatly altered in predictable ways after death, but that the impact of
from one location to the next. Thus, with better techniques, decomposition on soil microbial communities varied greatly
samples of soil microbes could yield valuable bio-geographic according on the time of year. Forensic investigations may
information to identify source of the soil samples. As another benefit from the authors' proposed use of the ecological
possible use, this may be implemented to learn more about succession of microbial communities, although more study of
the history of a criminal case's evidence. the impact of seasonality on decomposition is needed. The
ordination graphs suggest a small sample size (n 10 per
Distance-decay effects were shown by (35) [25-1000 m] treatment), however this was not indicated in the paper. As a
local scale connections between soil microbial samples [N = result, the findings should be taken with a grain of salt.
5 sites, N = 2-4 soil types and 5 replications]. Findings

Variations in the thanatomicrobiome that occur daily in success rate in matching foot microbiome samples to homes
the soil were investigated as a potential tool for determining is low from an evidentiary standpoint, it does show the
PMI (45). In order to demonstrate the everyday successional promise of these technologies for pinpoint bio-localization.
changes that occur in the soil around the cadavers, one male Microbiota samples from 12 cities in 7 countries were whole-
and two female cadavers were used. Fast development of genome sequenced as part of 2018 CAMDA Meta SUB
firmicutes was experienced between the bloat stage to the Forensic Challenge, and analysed the results (51). There were
advanced decay stage of corpus. These were used by 30 total samples in the CAMDA dataset, and 3 of them were
scientists to predict the PMI in Tennessee's summer hidden. The authors used machine learning methods to
condition; they advocate for future research to validate their determine where the microbiome samples had been collected.
findings with a larger sample size and in a variety of climates. Despite the promising results of applying machine learning to
biogeography (up to 90% accuracy), more proof is needed
While these findings don't prove anything new, they do before these methods can be used in a legally binding setting.
support the conclusions drawn (46) after they assessed the Ryan (2019) utilised a random forest classifier constructed
microbial communities linked to Human cadavers buried in from 311 city microbiome samples in a similar investigation
graves (n = 18). Researchers left the remains to decay for (52). They were able to correctly categorize 83.3% of the
varying amounts of time (three days to three years), mystery samples using their method. Using a novel approach
discovering that the relative abundance of Firmicutes in that is based on deep neural network classifiers (53),
surface bodies increased over time (from tenths of a percent researchers in the United States were able to improve their
at the beginning of decomposition to forty percent at the end). ability to predict the geo-site samples of fungus taken from
dust (N = 1300). American researchers forecast geographical
The effects of human cadaver decomposition on the distribution of fungal samples extracted from dust (N = 1300)
composition of soil microorganisms were studied (47). Soil was enhanced. According to the authors, their algorithms can
samples were collected from each geographical buffer [N = produce "good point predictions" on a worldwide dataset
14 for the 0 m, N = 17 for both the 1 & 5 m] and they found comprising samples from 28 countries; in a study covering
predictable reaction of evidence to dead body decay that the entire United States, over 50% of the geographic
differed depending on the distance from the buffer. There information errors fell within a hundred kilometers, and over
were no statistically significant variations between the 90% of the samples were correctly classified based on their
compositions of the bacterial communities at 1 and 5 meters, origin.
but there were substantial differences between the
communities at 0 meters and 5 meters (measured as beta The combination of machine learning and microbiomics
diversity). is still in its early stages; for applying these methods in a
forensic context, more research is needed to increase the
Bacterial alpha diversity was also found to be much sample size and improve classification accuracy.
lower in the 0 m samples, which the researchers attribute to
the increased nutritional supply from the corpses. This The fact that microbiome compositions vary vertically
research adds to the expanding corpus of spatial as well as horizontally is a further relevant geographical
compositional expertise in forensic microbiome applications. consideration. Humans at high and low elevations have been
found to have distinctive skin microbiomes. Human (n = 99)
However, more study is needed to establish the and swine (n = 82) skin microbiome samples were collected
sensitivity and reproducibility of results (48) before soil in Tibet (54). Several bacterial taxa (including
microbiome analysis may be employed in forensics. Spatial Carnobacterium sp., Arthrobacter sp., and Paenibacillus sp.)
and Temporal dynamics of the microbiome can be learned by were found to be enriched in samples taken at higher
more microbiome surveys in order to create methods to avoid elevations. The alpha diversity of the people samples from
these mistakes in the future. skin who are living at higher elevations was similarly much
lower. This hints at the possibility of using microbial samples
 The Potential of Machine Learning Across Various for the analysis from the skin as a future method for
Spatial Dimensions determining geolocation based on altitudinal parameters;
There has been an explosion in the number of however, further methodological improvement will be
techniques that have been created to sample and predict required in this area, as well. It will also be important to think
environmental microbiome profiles over a wide range of about how different skin microbiomes may change at
spatial dimensions and orientations (for example, between different stages of life.
houses, cities, states, and elevations) in order to trace the
origins of people and things. In general, classification is emphasized above
prediction in all of the models. To allow simultaneous
In spite of the fact that this study was conducted with a estimation of geographic coordinates from sampling data
sample size [n = 3 for each office], the results of the analysis larger geographical and temporal sample sizes are needed, as
of office microbiome samples utilising sampling plates are more rigorous approaches.
indicate that there is potential for further improvement (49).
The inhabitants of households (n = 1625 from 18 participants
in 10 residences) as well as the built environment that they
lived in were investigated (50). While the authors' 82.9%

 Individual Authentication identification in cases the investigator is unable to extract

There is mounting evidence that people can be significant quantity of DNA from human. However, there is
distinguished from one another using only the microbes that not enough evidence to support the conclusions. The
are autochthonous, or naturally present in their surroundings. sensitivity and specificity of model's must be enhanced and a
This has the potential to significantly affect forensic science, strategy must be developed to deal with the possibility of
for instance in cases where the investigator is unable to contamination. For the results to be useful in forensics, one
collect enough human DNA. However, it is still unclear if the must also get a deeper comprehension of the temporal and
diversity of microbes present in the human body is high spatial dynamics of microbes.
enough or stable enough to identify individuals within vast
populations or to locate them through time.  The Role of Biology in Defining Gender
In cases where large amounts of human DNA cannot be
Franzosa attempted to answer some of these problems recovered, microbiomics may be able to help with personal
by testing specific microbial profiles, matching with 25-105 identification by allowing for the differentiation of biological
microbial initial profiles and sampling site visits were sex. There has been prior work characterizing the populations
repeated (55). According to scientists, profiles are let them of airborne bacteria in indoor settings (59). The "aerobiome"
distinguish individuals at the time of the initial sampling, and was studied (60), who collected samples of airborne bacteria
they were able to uniquely identify 30% of the individuals and fungi from 91 student dorm rooms across campus. Using
many months later. Eighty percent of participants (n = 120) machine learning methods, they were able to determine, with
were correctly located using gut microbiota samples. after a 79% accuracy, whose gender the room's inhabitants belonged
year, now. Although promising, especially on shorter time to based on the ratio of their microbiota. Strangely, the
scales, the results are still plagued by considerable variation. microbial quantity was greatest in settings where males
As a result, more development is required, for example in dominated. The authors postulated that this could be because
methodology and sample effort, before such technologies men utilise less barriers against the elements, both natural and
may be beneficial in a forensic context. artificial, and because they shed more biological particles.
Despite the limited sample size (n = 5), high- Microbial communities of the human
determination melting examination of 16S rRNA gene thanatomicrobiome after the death they colonise the organs
samples of oral swabs was used to show its possibility of [thanatos, Greek for "death"](61), has been shown to have
discriminating among individuals (56). sex-related biological variations (62).
Individuals’ accurate identification (n = 12) using skin Using the V1-2 and V4 sections of the 16S rRNA gene,
samples of swab from various body regions (n = 14) given by the authors analysed amplicon signatures in cardiac tissue
Schmedes et al. (2018). Using nearest-1 neighbor from 10 deceased individuals and found significant variations
classification on the bacterial genome based on nucleotide among males, N = 6 and females, N = 4. Streptococcus sp was
diversity, they were able to achieve 96% accuracy by detected only in cardiac tissues of male whereas
sampling palms and 97% accuracy by sampling shirts. In Pseudomonas species was far more common in females. A
another recent study, the same method was used to track down more refined version of this method might be used to
participants (n = 51). Examined microbiome samples taken establish the biological sex of a deceased person's body and
from the manubrium (the uppermost section of the sternum), their place of origin for various body parts.
the ball of the foot, and the palmar surface of the hand (57).
When using a maximum nearest neighbor distance for According to the investigation of their pubic hair
diversity the researchers reached perfect classification microbiomes, "readily differentiated " male [n = 3] and
accuracy, indicating promising future work if the results can female [n = 4]participants. They discovered that the female
be replicated in investigations with bigger sample sizes. participants had their own strains of Lactobacillus spp (63).
Minor taxa were proposed as a crucial component in They also mentioned that pubic hair may be beneficial
differentiating individuals (58). Microbiome samples (n = 66) in forensics since it is protected from the elements and
from 11 participants were analysed during a 2-year period, colonized by niche-specific bacteria. Sample size being small
and 85% of the participants could be correctly identified. in this study makes it critical to draw any conclusions.
They also applied the same analysis techniques to the 89 Although this study was tiny, it was validated by another that
individual skin microbiome samples that were made used microbiota from pubic hair to determine the sex of
accessible to the public, correctly identifying 78 percent of individuals [n = 9]with ratio error of 0.027 0.058 and 0.017,
them. 0.052 respectively (64). All of these investigations have small
sample sizes, therefore more extensive validation studies are
However, for forensic uses, this precision is probably required before any firm conclusions can be formed.
not adequate. The scientists hypothesized that individual
identification is feasible, but that the estimation accuracy Interestingly, discovered that the lack of the bacterium
drops off significantly for bigger cohorts as the prevalence of species Alloiococcus in microbiome samples of skin from
individuals with strikingly similar microbiome patterns both male and female [n = 45] could be effective in
increases. Microbiomics has the potential and technological differentiating female biological sex (65). Certain bacterial
viability to be used as a forensic tool for determining personal species were found to be associated with individual traits

(such as biological sex) in the study. Using leave-one-out potential requires larger sample sets and more precise
cross-validation analysis to find out the microbiota from machine learning. It is unclear whether particular strains of
fingermarks if present or absent which were left behind on bacteria and other microorganisms are unique to women or
surfaces, they obtained a comparatively low accuracy in sex whether differences are due to environmental factors (such as
prediction of 67%. Further investigation of this strategy's the use of cosmetic items on the hands).
Fig 1 Possible Sources of Forensically Relevant Microbiomes
III. OVERVIEW OF POSSIBLE SOURCES OF included a bacterial community that was more homogeneous
FORENSICALLY RELEVANT MICROBIOMES with its owner than with anybody else's. A total of 22%
among the detected taxa on fingertips of the participants were
 Scanty Proof also present on their phones, while only 17% were common
The study of microbiological profiles that are significant to all participants' phones. About five percent more OTUs
to forensics and left behind on items and surfaces is gaining shared between a index finger of the persons and their
popularity. Example: a number of research (66, 67, 68, 69) specific smart phones compared to all the other phones in the
have demonstrated the prevalence of germs on common study combined. And between the index finger and the phone,
personal items like cell phones. In addition, it has been 82% of OTUs were exchanged. While these findings are
proven that human-associated objects like shoes and mobile encouraging, more research is needed to ensure their validity.
phones support unique microbiomes (70, 71). Mobile phones
have been studied (72), who looked into their potential as It was discovered that postmortem skin microbiomes
"personal microbiome sensors." Seventeen people were were strongly correlated with specific belongings. Medical
chosen, and samples of the touch screen, index finger, and equipment, eyeglasses, bottles, and coxswaining with 100%
thumb were taken. They showed that a person's mobile phone accuracy, while other objects like computers, there was an

association between skin microbiota samples and cell phones A recent study discovered distinct biomarkers that were
and remote controls with an accuracy of over 67%, which connected with the mode of death. Hospital-related deaths
suggests that, with some additional work, samples of were more likely to involve Xanthomonadaceae, while
microbiome of skin might be linked consistently to things suicides were more likely to involve Actinomyces sp. In most
near the scene. Up to 60 hours after death, the skin's cases, model accuracy improved as sample sizes grew larger
microbiome has been shown to remain stable and mostly (80).
unchanged from premortem levels (73).
The scientists noted that the number of anatomical sites
The microbial composition of various physiological analyzed and the machine learning algorithms employed both
fluids (n = 22) was studied. Different body fluids were tested, contribute to the prediction accuracy. According to the
and source-specific microbiological signatures were authors, this research provides essential groundwork for
discovered. The phylum Proteobacteria, for instance, has future efforts to include the machine learning process to
been linked to skin and semen sources, while the phylum create databases for reference that enable forecasts of deaths
Firmicutes has been found to be more common among saliva caused by microbes.
and vaginal secretions (74). Dobay and his associates
demonstrate that the samples harbour specific body site Kaszubinski et al. for postmortem cases used the data of
microbial fingerprints even after exposure to indoor microbes [n = 188 five body sites per case] to predict beta-
environments for a period of 30 days (75). Hanssen et al. dispersion and test for the association between method of
showed excellent findings for the microbially mediated death and cause of death. The study's authors proved that
categorization of bodily fluids albeit having a limited sample beta-dispersion and demographic information may
size [n = 6]. They were successful in classifying 94 percent differentiate between causes of mortality (81). In example,
of saliva samples after performing recognition of the pattern they discovered that 79% of deaths caused by cardiovascular
with Principal Component Scores using a linear discriminant illness or drugs were properly categorized. They concluded
analysis model (76). that models based on binary logistic regression were the most
useful for boosting model performance.Manner and cause of
Recent research looked into the possibility of death assessment was confirmed at the time by 88% which is
allochthonous microbiota (skin microbiomes from different a significant increase above the 62% accuracy achieved by
people) being transferred between people and surfaces (77). multinomial logistic regression models.
They discovered that physical contact, such as when people
shake hands, reliably transfers skin microbes. Microbiota can This study demonstrates the potential of postmortem
also spread through indirect touch, as seen when one person microbiomes as indicators of cause of death. Researchers
rubs a substrate and then exchanges it with another. stress the importance of larger samples. To train models with
high success rates before they can be utilized in practical
The authors claimed that this type of study could be forensic scenarios, it is anticipated that enormous databases
helpful in providing further evidence of sexual assault and will need to be developed.
other crimes involving physical touch. In addition, they
recommended that researchers investigate the effects of Researchers have looked into the significance of
contact pressure, friction, and time. microbiological testing in defining the reason for death i.e.,
sickness or physiological disruption due to injury in the body
 Cause and Method of Mortality leading to death. According to the author's study of autopsy
A death's "manner" is determined by an investigator. data (n = 42), microbiological analysis was successful in
Natural, accidental, suicide, homicide, and unexplained are determining the cause of death in 42% of the instances (82).
the five most common causes of death classified by medical
science (78). Recently, microbiome samples from 265 Increased CRP levels, for example, were singled out as
decedents in the United States, Finland, and Italy (79). The a possible indicator of death due to microbiological causes in
causes of death among the examined bodies ranged from the study. Both sudden infant death syndrome (SIDS) and
accidental [N = 88], to natural [N = 106], to homicidal [N = astronauts returning from space have been linked to increased
23], and to suicidal [N = 45]. According to findings by them, CRP levels (83, 84, 85). In addition, newborn circumcision
several types of death were linked to the bacterial families has been linked to potentially fatal bacterial illnesses such as
belonging to Lactobacillus, Enterobacteriaceae, infection and sepsis (86, 87).
Sediminibacterium, and Rhizobiales.
An example of a forensic microbiome application that
Upon further research, these associations have the can be used to determine the reason for death is the analysis
potential to be transformed into predictive markers that of ‘death using drowning’ which is ranked consistently
contribute to the process of determining the cause of death. among chief causes of fatalities which are unusual around the
The authors emphasize that Rhizobiales bacteria and world (88, 89). The study of diatoms, which are single-celled
Sedimini bacterium may signify pollution of the environment algae and considered as the ‘gold standard’ for the era, despite
which has to be regulated. An additional validation approach the fact that their accuracy has been questioned (90, 91).
towards improving the reliability through controlled
experiments is required to establish the mode of death.

Several studies have used real-time polymerase chain IV. POSTMORTEM LAG TIME
reaction (PCR) as a primer test to distinguish bacterial species
related with aquatic habitats i.e., Aeromonas spp. (92, 93, 94,  The Subatomic Structure of the Thanatobiome
95). These assays have been designed, and they have been In many cases, it is crucial to a criminal inquiry to
used, to strengthen the diagnosis of deaths caused by establish the PMI (the amount of time that has passed after a
drowning. These investigations provided support for the person's death). Many studies (99, 100) have looked at the
finding of the cause of death on the basis of rather rates of thanatomicrobiome to better predict PMI. In the postmortem
microbiota with high detection, such as 84% [N = 32] , 75% period, these communities proliferate and eventually colonise
[N = 20] and [N = 43], despite fact that levels of accuracy and the host (101).
sizes of the samples may again be considerably increased. Its
suggested that bioluminescent microorganisms could serve as Microbial communities may go through significant
potential indicators for mortality caused by drowning at sea. changes in organs that help determine PMI, according to
An easy and straightforward assay that concentrates on the preliminary investigations. Preliminary research using model
16S rRNA gene was developed for the purpose of organisms suggests this is doable (102).
bioluminescent colonies detection such as Vibrio fischeri and
Vibrio harveyi. Upon recent examination the expression of In order to develop a "microbial clock" that could
pulmonary surfactant protein (SPA) and the microbiota to forecast PMI over a period of 48 days of decomposition,
establish a marker for the identification of drowning as the Metcalf et al. sequenced 16S rRNA gene for archaeal and
cause of death (96). They compared the microbiota and bacterial groups as well as 18S rRNA gene for micro
histological appearance of experimental rats treated with eukaryotic communities (103). Their model yielded accurate
freshwater vs saltwater water, both during and after death. PMI predictions (3 days; n = 223). Nonetheless, care is
The authors counted 5513 marine OTUs and 5480 freshwater warranted when applying the data to 'real-world' scenarios
OTUs that were distinct from each other. In addition, they given that study was done under conditions which are
discovered that SP-A expression was higher in the lungs of controlled utilizing mice models. The decay of pig corpses
drowning victims than in those of those who had been was also the subject of another investigation. With additional
submerged postmortem. These results show promising methodological for experimentation improvement, their
possibilities for future applications and may have significant model predicted the PMI within 2 to 3 hours of the time of
forensic relevance (e.g., identifying the environment type and death with 94.4% accuracy (104). Postmortem microbiomes
the death time). Other research, as noted by Marella et al., were shown to not to have any changes, reflecting
have looked into coliforms, faecal and streptococcal bacteria antemortem microbiomes of 24-48 hrs after death, according
presence to aid cause of death due to drowning. The femoral to a wide-scale investigation of body microbiome samples
artery, vein, and ventricles are all sampled for these conducted (n = 188). Certain bacterial taxonomic groups were
microorganisms (97). also discovered to be significant predictors of health status by
the researchers (105).
Drowning victims are more likely to have faecal
bacteria present than those with other causes of death (98). The species of Hemophilus and Fusobacterium was
For instance, in their study of 22 cases of freshwater found to be twofold as common in people who are healthy,
drowning, found that 100% had faecal streptococcal presence but Rothia shown to be 0.09 times more common in those
and 90% had coliforms. Thirty healthy volunteers served as with heart disease. Clinical investigations of all types of
controls, and they all tested negative for faecal deaths, from those that are slow and natural to those that are
microorganisms. sudden and violent, could benefit from this in the future. It's
worth noting that the approach of bioinformatics used to
Subsequent research investigated if these bacteria might analyze OTUs and to give practical predictions is now
be detected in the drowning medium after death. Scientists deemed to be antiquated, although having been adequate at
obtained tissue samples from people who drowned (n = 5 in the time. Additional taxonomic detail may be available
freshwater and n = 5 in marine) or were submerged (n = 3). through Amplicon Sequence Variants (ASV) (106).
All victims who drowned had coliforms and streptococci
present, while those who were submerged after death did not. In order to estimate PMI researchers developed a
These results point to the possibility of using faecal coliforms algorithm based on data collected from skin microbiome
and streptococci as indicators of drowning. However, the samples taken from decomposing human cadavers. low error
small sample sizes necessitate extreme caution when rates for skin samples, an accuracy for 2 days estimation of
interpreting the results and calling for massively expanded PMI [n = 144 from 21 cadavers], which was a significant
sample sizes in subsequent research. change over past attempts (for example, using entomological
inspection). By sequencing 16S rRNA amplicons, researchers
were able to discover that models created at the phylum
taxonomic class levels produced accurate and dependable
predictions of PMI. This evidence supported research and
showed its potential forensic applications (107, 108).

An additional study reported PMIs between 29.5 and because PMI is based on the microbial population that was
240 hours when utilising 454 pyrosequencing to analyse the present in the gut at the time of discovery. The reconstructed
postmortem microbiome of numerous important organs. microbiome’s can pass easily the Daubert standard, which
These included organs like brain, heart, liver, and spleen. was set by the United States Supreme Court (119) it has been
Postmortem microbial communities were shown to be recognized as scientific evidence towards the bearing
dominated by anaerobic and spore-forming Firmicute foundation. This includes widespread recognition among
bacteria, specifically Clostridium sp. Additional evidence for scientific community, acceptable rate of error, and a variety
the future application of microbiomics in PMI estimation was of other criteria and so on.
provided and demonstrated that species of the Clostridium
dominated at lengthy PMIs (109). However, microbiome comparison techniques may be
more useful for criminal investigations, but they are more
 Animal-Based Microbiome-Based Localization challenging to build to a standard that would meet the
Several studies have demonstrated that the microbiome Daubert threshold. In the same way the human DNA analysis
of animals from different taxonomic groupings and settings in forensics compares the trace evidence DNA profile (from
is distinct. Genetics, geography, and altitude all plays a role known samples and known locations) in a database, the
in shaping gut microbiomes of numerous animals. all have productive means to use such tools would be in "one-to-
distinct gut microbiomes (110, 111). Skin microbiomes of many" configuration, the frequency of a profile in the relevant
many species have been shown to be distinct (112, 113, 114, population is calculated in order to elaborate to encounter the
115, 116, 117). This includes the skin microbiome of dogs probability chances of profile [i.e., initiating from a location
Canis lupus familiaris. Humans and their pets have similar or person unreal].
microbial ecosystems, according to research (118).
However, there are currently very few microbiome
Such skills hint at the possible viability of tying a human databases that may be used for forensic purposes and
based on shared microbes with animals, pending more comparison to minute evidence in forensic settings, their
research and methodological improvement. Although further absence necessitates a "one-to-one" arrangement. Samples
research is required, forensic pathways may be able to link from the scene of the crime, the alibi location, and other
specific environments or occupations such as animal relevant areas are matched to samples from the suspect's
industries with profiles of trace microbes collected from other clothing and other items.
species. Detecting animal-specific microbiota on a suspect's
or victim's skin or clothing could be helpful if there isn't In comparison to other methods, this one is less helpful
required DNA of animals [i.e., from somatic and germ cells] because it can only provide either exclusionary or
evidence. As a supplement to more conventional forensic comparative results, such as "sample A and sample B show
evidence, this profile might potentially be tracked to the more similarities than compared to sample C". The European
interaction site with animal or animal-based contexts such as Network of Forensic Science Institutes recommends the
equine stables, Animal shops, or safari park. However, at the Likelihood Ratio framework (120), and we are able to utilize
present time this strategy is largely theoretical, thus more this to statistically evaluate the evidence even for "one-to-
investigation into its viability is required. one" research (121).
V. DISCUSSION Forensic technologies based on comparative

microbiomes still need to overcome three other significant
Almost no current criminal investigations or legal obstacles. In the first case, a sample may contain substrates
proceedings make use of forensics based on the microbiome. or DNA from multiple individuals or geographic areas.
This can be better understood by classifying the various Mixtures of DNA analysis is challenging with relatively
applications of microbial forensics can be categories for straightforward profiles of DNA, of humans much less with
better understanding into either issue in reconstruction, such microbiome DNA profiles. The DNA profiles of the various
as the PMI and cause of the death, which focuses on "what microbiomes in a mixture are generally difficult to deduce,
happened?" and help shed light on the crime's content, or and it is often hard to know for sure how many different
comparison issues are geo-sites and personal identification it microbiomes are present. 2016's illustrious Independent
includes similarity for these 2 DNA profiles? It may connect analysis of complex DNA of human mixes by forensic
or disconnect a suspect from an entity or a place (for example, professionals is not a credible methodology, according to a
crime evidence). The applications of Forensic Reconstruction report from the US President's Council of Advisors on
are simpler and easier to develop because they focus on issues Science and Technology (122). As a result, a number of
including PMI and the cause of death. If sufficient time and computer programs were created to objectively evaluate
effort are spent identifying characteristics of microbes related complicated human DNA mixes, and these are gradually
with each possible groups of temporal, spatial conditions and gaining validation and acceptance for regular forensic use.
environmental, then reconstruction can be made relatively Years from now, this foundation may be used to develop
straightforward. If PMI is extrapolated, precision of high software for the impartial examination of microbial mixtures
degree and inevitability from the discovery of a body buried of DNA. Temporal fluctuation is a further substantial
deep around one meter in a desert in the summertime, shows challenge. While human DNA tends to remain stable over
that the gut microbial population has been specified for the time, microbial populations can vary, typically in response to
environmental conditions at temporal succession. This is variations in environmental factors like moisture and pH

(123). When comparing two samples collected at separate There are still a lot of challenges to be conquered, such
times under very diverse environmental conditions, it is as small study samples, contamination issues, model over-
necessary to first account for the temporal changes in specification and misspecification, the accuracy of
community structure. Experimental research on this area is predictions made by machine learning techniques, the
scarce; what little there is has mostly focused on using complexity of temporal and spatial variations in the dynamics
different carbon sources to "converge" (124) the diverse of the environmental microbiome, along risks and ethical
microbial communities. concerns (126). However, there is still a lot of potential for
success in overcoming these challenges. The case of the
The tertiary and potentially insurmountable obstacle is Phantom of Heilbronn indicates that evidence from human
difficulty in transfer of DNA and Its identification from trace DNA based which is somewhat known, is not completely
evidence are belonging to a exact person is now rarely correct (127). The majority of the previously published work
borderline, because to the broad legitimacy and acceptance of relied on 16S or targeted sequencing procedures, both which
human DNA evidence in the courts during the past decade. are known to have limitations for taxonomic resolution.
Metagenomics techniques (128, 129) and/or techniques that
Instead, it is increasingly common for the defense to use longer reads (130, 131) have the potential to be an
question how the DNA was initially deposited, arguing that it improvement upon this body of work. Furthermore, strict,
could have been brought to the scene through lawful means titrated requirements for assuring the accuracy of these
or through DNA transfer (such as when the suspect who is sequencing approaches throughout field-based testing and
innocent shook hands with the real perpetrator). Usage of deployment would be beneficial (132). Given this, it's
probability ratio approach with activity level propositions can important to keep in mind that many of the literature
help get around this problem in some cases (125), but it's still applications are more like ideas of proof rather than actual
an open question whether or not this will work reliably, and forensic uses.
more work needs to be done before microbiomes can be used
and accepted in the legal system. " It is abundantly evident that we are still in a moment
of discovery and revelation, as Ogilvie and Jones write, "for
VI. CONCLUSION an observation of the inherent of microbial ecosystem, newer
methods and technologies are being provided."
The field of microbiomics has blossomed over the past
decade thanks to developments in genome sequencing and ACKNOWLEDGMENTS
bioinformatics, resulting in an expanding suite of instruments
designed to investigate the vast diversity of microorganisms I would like to express my profound gratitude to
that inhabit human bodies and the surrounding environment. Centurion University of Technology and Management,
Based on the evidence analysed in this study, microbiomics Andhra Pradesh Campus for providing infrastructure and
has the potential to become a useful and exciting forensic other facilities.
science with many potential applications, including but not
limited to the following: identifying individuals, estimating  Author’s Contributions
PMI, detecting substrate provenance, and gathering trace Vataparthi Pravallika: Preparation of Manuscript, Collection
evidence. These developments could eventually allow of Data and Formatting
forensic investigators to employ different types of Dr. Naga Jogayya. K: Grammer Checking, Plagiarism check,
microbiomic data, such as that produced by and Final Formatting
thanatomicrobiome analysis, to answer problems connected Syamala Alana Teja: Proof Reading
to criminal investigations, or at least to use these data in Dr. Satheesh Ampolu: Final Drafting and Proofreading
conjunction with other forensic procedures.
 Funding
Nutrient intake and the subsequent generation of No Funding
breakdown products like metabolites are only two examples
of the intricate interactions and co-adaptation processes that  Declarations
occur between humans and their microbiomes over the course Conflict of interest: The authors declare that they have
of a lifetime. The microbiome's make-up and dynamics shift no known financial or interpersonal conflicts that might be
drastically after death as a result of these alterations in perceived as having influenced the review presented in this
interaction. Conceptually, methodologically, and paper.
computationally, understanding these colonisations and
fluctuations, as well as antemortem microbial dynamics, is REFERENCES
extremely difficult. There is potential for further development
of forensic resources through studies of related microbiomes [1]. MacCallum, W. G., and Hastings, T. W. (1899). A case
in forensics setting, as well as through a deeper investigation of acute endocarditis caused by Micrococcus
of fungal and viral communities. zymogenes (nov. spec.), with a description of the
microorganism. J. Exp. Med. 4, 521–534. doi:
10.1084/jem.4.5-6.521

[2]. Smith, T. F., and Waterman, M. S. (1992). The [16]. Sangero, B., and Halpert, M. (2007). Why a
continuing case of the Florida dentist. Science 256, conviction should not be based on a single piece of
1155–1156. doi: 10.1126/science.256.5060.1155 evidence: a proposal for reform. Jurimetrics 48, 43–
[3]. van Belkum, A. (1994). DNA fingerprinting of 94.
medically important microorganisms by use of PCR. [17]. Ingemann-Hansen, O., Brink, O., Sabroe, S.,
Clin. Microbiol. Rev. 7, 174–184. doi: Sørensen, V., and Charles, A. V. (2008). Legal aspects
10.1128/cmr.7.2.174-184.1994 of sexual violence—does forensic evidence make a
[4]. Bruce, R. G., and Dettmann, M. E. (1996). difference? Forensic Sci. Int. 180, 98–104. doi:
Palynological analyses of Australian surface soils and 10.1016/j.forsciint.2008.07.009
their potential in forensic science. Forensic Sci. Int. [18]. LaPorte, G. (2017). Wrongful convictions and DNA
81, 77–94. doi: 10.1016/s0379-0738(96)01973-1 exonerations: Understanding the role of forensic
[5]. Bryant, V. M., and Mildenhall, D. C. (1998). Forensic science. NIJ J. 279:250705.
palynology: a new way to catch crooks. Contrib. Ser. [19]. Walsh, K., Hussemann, J., Flynn, A., Yahner, J., and
Am. Assoc. Stratigraph. Palynol. 33, 145–155. Golian, L. (2017). Estimating the Prevalence of
[6]. Budowle, B., Schutzer, S. E., Einseln, A., Kelley, L. Wrongful Convictions. Final Report 251115.
C., Walsh, A. C., Smith, J. A., et al. (2003). Building (Washington, DC: Urban Institute), 1–13.
microbial forensics as a response to bioterrorism. [20]. Innocent Project (2020). Exonerate the Innocent.
Science 301, 1852–1853. doi: Available online at: https://www.
10.1126/science.1090083 innocenceproject.org/exonerate/ (accessed September
[7]. Carter, D. O., Tomberlin, J. K., Benbow, M. E., and 18, 2020).
Metcalf, J. L. (eds) (2017). Forensic Microbiology. [21]. Desmond, A. U., Nicholas, O., and Emmanuel, O. O.
Hoboken, NJ: John Wiley & Sons. (2018). Microbial forensics: forensic relevance of the
[8]. Berglund, E. C., Kiialainen, A., and Syvänen, A. C. individual person’s microbial signature. Int. J. Life
(2011). Next-generation sequencing technologies and Sci. Scient. Res. 2455, 2037–2043.
applications for human genetic history and forensics. [22]. Sender, R., Fuchs, S., and Milo, R. (2016). Revised
Investig. Genet. 2:23. doi: 10.1186/2041-2223-2-23 estimates for the number of human and bacteria cells
[9]. Kuiper, I. (2016). Microbial forensics: next-generation in the body. PLoS Biol. 14:e1002533. doi:
sequencing as catalyst. EMBO Rep. 17, 1085–1087. 10.1371/journal.pbio.1002533
doi: 10.15252/embr.201642794 [23]. Vázquez-Baeza, Y., Callewaert, C., Debelius, J.,
[10]. Clarke, T. H., Gomez, A., Singh, H., Nelson, K. E., and Hyde, E., Marotz, C., Morton, J. T., et al. (2018).
Brinkac, L. M. (2017). Integrating the microbiome as Impacts of the human gut microbiome on therapeutics.
a resource in the forensics toolkit. Forensic Sci. Int. Annu. Rev. Pharmacol. Toxicol. 58, 253–270.
Genet. 30, 141–147. doi: [24]. Noel, S., Martina-Lingua, M. N., Bandapalle, S.,
10.1016/j.fsigen.2017.06.008 Pluznick, J., Hamad, A. R. A., Peterson, D. A., et al.
[11]. Hampton-Marcell, J. T., Lopez, J. V., and Gilbert, J. A. (2014). Intestinal microbiota-kidney cross talk in
(2017). The human microbiome: an emerging tool in acute kidney injury and chronic kidney disease.
forensics. Microb. Biotechnol. 10, 228–230. doi: Nephron Clin. Pract. 127, 139–143. doi:
10.1111/1751-7915.12699 10.1159/000363209
[12]. Statnikov, A., Henaff, M., Narendra, V., Konganti, K., [25]. Metcalf, J. L., Xu, Z. Z., Bouslimani, A., Dorrestein,
Li, Z., Yang, L., et al. (2013). A comprehensive P., Carter, D. O., and Knight, R. (2017). Microbiome
evaluation of multicategory classification methods for tools for forensic science. Trends Biotechnol. 35, 814–
microbiomic data. Microbiome 1:11. 823. doi: 10.1016/j.tibtech.2017.03.006
[13]. Capasso, L., Vento, G., Loddo, C., Tirone, C., [26]. Schmedes, S. E., Woerner, A. E., and Budowle, B.
Iavarone, F., Raimondi, F., et al. (2019). Oxidative (2017). Forensic human identification using skin
stress and bronchopulmonary dysplasia: evidences microbiomes. Appl. Environ. Microbiol. 83:e01672-
from microbiomics, metabolomics and proteomics. 17.
Front. Pediatr. 7:30. doi: 10.3389/fped.2019.00030 [27]. Richardson, M., Gottel, N., Gilbert, J. A., and Lax, S.
[14]. Office for National Statistics (2019). Homicide in (2019). Microbial similarity between students in a
England and Wales: Year Ending March 2019. common dormitory environment reveals the forensic
Available online at: potential of individual microbial signatures. mBio
https://www.ons.gov.uk/peoplepopulationandcommu 10:e1054-19.
nity/crimeandjustice/articles/homicideinenglandandw [28]. Phan, K., Barash, M., Spindler, X., Gunn, P., and
ales/yearendingmarch2019 (accessed February 14, Roux, C. (2020). Retrieving forensic information
2020). about the donor through bacterial profiling. Int. J.
[15]. Bureau for Justice Statistics. (2019). What is the Legal Med. 134, 21–29. doi: 10.1007/s00414-019-
Probability of Conviction for Felony Defendants? 02069-2
Available online at: [29]. Goudarzi, M., Mak, T. D., Jacobs, J. P., Moon, B. H.,
https://www.bjs.gov/index.cfm?ty=qa&iid=403 Strawn, S. J., Braun, J., et al. (2016). An integrated
(accessed February 14, 2020). multi-omic approach to assess radiation injury on the
host-microbiome axis. Radiat. Res. 186, 219–234. doi:
10.1667/rr14306.1

[30]. Komaroff, A. L. (2018). The microbiome and risk for [43]. Keet, J. H., Ellis, A. G., Hui, C., and Le Roux, J. J.
atherosclerosis. JAMA 319, 2381–2382. doi: (2019). Strong spatial and temporal turnover of soil
10.1001/jama.2018.5240 bacterial communities in South Africa’s hyperdiverse
[31]. Morgan, R. M., and Levin, E. A. (2019). A crisis for fynbos biome. Soil Biol. Biochem. 136:107541. doi:
the future of forensic science: lessons from the UK of 10.1016/j.soilbio.2019.107541
the importance of epistemology for funding research [44]. Carter, D. O., Metcalf, J. L., Bibat, A., and Knight, R.
and development. Forensic Sci. Int. 1, 243–252. doi: (2015). Seasonal variation of postmortem microbial
10.1016/j.fsisyn.2019.09.002 communities. Forensic Sci. Med. Pathol. 11, 202–207.
[32]. Afshinnekoo, E., Meydan, C., Chowdhury, S., Jaroudi, doi: 10.1007/s12024-015-9667-7
D., Boyer, C., Bernstein, N., et al. (2015). Geospatial [45]. Adserias-Garriga, J., Hernández, M., Quijada, N. M.,
resolution of human and bacterial diversity from city- Lázaro, D. R., Steadman, D., and Garcia-Gil, J.
scale metagenomics. Cell Syst. 1, 72–87. (2017). Daily thanatomicrobiome changes in soil as an
[33]. Rosenfeld, J., Reeves, D., Brugler, M. R., Narechania, approach of postmortem interval estimation: an
A., Simon, S., Kolokotronis, S., et al. (2016). Genome ecological perspective. Forensic Sci. Int. 278, 388–
assembly, annotation, and urban phylogenomics of the 395. doi: 10.1016/j.forsciint.2017.07.017
bedbug (Cimex lectularius). Nat. Commun. 7:10164. [46]. Finley, S. J., Pechal, J. L., Benbow, M. E., Robertson,
[34]. Danko, D. C., Bezdan, D., Afshinnekoo, E., B. K., and Javan, G. T. (2016). Microbial signatures of
Ahsanuddin, S., Bhattacharya, C., Butler, D. J., et al. cadaver gravesoil during decomposition. Microb.
(2019b). Global genetic cartography of urban Ecol. 71, 524–529. doi: 10.1007/s00248-015-0725-1
metagenomes and anti-microbial resistance. bioRxiv [47]. Singh, B., Minick, K. J., Strickland, M. S., Wickings,
[Preprint]. doi: 10.1101/724526 K. G., Crippen, T. L., Tarone, A. M., et al. (2018).
[35]. Habtom, H., Pasternak, Z., Matan, O., Azulay, C., Temporal and spatial impact of human cadaver
Gafny, R., and Jurkevitch, E. (2019). Applying decomposition on soil bacterial and arthropod
microbial biogeography in soil forensics. Forensic Sci. community structure and function. Front. Microbiol.
Int. Genet. 38, 195–203. doi: 8:2616. doi: 10.3389/fmicb.2017.02616
10.1016/j.fsigen.2018.11.010 [48]. Young, J. M., Austin, J. J., and Weyrich, L. S. (2017).
[36]. Macdonald, C. A., Ang, R., Cordiner, S. J., and Soil DNA metabarcoding and high-throughput
Horswell, J. (2011). Discrimination of soils at regional sequencing as a forensic tool: considerations, potential
and local levels using bacterial and fungal T-RFLP limitations and recommendations. FEMS Microbiol.
profiling. Ecol. 93:fiw207. doi: 10.1093/femsec/fiw207
[37]. Jesmok, E. M., Hopkins, J. M., and Foran, D. R. [49]. Chase, J., Fouquier, J., Zare, M., Sonderegger, D. L.,
(2016). Next-generation sequencing of the bacterial Knight, R., Kelley, S. T., et al. (2016). Geography and
16S rRNA gene for forensic soil comparison: a location are the primary drivers of office microbiome
feasibility study. composition. mSystems 1:e00022-16.
[38]. Demanèche, S., Schauser, L., Dawson, L., [50]. Lax, S., Smith, D. P., Hampton-Marcell, J., Owens, S.
Franqueville, L., and Simonet, P. (2017). Microbial M., Handley, K. M., Scott, N. M., et al. (2014).
soil community analyses for forensic science: Longitudinal analysis of microbial interaction
application to a blind test. Forensic Sci. Int. 270, 153– between humans and the indoor environment. Science
158. doi: 10.1016/j.forsciint.2016.12.004 345, 1048–1052. doi: 10.1126/science.1254529
[39]. Young, J. M., Weyrich, L. S., Breen, J., Macdonald, L. [51]. Walker, A. R., and Datta, S. (2019). Identification of
M., and Cooper, A. (2015). Predicting the origin of soil city specific important bacterial signature for the
evidence: high throughput eukaryote sequencing and MetaSUB CAMDA challenge microbiome data. Biol.
MIR spectroscopy applied to a crime scene scenario. Direct 14:11.
Forensic Sci. Int. 251, 22–31. doi: [52]. Ryan, F. J. (2019). Application of machine learning
10.1016/j.forsciint.2015.03.008 techniques for creating urban microbial fingerprints.
[40]. Sanachai, A., Katekeaw, S., and Lomthaisong, K. Biol. Direct 14:13.
(2016). Forensic soil investigation from the 16S rDNA [53]. Grantham, N. S., Reich, B. J., Laber, E. B., Pacifici,
profiles of soil bacteria obtained by denaturing K., Dunn, R. R., Fierer, N., et al. (2019). Global
gradient gel electrophoresis. Chiang Mai J. Sci. 43, forensic geolocation with deep neural networks. arXiv
748–755. [Preprint]. Available online at:
[41]. Pasternak, Z., Luchibia, A. O., Matan, O., Dawson, L., https://arxiv.org/abs/1905.11765 (accessed September
Gafny, R., Shpitzen, M., et al. (2019). Mitigating 1, 2020).
temporal mismatches in forensic soil microbial [54]. Zeng, B., Zhao, J., Guo, W., Zhang, S., Hua, Y., Tang,
profiles. Aust. J. Forensic Sci. 51, 685–694. doi: J., et al. (2017). High-altitude living shapes the skin
10.1080/00450618.2018.1450897 microbiome in humans and pigs. Front. Microbiol.
[42]. Pasternak, Z., Al-Ashhab, A., Gatica, J., Gafny, R., 8:1929.doi: 10.3389/fmicb.2017.01929
Avraham, S., Minz, D., et al. (2013). Spatial and [55]. Franzosa, E. A., Huang, K., Meadow, J. F., Gevers, D.,
temporal biogeography of soil microbial communities Lemon, K. P., Bohannan, B. J., et al. (2015).
in arid and semiarid regions. PLoS One 8:e69705. doi: Identifying personal microbiomes using metagenomic
10.1371/journal.pone. 0069705 codes. Proc. Natl. Acad. Sci. U.S.A. 112, 2930–2938.

[56]. Wang, S., Song, F., Wang, Y., Huang, Y., Xie, B., and [68]. Koscova, J., Hurnikova, Z., and Pistl, J. (2018).
Luo, H. (2019). High resolution melting analysis Degree of bacterial contamination of mobile phone
(HRM) based on 16SrRNA as a tool for personal and computer keyboard surfaces and efficacy of
identification with the human oral microbiome. disinfection with chlorhexidine digluconate and
Forensic Sci. Int. Genet. 7, 161–163. doi: triclosan to its reduction. Int. J. Environ. Res. Public
10.1016/j.fsigss.2019.09.063 Health 15:2238. doi: 10.3390/ijerph15102238
[57]. Woerner, A. E., Novroski, N. M., Wendt, F. R., [69]. Kurli, R., Chaudhari, D., Pansare, A. N., Khairnar, M.,
Ambers, A., Wiley, R., Schmedes, S. E., et al. (2019). Shouche, Y. S., and Rahi, P. (2018). Cultivable
Forensic human identification with targeted microbial diversity associated with cellular phones.
microbiome markers using nearest neighbor Front. Microbiol. 9:1229. doi:
classification. Forensic Sci. Int. Genet. 38, 130–139. 10.3389/fmicb.2018.01229
doi: 10.1016/j.fsigen.2018.10.003 [70]. Lax, S., Hampton-Marcell, J. T., Gibbons, S. M.,
[58]. Watanabe, H., Nakamura, I., Mizutani, S., Kurokawa, Colares, G. B., Smith, D., Eisen, J. A., et al. (2015).
Y., Mori, H., Kurokawa, K.,et al. (2018). Minor taxa Forensic analysis of the microbiome of phones and
in human skin microbiome contribute to the personal shoes. Microbiome 3:21.
identification. PLoS One 13:e0199947. doi: [71]. Coil, D. A., Neches, R. Y., Lang, J. M., Jospin, G.,
10.1371/journal.pone.0199947 Brown, W. E., Cavalier, D.,et al. (2019). Bacterial
[59]. Chan, P. L., Yu, P. H. F., Cheng, Y. W., Chan, C. Y., communities associated with cell phones and shoes.
and Wong, P. K. (2009). Comprehensive PeerJ 8:e9235. doi: 10.7717/peerj.9235
characterization of indoor airborne bacterial profile. J. [72]. Meadow, J. F., Altrichter, A. E., and Green, J. L.
Environ. Sci. 21, 1148–1152. doi: 10.1016/s1001- (2014). Mobile phones carry the personal microbiome
0742(08)62395-5 of their owners. PeerJ 2:e447. doi: 10.7717/peerj.447
[60]. Luongo, J. C., Barberán, A., Hacker-Cary, R., Morgan, [73]. Kodama, W. A., Xu, Z., Metcalf, J. L., Song, S. J.,
E. E., Miller, S. L., and Fierer, N. (2017). Microbial Harrison, N., Knight, R., et al. (2019). Trace evidence
analyses of airborne dust collected from dormitory potential in postmortem skin microbiomes: from death
rooms predict the sex of occupants. Indoor Air 27, scene to morgue. J. Forensic Sci. 64, 791–798. doi:
338–344. doi: 10.1111/ina.12302 10.1111/1556-4029.13949
[61]. Zhou, W., and Bian, Y. (2018). Thanatomicrobiome [74]. Salzmann, A. P., Russo, G., Aluri, S., and Haas, C.
composition profiling as a tool for forensic (2019). Transcription and microbial profiling of body
investigation. Forensic Sci. Res. 3, 105–110. doi: fluids using a massively parallel sequencing approach.
10.1080/20961790.2018.1466430 Forensic Sci. Int. Genet. 43:102149. doi:
[62]. Bell, C. R., Wilkinson, J. E., Robertson, B. K., and 10.1016/j.fsigen.2019.10 2149
Javan, G. T. (2018). Sex-related differences in the [75]. Dobay, A., Haas, C., Fucile, G., Downey, N.,
thanatomicrobiome in postmortem heart samples Morrison, H. G., Kratzer, A., et al. (2019).
using bacterial gene regions V1-2 and V4. Lett. Appl. Microbiome-based body fluid identification of
Microbiol. 67, 144–153. doi: 10.1111/lam.13005 samples exposed to indoor conditions. Forensic Sci.
[63]. Tridico, S. R., Murray, D. C., Addison, J., Kirkbride, Int. Genet. 40, 105–113. doi: 10.1016/j.fsigen.
K. P., and Bunce, M. (2014). Metagenomic analyses 2019.02.010
of bacteria on human hairs: a qualitative assessment [76]. Hanssen, E. N., Avershina, E., Rudi, K., Gill, P., and
for applications in forensic science. Investig. Genet. Snipen, L. (2017). Body fluid prediction from
5:16. microbial patterns for forensic application. Forensic
[64]. Williams, D. W., and Gibson, G. (2017). Sci. Int. Genet. 30, 10–17. doi:
Individualization of pubic hair bacterial communities 10.1016/j.fsigen.2017.05.009
and the effects of storage time and temperature. [77]. Neckovic, A., van Oorschot, R. A., Szkuta, B., and
Forensic Sci. Int.Genet. 26, 12–20. doi: Durdle, A. (2020). Investigation of direct and indirect
10.1016/j.fsigen.2016.09.006 transfer of microbiomes between individuals. Forensic
[65]. Phan, K., Barash, M., Spindler, X., Gunn, P., and Sci. Int. Genet. 45:102212. doi:
Roux, C. (2020). Retrieving forensic information 10.1016/j.fsigen.2019.102212
about the donor through bacterial profiling. Int. J. [78]. Advenier, A. S., Guillard, N., Alvarez, J. C., Martrille,
Legal Med. 134, 21–29. doi: 10.1007/s00414-019- L., and Lorin de la Grandmaison, G. (2016).
02069-2 Undetermined manner of death: an autopsy series. J.
[66]. Koroglu, M., Gunal, S., Yildiz, F., Savas, M., Ozer, A., Forensic Sci. 61(Suppl. 1), S154–S158.
and Altindis, M. (2015). Comparison of keypads and [79]. Lutz, H., Vangelatos, A., Gottel, N., Speed, E.,
touch-screen mobile phones/devices as potential risk Osculati, A., Visona, S., et al. (2019). Manner of death
for microbial contamination. J. Infect. Dev. Ctries. 9, and demographic effects on microbial community
1308–1314. doi:10.3855/jidc.6171 composition in organs of the human cadaver. bioRxiv
[67]. Kõljalg, S., Mändar, R., Sõber, T., Rööp, T., and [Preprint]. doi: 10.1101/752576
Mändar, R. (2017). High level bacterial contamination
of secondary school students’ mobile phones. Germs
7, 73–77. doi: 10.18683/germs.2017.1111

[80]. Zhang, Y., Pechal, J. L., Schmidt, C. J., Jordan, H. R., [92]. Aoyagi, M., Iwadate, K., Fukui, K., Abe, S., Sakai, K.,
Wang, W. W., Benbow, M. E., et al. (2019). Machine Maebashi, K., et al. (2009). A novel method for the
learning performance in a microbial molecular diagnosis of drowning by detection of Aeromonas
autopsy context: a cross-sectional postmortem human sobria with PCR method. Leg. Med. 11, 257–259. doi:
population study. PLoS One 14:e0213829. doi: 10.1016/j.legalmed.2009.07.003
10.1371/journal.pone.0213829 [93]. Uchiyama, T., Kakizaki, E., Kozawa, S., Nishida, S.,
[81]. Kaszubinski, S. F., Pechal, J. L., Smiles, K., Schmidt, Imamura, N., and Yukawa, N. (2012). A new
C. J., Jordan, H. R., Meek, M. H., et al. (2020). molecular approach to help conclude drowning as a
Dysbiosis in the dead: human postmortem cause of death: simultaneous detection of eight
microbiome beta-dispersion as an indicator of manner bacterioplankton species using real-time PCR assays
and cause of death. Front. Microbiol. 11:555347. doi: with TaqMan probes. Forensic Sci. Int. 222, 11–26.
10.3389/fmicb.2020.555347 doi: 10.1016/j.forsciint.2012.04.029
[82]. Christoffersen, S. (2015). The importance of [94]. Rutty, G. N., Bradley, C. J., Biggs, M. J., Hollingbury,
microbiological testing for establishing cause of death F. E., Hamilton, S. J., Malcomson, R. D., et al. (2015).
in 42 forensic autopsies. Forensic Sci. Int. 250, 27–32. Detection of bacterioplankton using PCR probes as a
doi: 10.1016/j.forsciint.2015.02.020 diagnostic indicator for drowning; the Leicester
[83]. Rambaud, C., Guibert, M., Briand, E., Grangeot- experience. Leg. Med. 17, 401–408. doi:
Keros, L., Coulomb-L’Herminé, A., and Dehan, M. 10.1016/j.legalmed.2015.06.001
(1999). Microbiology in sudden infant death [95]. Voloshynovych, V. M., Kasala, R. O., Stambulska, U.
syndrome (SIDS) and other childhood deaths. FEMS Y., and Voloshynovych, M. S. (2019). Determination
Immunol. Med. Microbiol. 25, 59–66. doi: the presence of amplification products of 16s rRNA
10.1111/j.1574-695x.1999.tb01327.x microcystis aeruginosa as a biomarker of drowning.
[84]. Szydlowski, L., Skierska, A., Markiewicz-Loskot, G., Rom. J. Leg. Med. 27, 16–21. doi:
Mazurek, B., Morka, A., and Undas, A. (2013). The 10.4323/rjlm.2019.16
role of Interleukin-6, its- 174 G > C polymorphism [96]. Lee, S. Y., Woo, S. K., Lee, S. M., Ha, E. J., Lim, K.
and C-reactive protein in idiopathic cardiac H., Choi, K. H., et al. (2017). Microbiota composition
arrhythmias in children. Adv. Med. Sci. 58, 320–325. and pulmonary surfactant protein expression as
doi: 10.2478/ams-2013-0003 markers of death by drowning. J. Forensic Sci. 62,
[85]. Garrett-Bakelman, F. E., Darshi, M., Green, S. J., Gur, 1080–1088. doi: 10.1111/1556-4029.13347
R. C., Lin, L., Macias, B. R., et al. (2019). The NASA [97]. J. Forensic Sci. 56, 61–69. doi: 10.1111/j.1556-
Twins Study: a multidimensional analysis of a year- 4029.2010.01542.x Marella, G. L., Feola, A.,
long human spaceflight. Science 364:eaau8650. Marsella, L. T., Mauriello, S., Giugliano, P., and
[86]. Elhaik, E. (2016). a “Wear and tear” Hypothesis to Arcudi, G. (2019). Diagnosis of drowning, an
explain sudden infant death syndrome. Front. Neurol. everlasting challenge in forensic medicine: review of
7:180. doi: 10.3389/fneur.2016.00180 the literature and proposal of a diagnostic algorithm.
[87]. Elhaik, E. (2019). Neonatal circumcision and Acta Med. 35, 900–919.
prematurity are associated with sudden infant death [98]. Lucci, A., Campobasso, C. P., Cirnelli, A., and
syndrome (SIDS). J. Clin. Transl. Res. 4, 136–151. Lorenzini, G. (2008). A promising microbiological
[88]. Domínguez, A. M., Pino, J., García, A., García, I., and test for the diagnosis of drowning. Forensic Sci. Int.
González, I. (2018). PW 1649 Drowning prevention 182, 20–26. doi: 10.1016/j.forsciint.2008. 09.004
campaigns, do they really work? Injury Prev. [99]. Javan, G. T., Finley, S. J., Abidin, Z., and Mulle, J. G.
24:A153. (2016). The thanatomicrobiome: a missing piece of
[89]. Cenderadewi, M., Franklin, R. C., Peden, A. E., and the microbial puzzle of death. Front. Microbiol. 7:225.
Devine, S. (2019). Pattern of intentional drowning doi: 10.3389/fmicb.2016.00225
mortality: a total population retrospective cohort study [100]. Burcham, Z. M., Pechal, J. L., Schmidt, C. J., Bose, J.
in Australia, 2006–2014. BMC Public Health 19:207. L., Rosch, J. W., Benbow, M. E., et al. (2019).
doi: 10.1186/s12889-019-6476-z Bacterial community succession, transmigration, and
[90]. Kakizaki, E., Kozawa, S., Sakai, M., and Yukawa, N. differential gene transcription in a controlled
(2009). Bioluminescent bacteria have potential as a vertebrate decomposition model. Front. Microbiol.
marker of drowning in seawater: two immersed 10:745. doi: 10.3389/fmicb.2019.00745.
cadavers retrieved near estuaries. Leg. Med. 11, 91– [101]. Javan, G. T., Finley, S. J., Tuomisto, S., Hall, A.,
96. doi: 10.1016/j.legalmed.2008.10.004 Benbow, M. E., and Mills, D. (2019). An
[91]. Huys, G., Coopman, V., Van Varenbergh, D., and interdisciplinary review of the thanatomicrobiome in
Cordonnier, J. (2012). Selective culturing and genus- human decomposition. Forensic Sci. Med. Pathol. 15,
specific PCR detection for identification of 75–83. doi: 10.1007/s12024-018-0061-0
Aeromonas in tissue samples to assist the medico- [102]. Adserias-Garriga, J., Hernández, M., Quijada, N. M.,
legal diagnosis of death by drowning. Forensic Sci. Lázaro, D. R., Steadman, D., and Garcia-Gil, J.
Int. 221, 11–15. doi: 10.1016/j.forsciint.2012.03.017 (2017). Daily thanatomicrobiome changes in soil as an
approach of postmortem interval estimation: an
ecological perspective. Forensic Sci. Int. 278, 388–
395. doi: 10.1016/j.forsciint.2017.07.017.

[103]. Metcalf, J. L., Parfrey, L. W., Gonzalez, A., Lauber, C. [115]. Torres, S., Clayton, J. B., Danzeisen, J. L., Ward, T.,
L., Knights, D., Ackermann, G., et al. (2013). A Huang, H., Knights, D., et al. (2017). Diverse bacterial
microbial clock provides an accurate estimate of the communities exist on canine skin and are impacted by
postmortem interval in a mouse model system. eLife cohabitation and time. PeerJ 5:e3075. doi:
2:e01104. 10.7717/peerj.3075
[104]. Pechal, J. L., Crippen, T. L., Benbow, M. E., Tarone, [116]. Engel, K., Sauer, J., Jünemann, S., Winkler, A.,
A. M., Dowd, S., and Tomberlin, J. K. (2014). The Wibberg, D., Kalinowski, J., et al. (2018). Individual-
potential use of bacterial community succession in and species-specific skin microbiomes in three
forensics as described by high throughput different estrildid finch species revealed by 16S
metagenomic sequencing. Int. J. Legal Med. 128, amplicon sequencing. Microb. Ecol. 76, 518–529. doi:
193–205. doi: 10.1007/s00414-013-0872-1 10.1007/s00248-017-1130-8
[105]. Pechal, J. L., Schmidt, C. J., Jordan, H. R., and [117]. Russo, C. D., Weller, D. W., Nelson, K. E., Chivers, S.
Benbow, M. E. (2018). A large-scale survey of the J., Torralba, M., and Grimes, D. J. (2018). Bacterial
postmortem human microbiome, and its potential to species identified on the skin of bottlenose dolphins
provide insight into the living health condition. Sci. off southern California via next generation sequencing
Rep. 8:5724. techniques. Microb. Ecol. 75, 303–309. doi:
[106]. Callahan, B. J., McMurdie, P. J., and Holmes, S. P. 10.1007/s00248-017-1071-2
(2017). Exact sequence variants should replace [118]. Song, S. J., Lauber, C., Costello, E. K., Lozupone, C.
operational taxonomic units in marker-gene data A., Humphrey, G., Berg-Lyons, D., et al. (2013).
analysis. ISMEJ. 11, 2639–2643. doi: Cohabiting family members share microbiota with one
10.1038/ismej.2017.119 another and with their dogs. eLife 2:e00458.
[107]. J. Forensic Sci. 61, 607–617. doi: 10.1111/1556- [119]. Daubert v. Merrell Dow Pharmaceuticals Inc (1993).
4029.13049 Johnson, H. R., Trinidad, D. D., Guzman, 509 U.S. p. 579. Available online at:
S., Khan, Z., Parziale, J. V., DeBruyn, https://supreme.justia.com/cases/federal/us/509/579/
[108]. Belk, A., Xu, Z. Z., Carter, D. O., Lynne, A., Bucheli, (accessed September 1, 2020).
S., Knight, R., et al. (2018). Microbiome data [120]. Willis, S. M., McKenna, L., McDermott, S., O’Donell,
accurately predicts the postmortem interval using G., Barrett, A., Rasmusson, B., et al. (2015). ENFSI
random forest regression models. Genes 9, 104–128. Guideline for Evaluative Reporting in Forensic
doi: 10.3390/genes9020104 Science. Wiesbaden: European Network of Forensic
[109]. Javan, G. T., Finley, S. J., Smith, T., Miller, J., and Science Institutes.
Wilkinson, J. E. (2017). Cadaver thanatomicrobiome [121]. Habtom, H., Pasternak, Z., Matan, O., Azulay, C.,
signatures: the ubiquitous nature of Clostridium Gafny, R., and Jurkevitch, E. (2019). Applying
species in human decomposition. Front. Microbiol. microbial biogeography in soil forensics. Forensic Sci.
8:2096. doi: 10.3389/fmicb.2017. 02096 Int. Genet. 38, 195–203. doi:
[110]. Zhou, X., Jiang, X., Yang, C., Ma, B., Lei, C., Xu, C., 10.1016/j.fsigen.2018.11.010
et al. (2016). Cecal microbiota of Tibetan Chickens [122]. President’s Cou Pasternak, Z., Luchibia, A. O., Matan,
from five geographic regions were determined by 16S O., Dawson, L., Gafny, R., Shpitzen, M., et al. (2019).
rRNA sequencing. Microbiologyopen 5, 753–762. Mitigating temporal mismatches in forensic soil
doi: 10.1002/mbo3.367 microbial profiles. Aust. J. Forensic Sci. 51, 685–694.
[111]. Zhao, J., Yao, Y., Li, D., Xu, H., Wu, J., Wen, A., et al. doi: 10.1080/00450618.2018.1450897
(2018). Characterization of the gut microbiota in six [123]. ncil of Advisors on Science and Technology (US)
geographical populations of Chinese Rhesus (2016). Report to the President. Forensic Science in
macaques (Macaca mulatta), implying an adaptation Criminal Courts: Ensuring scientific validity of
to high-altitude environment. Microb. Ecol. 76, 565– Feature-Comparison Methods. Washington, DC:
577. doi: 10.1007/s00248-018-1146-8 Executive Office of the President of the United States.
[112]. McKenzie, V. J., Bowers, R. M., Fierer, N., Knight, R., [124]. Pasternak, Z., Al-Ashhab, A., Gatica, J., Gafny, R.,
and Lauber, C. L. (2012). Co-habiting amphibian Avraham, S., Minz, D., et al. (2013). Spatial and
species harbor unique skin bacterial communities in temporal biogeography of soil microbial communities
wild populations. ISME J. 6, 588–596. doi: in arid and semiarid regions. PLoS One 8:e69705. doi:
10.1038/ismej.2011.129 10.1371/journal.pone. 0069705
[113]. Avena, C. V., Parfrey, L. W., Leff, J. W., Archer, H. M., [125]. Mayuoni-Kirshenbaum, L., Waiskopf, O., Finkelstein,
Frick, W. F., Langwig, K. E., et al. (2016). N., and Pasternak, Z. (2020). How did the DNA of a
Deconstructing the bat skin microbiome: influences of suspect get to the crime scene? A practical study in
the host and the environment. Front. Microbiol. DNA transfer during lock-picking. Aust. J. Forensic
7:1753. doi: 10.3389/fmicb.2016.01753 Sci. 52, 1–11. doi: 10.1080/00450618.2020.1793384
[114]. Erwin, P. M., Rhodes, R. G., Kiser, K. B., Keenan- [126]. Shamarina, D., Stoyantcheva, I., Mason, C. E., Bibby,
Bateman, T. F., McLellan, W. A., and Pabst, D. A. K., and Elhaik, E. (2017). Communicating the
(2017). High diversity and unique composition of gut promise, risks, and ethics of large-scale, open space
microbiomes in pygmy (Kogia breviceps) and dwarf microbiome and metagenome research. Microbiome
(K. sima) sperm whales. Sci. Rep. 7:7205. 5, 132–141.

[127]. Daniel, R., and van Oorschot, R. A. (2011). An

investigation of the presence of DNA on unused
laboratory gloves. Forensic Sci. Int. 3, e45–e46.
[128]. Ogilvie, L. A., and Jones, B. V. (2015). The human gut
virome: a multifaceted majority. Front. Microbiol.
6:918. doi: 10.3389/fmicb.2015.00918
[129]. McIntyre, A. B. R., Ounit, R., Afshinnekoo, E., Prill,
R. J., Hénaff, E., Alexander, N., et al. (2017).
Comprehensive benchmarking and ensemble
approaches for metagenomic classifiers. Genome
Biol. 18:182.
[130]. Danko, D. C., Bezdan, D., Afshinnekoo, E.,
Ahsanuddin, S., Bhattacharya, C., Butler, D. J., et al.
(2019b). Global genetic cartography of urban
metagenomes and anti-microbial resistance. bioRxiv
[Preprint]. doi: 10.1101/724526
[131]. Foox, J., Tighe, S. W., Nicolet, C. M., Zook, J. M.,
Herbert, Z. T., Warner, D., et al. (2020). Multi-
platform assessment of DNA sequencing performance
using human and bacterial reference genomes in the
ABRF next-generation sequencing study. bioRxiv
[Preprint]. doi: 10.1101/2020.07.23.218602
[132]. McIntyre, A. B. R., Alexander, N., Grigorev, K.,
Bezdan, D., Sichtig, H., Chiu, C. Y., et al. (2019).
Single-molecule sequencing detection of N6-
methyladenine in microbial reference materials. Nat.
Commun. 10:579.

A Review of Forensic Microbes in Crime Investigations

Uploaded by

Copyright:

Available Formats

A Review of Forensic Microbes in Crime Investigations

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

A Review of Forensic Microbes in Crime Investigations

Uploaded by

Copyright:

Available Formats

Volume 9, Issue 6, June – 2024 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165 https://doi.org/10.38124/ijisrt/IJISRT24JUN963

A Review of Forensic Microbes in

Abstract:- Advances in genetic sequencing technology, I. INTRODUCTION

IJISRT24JUN963 www.ijisrt.com 3088

IJISRT24JUN963 www.ijisrt.com 3089

IJISRT24JUN963 www.ijisrt.com 3090

 Individual Authentication identification in cases the investigator is unable to extract

IJISRT24JUN963 www.ijisrt.com 3091

Fig 1 Possible Sources of Forensically Relevant Microbiomes

IJISRT24JUN963 www.ijisrt.com 3092

IJISRT24JUN963 www.ijisrt.com 3093

IJISRT24JUN963 www.ijisrt.com 3094

V. DISCUSSION Forensic technologies based on comparative

IJISRT24JUN963 www.ijisrt.com 3095

IJISRT24JUN963 www.ijisrt.com 3096

IJISRT24JUN963 www.ijisrt.com 3097

IJISRT24JUN963 www.ijisrt.com 3098

IJISRT24JUN963 www.ijisrt.com 3099

IJISRT24JUN963 www.ijisrt.com 3100

IJISRT24JUN963 www.ijisrt.com 3101

[127]. Daniel, R., and van Oorschot, R. A. (2011). An

IJISRT24JUN963 www.ijisrt.com 3102

You might also like