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This review discusses microbial forensics as an emerging science that finds application in protecting human health. It is important to distinguish naturally acquired infections from those caused by the intentional release of microorganisms to the environment. This information is crucial in formulating procedures against the spread of infectious diseases and prosecuting persons who may be involved in acts of biocrime, bioterrorism, or biowarfare. A comparison between epidemiological investigations and microbial forensic investigations is provided. In addition, a discussion on how microbial forensics strengthens health systems is included in this review. Microbial forensic investigations and epidemiologic examinations employ similar concepts and involve identifying and characterising the microbe of interest. Both fields require formulating an appropriate case definition, determining a pathogen's mode of transmission, and identifying the source(s) of infection. However, the two subdisciplines differ in their objectives. An epidemiological investigation aims to identify the pathogen's source to prevent the spread of the disease. Microbial forensics focuses on source-tracking to facilitate the prosecution of persons responsible for the spread of a pathogen. Both fields use molecular techniques in analysing and comparing DNA, gene products, and biomolecules to identify and characterise the microorganisms of interest. We included case studies to show methods used in microbial forensic investigations, a brief discussion of the public significance of microbial forensic systems, and a roadmap for establishing a system at a national level. This system is expected to strengthen a country's capacity to respond to public health emergencies. Several factors must be considered in establishing national microbial forensic systems. First is the inherent ubiquity, diversity, and adaptability of microorganisms that warrants the use of robust and accurate molecular typing systems. Second, the availability of facilities and scientists who have been trained in epidemiology, molecular biology, bioinformatics, and data analytics. Human resources and infrastructure are critical requirements because formulating strategies and allocating resources in times of infectious disease outbreaks must be data-driven. Establishing and maintaining a national microbial forensic system to strengthen capacities in conducting forensic and epidemiological investigations should be prioritised by all countries, accompanied by a national policy that sets the legislative framework and provides for the system's financial requirements.

Recent advances in genetic data generation, through massive parallel sequencing (MPS), storage and analysis have fostered significant progresses in microbial forensics (or forensic microbiology). Initial applications in circumstances of biocrime, bioterrorism and epidemiology are now accompanied by the prospect of using microorganisms (i) as ancillary evidence in criminal cases; (ii) to clarify causes of death (e.g., drownings, toxicology, hospital-acquired infections, sudden infant death and shaken baby syndromes); (iii) to assist human identification (skin, hair and body fluid microbiomes); (iv) for geolocation (soil microbiome); and (v) to estimate postmortem interval (thanatomicrobiome and epinecrotic microbial community). When compared with classical microbiological methods, MPS offers a diverse range of advantages and alternative possibilities. However, prior to its implementation in the forensic context, critical efforts concerning the elaboration of standards and guidelines consolidated by the creation of robust and comprehensive reference databases must be undertaken.

Estimation of post-mortem time interval (PMI) is a key parameter in the forensic investigation which poses a huge challenge to the medico-legal experts. The succession of microbes within different parts of the human body after death has shown huge potential in the determination of PMI. Human body harbors trillions of microorganisms as commensals. With the death of an individual when biological functions are stopped, these microorganisms behave contrarily along with the invasion of degrading microbes from the environment. Human cadaver becomes a rich source of nutrients due to autolysis of cells, which attracts various invading microorganisms as well as macroorganisms. At different stages of degradation, the succession of microorganisms differs significantly which can be explored for accurate PMI estimation. With the advent of microbial genomics technique and reduction in the cost of DNA sequencing, thanatomicrobiome and epinecrotic community analysis have gained huge attention in PMI estimation. The article summarizes different sources of microorganisms in a human cadaver, their succession pattern, and analytical techniques for application in the field of microbial forensics.Key points• Thanatomicrobiome and epinecrotic microbiome develop in postmortem human body.• Lack of metabolic, immune, neuroendocrine systems facilitate microbial succession.• Analysis of postmortem microbial communities predicts accurate PMI.

Airborne microbes (bacteria, archaea, protists, and fungi) were surveyed over a 7-y period via high-throughput massive sequencing of 16S and 18S rRNA genes in rain and snow samples collected fortnightly at a high-elevation mountain Long-Term Ecological Research (LTER) Network site (LTER-Aigüestortes, Central Pyrenees, Spain). This survey constitutes the most comprehensive mountain-top aerobiology study reported to date. The air mass origins were tracked through modeled back-trajectories and analysis of rain water chemical composition. Consistent microbial seasonal patterns were observed with highly divergent summer and winter communities recurrent in time. Indicative microbial taxa were unveiled as a forensic signature, and ubiquitous taxa were observed as common atmosphere inhabitants, highlighting aerosols as a potentially successful mechanism for global microbial dispersal. Source-tracking analyses identified freshwater, cropland, and urban biomes as the most important sources for airborne bacteria in summer, while marine and forest biomes prevailed in winter, in agreement with air mass retrotrajectories and the prevailing general and regional atmospheric circulation.

Microbial diversity's potential has been investigated in medical and therapeutic studies throughout the last few decades. However, its usage in forensics is increasing due to its effectiveness in circumstances when traditional approaches fail to provide a decisive opinion or are insufficient in forming a concrete opinion. The application of human microbiome may serve in detecting the type of stains of saliva and vaginal fluid, as well as in attributing the stains to the individual. Similarly, the microbiome makeup of a soil sample may be utilised to establish geographic origin or to associate humans, animals, or things with a specific area, additionally microorganisms influence the decay process which may be used in depicting the Time Since death. Further in detecting the traces of the amount and concentration of alcohol, narcotics, and other forensically relevant compounds in human body or visceral tissues as they also affect the microbial community within human body. Beside these, there is much more scope of microbiomes to be explored in terms of forensic investigation, this review focuses on multidimensional approaches to human microbiomes from a forensic standpoint, implying the potential of microbiomes as an emerging tool for forensic investigations such as individual variability via skin microbiomes, reconstructing crime scene, and linking evidence to individual. •Microbiome analysis holds great promise for providing insights into forensically important issues.•The use of microbiomes in Forensic investigations are highlighted in this article.•The various opportunities and difficulties in the field of Microbial forensics are also outlined, along with solutions.

Recent advances in DNA sequencing technology have allowed the collection of high-dimensional data from human-associated microbial communities on an unprecedented scale. A major goal of these studies is the identification of important groups of microorganisms that vary according to physiological or disease states in the host, but the incidence of rare taxa and the large numbers of taxa observed make that goal difficult to obtain using traditional approaches. Fortunately, similar problems have been addressed by the machine learning community in other fields of study such as microarray analysis and text classification. In this review, we demonstrate that several existing supervised classifiers can be applied effectively to microbiota classification, both for selecting subsets of taxa that are highly discriminative of the type of community, and for building models that can accurately classify unlabeled data. To encourage the development of new approaches to supervised classification of microbiota, we discuss several structures inherent in microbial community data that may be available for exploitation in novel approaches, and we include as supplemental information several benchmark classification tasks for use by the community.

•Microorganisms have been use as weapons since pre-historic times.•Biowarfare is the intentional use of biological agents as weapons in war scenarios.•Bioterrorism is the intentional use of biological agents against a civilian population.•Biocrime is the intentional use of biological agents against a specific individual.•Microbial forensics can be applied to solve cases of BW, BT, and BC. Microbial Forensics is a field that continues to grow in interest and application among the forensic community. This review, divided into two sections, covers several topics associated with this new field. The first section presents a historic overview concerning the use of microorganisms (or its product, i.e. toxins) as harmful biological agents in the context of biological warfare (biowarfare), bioterrorism, and biocrime. Each case is illustrated with the examination of case reports that span from prehistory to the present day. The second part of the manuscript is devoted to the role of MF and highlights the necessity to prepare for the pressing threat of the harmful use of biological agents as weapons. Preventative actions, developments within the field to ensure a timely and effective response and are discussed herein.

ABSTRACT Forensics aims at using physical evidence to solve investigations with science-based principles, thus operating within a theoretical framework. This however is often rather weak, the exception being DNA-based human forensics that is well anchored in theory. Soil is a most commonly encountered, easily and unknowingly transferred evidence but it is seldom employed as soil analyses require extensive expertise. In contrast, comparative analyses of soil bacterial communities using nucleic acid technologies can efficiently and precisely locate the origin of forensic soil traces. However, this application is still in its infancy, and is very rarely used. We posit that understanding the theoretical bases and limitations of their uses is essential for soil microbial forensics to be judiciously implemented. Accordingly, we review the ecological theory and experimental evidence explaining differences between soil microbial communities, i.e. the generation of beta diversity, and propose to integrate a bottom-up approach of interactions at the microscale, reflecting historical contingencies with top-down mechanisms driven by the geographic template, providing a potential explanation as to why bacterial communities map according to soil types. Finally, we delimit the use of soil microbial forensics based on the present technologies and ecological knowledge, and propose possible venues to remove existing bottlenecks. The authors describe the application and current limitations of molecular microbial ecology to soil forensics, and provide the ecological theory background on which the approach relies, linking microscale and macroscale processes.

For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR–Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don’t exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check. [Display omitted] •The CRISPR/Cas system is an innovative technology with numerous applications in several fields.•The intentional misuse of the CRISPR/Cas system could be disastrous for society and the environment.•Dual use of this technology would be challenging for law enforcement to combat.•Several countries have laws and guidelines to regulate genome editing through multiple agencies.

Background The increasing numbers of sexual violence and unresolved rape cases require alternative approaches with higher evidential value to complement existing forensic tools. Predicting recent intercourse is crucial in forensic casework on sexual assaults. In this work, we assessed whether sexual intercourse can be predicted based on the vaginal microbiome and compared it to the gold standard method of semen detection. Results Using a prediction model based on microbiome of 3043 women, intercourse was predicted with 71% accuracy in a balanced cross-validation machine learning setting. This prediction model was validated in a longitudinal intervention study and tested on forensic sexual assault cases. The developed predictor could accurately establish intercourse in 82% of the studied cases. Yet, underwear was found to hold an even greater evidential value and replace the more invasive vaginal sampling for semen detection in some cases with an accuracy of 95%. This was substantiated through a retrospective analysis of 207 forensic sexual assault cases. Conclusions Taken together, this study revealed that the vaginal microbiome is better at predicting recent sexual intercourse, while the victim’s underwear has a clear value as additional biological trace evidence for semen detection. These findings are particularly useful in cases with delayed reporting and are obtained with less invasive sampling. 4mnRMKAz3tKRXghF2Tr5gx Video Abstract