Microbial Forensics Research Articles

Bacteriologic and Genomic Investigation of Bacillus anthracis Isolated from World War II Site, China.

Records suggest Bacillus anthracis was used in biowarfare during World War II, but evidence remains limited. We isolated B. anthracis from soil at the remains of a World War II-era laboratory in China. Phenotypic and genomic analyses confirmed the finding, highlighting the value of microbial forensics in biothreat investigation.

Microbial Forensics and Bioterrorism: Review of challenges, Investigation, and Future Directions

This review describes how the field of microbial forensic can contribute in investigation by examining and characterizing microbial evidences. Analysis of microbial evidence to assist in accreditation of the bioagent to a source and reconstruction of the crime scene. Bioterrorism is intentional dispersal of bio-warfare agents in a population to cause illness or death. This review includes microbial forensic techniques, including DNA-based methods. Additionally, it describes the applications in microbial forensics in; identification of source and tracking, crime scene reconstruction, bio safety and biosecurity measures. This aims to provide a detailed understanding, of microbial forensics and its application in combating bioterrorism.

A Custom qPCR Assay to Simultaneously Quantify Human and Microbial DNA.

To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10-8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations.

Microbial Forensics: Comparison of MLVA Results According to NGS Methods, and Forensic DNA Analysis Using MLVA

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled <i>in silico</i> anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General’s Mechanism(UNSGM)’s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were <i>B. anthracis</i> through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of <i>B. anthracis</i> were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of <i>B. anthracis</i>, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as <i>B. anthracis</i> strain A4564 which is associated with injectional anthrax isolates in heroin users.

Microbial forensics: are we going on right path for management of crops biosecurity in Pakistan?

In any country biological terrorism outbreaks on crops could reduce the quality and quantity of agricultural products, which in turn decreases the consumer’s confidence, nutrition loss, resulting in worse impacts on the nation’s economy, international souks and is subsequently harmful for the environment. Awareness for a crop bioterror incident begins with the start of the threat symptoms, which must lead to the progress of a plan to thwart. However, most of Pakistani scientists and crop producers have not focused on the option of deliberative plant pathogen introduction but instead they use their expertise for the prevention of natural introductions and the improvement of cost-effective strategies for disease management. It is also an interesting fact that under the mask of genetically modified feeds, high yield varieties and hybrids; we are also importing bio terrorism. Are we going on the right path? So, it is time that we should review the global microbial knowledge and should apply it according to our needs. In ongoing topics of present article, we will review the microbial forensics status globally in comparison with Pakistan. We will also suggest how to fill these gaps. Keywords: Biological terrorism; Microbial forensics; Pathogen; Plants; Crops; Biosecurity

Microbial genomics: a potential toolkit for forensic investigations.

Microbial forensics is a new discipline of science that analyzes evidence related to biological crime through the uniqueness and abundance of microorganisms and their toxins. Microorganisms remain alive longer than any other trace of biological evidence, such as DNA, fingerprints, and fibers, because of the protective cell membrane or capsules. Microbiological research has opened up various possibilities for forensic investigations of microbial flora. Current molecular technologies, including DNA sequencing, whole-genome sequencing, metagenomics, DNA fingerprinting, and molecular phylogeny, provide valid results for forensic investigations. Recent advancements in genome sequencing technologies, genetic data generation, and bioinformatic tools have significantly improved microbial sampling methods and forensic analyses. In this review, we discuss the applications of microbial genomic tools and technologies in forensic investigations, including human identification, geolocation, and causes of death.

The role of ecogenomics in environmental and forensic discoveries

Ecogenomics is the application of genetic information and environmental science to understand the genetic diversity and functional potential of microbial communities in various environments. It involves the analysis of DNA and RNA sequences to gain insights into the structure, function, and dynamics of ecosystems. In the context of the environment, ecogenomics plays a crucial role in understanding the impact of human activities on natural ecosystems. By studying the genetic composition of microbial communities, researchers can assess the health and resilience of ecosystems, identify key species involved in nutrient cycling and energy flow, and monitor the effects of pollution and climate change. Ecogenomics also has significant applications in forensic research. By analyzing the microbial DNA present at crime scenes, forensic scientists can gain valuable information about the individuals involved. Microbes are ubiquitous and can be transferred from person to person, leaving behind a unique microbial signature that can be used for identification purposes. This approach, known as microbial forensics, can complement traditional forensic techniques and provide additional evidence in criminal investigations. By harnessing the power of genomics, researchers can uncover hidden microbial diversity, track ecosystem changes, and provide valuable insights into criminal investigations.

A systematic approach to microbial forensics.

The coronavirus disease 2019 pandemic accelerated developments in biotechnology that underpin infection science. These advances present an opportunity to refresh the microbial forensic toolkit. Integration of novel analytical techniques with established forensic methods will speed up acquisition of evidence and better support lines of enquiry. A critical part of any such investigation is demonstration of a robust causal relationship and attribution of responsibility for an incident. In the wider context of a formal investigation into agency, motivation and intent, the quick and efficient assembly of microbiological evidence sets the tone and tempo of the entire investigation. Integration of established and novel analytical techniques from infection science into a systematic approach to microbial forensics will therefore ensure that major perspectives are correctly used to frame and shape the evidence into a clear narrative, while recognizing that forensic hypothesis generation, testing and refinement comprise an iterative process. Development of multidisciplinary training exercises that use this approach will enable translation into practice and efficient implementation when the need arises.

Molecular Approaches in Soil Microbial Analysis: Forensic Perspective

The growing field of soil microbial forensics provides the legal answer to how microorganisms play a crucial role in criminal investigations. It is an advanced crossdisciplinary science capable of offering significant physical evidence with considerable interest in criminal investigations, environmental crime, and counterterrorism. Microbial forensics of soil consists of different techniques to identify and evaluate microbial abundance, their complexity, and their interaction with soil and surrounding objects. The present review highlights various microbial analysis techniques such as Terminal restriction fragment length polymorphism, Temperature/Denaturing Gradient Gel Electrophoresis, Amplified Ribosomal DNA Restriction Analysis, Length Heterogeneity PCR, Phospholipid-derived fatty acids, Fluorescence in situ hybridization, Stable-isotope probing and metagenomics using nextgeneration sequencing. This article also summarises the challenges faced in soil microbial forensics, various statistical approaches, reference databases commonly used in forensic soil studies, and different methodological approaches used in forensic laboratories. Literature was studied using various online bibliographic databases like Google Scholar, Web of Science, Pub Med, Scopus, and several other search engines. Conclusive evidence generated by this paper signifies the importance of soil microbes to assist forensic scientists and researchers in selecting adequate methods to differentiate soil samples. The present comparative study concluded that targeted molecular analysis of microbes as a forensic soil typing tool has a lot of potential and should be investigated further.

Introducing the New Editorial Team

Introducing the New Editorial Team

Microbial forensics: A potential tool for investigation of bioterrorism

Microbial forensics: A potential tool for investigation of bioterrorism

A bibliometric analysis of microbial forensics from 1984 to 2022: progress and research trends.

Microbial forensics is a rapidly evolving discipline that has gained significant momentum in recent years. The study evaluated relevant results over the last four decades from 1984 to 2022 all over the world, aiming to analyze the growing trends and research orientations of microbial forensics. Using "microbial forensics" as the search topic in the Web of Science Core Collection, the systematic retrieval identified 579 documents relevant to the field and draw many statistical tables and maps to make the retrieval results visible. According to further bibliometric analysis, there are an increasing number of publications related to microbial forensics from the overall trend, with the highest number of publications recorded in 2021. In terms of the total number of articles, the USA and China were both the leading contributors to the field among 40 countries. The field has developed rapidly in recent years based on the development of next-generation sequencing. Over the course of its development, there are rich keywords in the research of scholars, which focus on diversity and identification. Moreover, despite the early hot topic being PCR (the use of PCR to probe microorganisms), in recent years, the topics, markers, and the potential application of microorganisms in forensic practice have become hot, which also indicates the future research directions of microbial forensic.

Oral Microbial Identity: A Potential Tool for Forensic Science

Microbial forensics is a feld that uses scientifc methods to analyze the evidence of bioterrorism, bio crimes, hoaxes, or the unintentional release of a biological agent or microbial toxin to determine who is to blame. It is now possible to characterize microorganisms for various human forensic applications, including human identifcation, body fluid characterization, postmortem interval estimation, and bio crimes involving the tracking of infectious agents. Big applause to technological advancements, remarkably massively parallel sequencing and bioinformatics. The oral microbiome contains microbiota markers that vary between individuals, emphasizing the possibility that it is highly individualized and perhaps even particular to each person. As a result, the oral microbial evidence found at crime scenes may lead to new information. This comprehensive review will discuss the state of the art of using the oral microbiome as a signature to conclude in the forensics sector.

Investigating the Post-Mortem Interval (PMI) with Forensically Important Necrobiomes

Estimating the post-mortem time interval (PMI) is a crucial component of the forensic investigation and is extremelydifficult for medico-legal experts to do. After death, the succession of microbes in various parts of the human bodyhas enormous potential for predicting PMI. The human body is home to trillions of commensal microorganisms.These microbes behave in a different way when biological processes stop, which coincides with the death of anindividual and the invasion of deteriorating microbes from the environment. Due to cell autolysis, which draws avariety of invasive macro- and microorganisms, human cadavers become a rich source of nutrients. The successionof microorganisms differs significantly at various stages of degradation, which can be investigated for precisePMI estimation. Necrobiome analysis has drawn a lot of attention in PMI estimation due to the developmentof microbial genomics technology and decrease in the price of DNA sequencing. The review article provides asummary of the various microorganism sources, their successional pattern, and analytical methods that can beused in the field of microbial forensics.

Microbial forensics: A potential tool for investigation and response to bioterrorism

Bioterrorism is the intentional dissemination of bio-warfare agents in a population to cause illness or death. Microbial forensics plays an essential role in the investigation of bio-terror attacks. This is a new field of forensic science that is still in the early stages of growth and faces huge theoretical and ethical obstacles. There is a profound concern with current biotechnological developments that bacteria and viruses may be genetically engineered to modify them as biological weapons. Bioterrorism, like other nuclear weapons, has the potential to cause humanitarian disasters. This paper summarizes different detection methods of bio-warfare agents besides highlighting the various phases of bioterror attacks through the investigatory lens of microbial forensics.

Supervised Machine Learning Enables Geospatial Microbial Provenance.

The recent increase in publicly available metagenomic datasets with geospatial metadata has made it possible to determine location-specific, microbial fingerprints from around the world. Such fingerprints can be useful for comparing microbial niches for environmental research, as well as for applications within forensic science and public health. To determine the regional specificity for environmental metagenomes, we examined 4305 shotgun-sequenced samples from the MetaSUB Consortium dataset—the most extensive public collection of urban microbiomes, spanning 60 different cities, 30 countries, and 6 continents. We were able to identify city-specific microbial fingerprints using supervised machine learning (SML) on the taxonomic classifications, and we also compared the performance of ten SML classifiers. We then further evaluated the five algorithms with the highest accuracy, with the city and continental accuracy ranging from 85–89% to 90–94%, respectively. Thereafter, we used these results to develop Cassandra, a random-forest-based classifier that identifies bioindicator species to aid in fingerprinting and can infer higher-order microbial interactions at each site. We further tested the Cassandra algorithm on the Tara Oceans dataset, the largest collection of marine-based microbial genomes, where it classified the oceanic sample locations with 83% accuracy. These results and code show the utility of SML methods and Cassandra to identify bioindicator species across both oceanic and urban environments, which can help guide ongoing efforts in biotracing, environmental monitoring, and microbial forensics (MF).

A Short Review of Forensic Microbiology

Background: Microbial forensics is a multidisciplinary area, which has been recently considered an effective tool in forensic investigations. This growing field of forensics covers a wide spectrum of different branches of science, including biology, chemistry, physics, geology, mathematics, and computer sciences, leading to a practical approach that can be applied in several areas such as bioterrorist actions, environmental issues, emerging and reemerging diseases, as well as reliable trace evidence at a crime scene. Methods: The information has been gathered via Google Scholar using several keywords, including forensic microbiology, bioterrorism, forensic investigation, and trace evidence. The data were from reliable articles and books published over 50 years. This paper is a short review of forensic microbiology with a bioinformatics perspective to use in different fields such as the court. Results: It is known that using either microorganisms or their toxins is a low-cost potential tool with serious morbidity and mortality rates that can spread all around the world by food or water supplies or even through the air, making them a perfect candidate bioweapon with minimum traceability. Studies have indicated that environmental conditions plus biological and abiotic factors would affect the following analysis and the final validation, which is an essential step in the forensic investigation due to its highly effective role in the court vote. To face different challenges, law enforcement has the infrastructure for attribution and deterrence (e.g., following the exact microbial forensics program) so that it can be used in court. Developing more reproducible, sensitive, and accurate methods, preparing a wide reliable database, and devoting the right amount of budget will help improve the whole forensic procedure in the legal system. Conclusions: The current paper is a short review of how forensic scientists can use microbial features on a crime scene to clarify and enhance the procedure to solve different criminal cases.

Microbial Forensics: A Present to Future Perspective on Genomic Targets, Bioinformatic Challenges, and Applications

Introduction: The human microbiome is a compendium of all organisms on and within the human body (i.e., bacteria, archaea, fungi, and eukaryotes), where the diversity of taxa as well as their relative abundance contribute to the highly individualizing nature of an individual's microbial signature. Discussion: The effect of individual characteristics such as lifestyle, genetics, health, and geographic provenance on the human microbiome contribute to both the individuality and forensic usefulness of microbiome samples for the purposes of human identification, body fluid attribution, estimation of the post-mortem interval, or geolocation. Further, the use of technologies such as massively parallel sequencing and long-read sequencing, in combination with bioinformatic tools and machine learning models, allow for quick and accurate characterization of individual human microbiome samples. Conclusion: However, before microbial profiling can be implemented into routine forensic casework, extensive standardization and validation of sample types, collection procedures and analysis pipelines must be established.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10fg genomic DNA and 1CFUml-1 bacterial culture. No change in the detection threshold (1CFUml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Evaluating the persistence and stability of a DNA-barcoded microbial system in a mock home environment.

Recent advancements in engineered microbial systems capable of deployment in complex environments have enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures, enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system composed of germination-deficient Bacillus subtilis and Saccharomyces cerevisiae spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR-associated proteins (Cas) detection (Sherlock). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together, we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications. Graphical Abstract.