RESUMEN

High throughput sequencing is improving the efficiency of monitoring diatoms, which inhabit and support aquatic ecosystems across the globe. In this study, we explored the potential of a standard V4 515F-806RB primer pair in recovering diatom plastid 16S rRNA sequences. We used PhytoREF to classify the 16S reads from our freshwater biofilm field sampling from three stream segments across two streams in south-eastern Australia and retrieved diatom community data from other, publicly deposited, Australian 16S amplicon datasets. When these diatom operational taxonomic units (OTUs) were traced using the default RDPII and NCBI databases, 68% were characterized as uncultured cyanobacteria. We analysed the 16S rRNA sequences from 72 stream biofilm samples, separated the chloroplast OTUs, and classified them using the PhytoREF database. After filtering the reads attributed to Bacillariophyta (relative abundance >1%), 71 diatom OTUs comprising more than 90% of the diatom reads in each stream biofilm sample were identified. Beta-diversity analyses demonstrated significantly different diatom assemblages and discrimination among river segments. To further test the approach, the diatom OTUs from our biofilm sampling were used as reference sequences to identify diatom reads from other Australian 16S rRNA datasets in the NCBI-SRA database. Across the three selected public datasets, 67 of our 71 diatom OTUs were detected in other Australian ecosystems. Our results show that diatom plastid 16S rRNA genes are readily amplified with existing 515F-806RB primer sets. Therefore, the volume of existing 16S rRNA amplicon datasets initially generated for microbial community profiling can also be used to detect, characterize, and map diatom distribution to inform phylogeny and ecological health assessments, and can be extended into a range of ecological and industrial applications. To our knowledge, this study represents the first attempt to classify freshwater samples using this approach and the first application of PhytoREF in Australia.

RESUMEN

16S rRNA profiling of bacterial communities may have forensic utility in the identification or association of individuals involved with criminal activities. Microbial profiling of evidence may, in the future, be performed within environments currently utilised for human DNA recovery, such as a forensic biology laboratory. It would be important to establish the background microbiome of such an environment to determine the potential presence of human or environmental microbial signatures to assist forensic scientists in the appropriate interpretation of target microbial communities. This study sampled various surfaces of an Evidence Recovery Laboratory (ERL) on three occasions including (a) before a monthly deep-clean, (b) immediately following the deep-clean, and (c) immediately after the laboratory's use by a single participant for the purposes of routine item examinations. Microbial profiles were also generated for the involved participant and researcher for comparison purposes. Additionally, human nuclear DNA was profiled for each of the samples collected, using standard forensic profiling techniques, to provide a prospective link to the presence or absence of a background microbial signature within the ERL after its use. Taxonomic distributions across ERL samples revealed no consistent signature of any of the items sampled over time, however, major phyla noted within all ERL samples across the three timepoints were consistent with those found in human skin microbiomes. PCoA plots based on the Unweighted Unifrac metric revealed some clustering between participant microbial reference samples and surfaces of the ERL after use, suggesting that despite a lack of direct contact, and adherence to standard operating procedures (SOPs) suitable for human DNA recovery, microbiomes may be deposited into a forensic setting over time. The reference samples collected from the involved participant and researcher generated full STR profiles. Human DNA was observed to varying degrees in samples taken from the ERL across each of the sampling timepoints. There was no correlation observed between samples that contained or did not contain detectable quantities of human nuclear DNA and microbial profile outputs.

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RESUMEN

Microbial profiling within forensic science is an emerging field that may have applications in the identification of individuals using microbial signatures. It is important to determine if microbial transfer may occur within a forensic laboratory setting using current standard operating procedures (SOPs) for nuclear DNA recovery, to assess the suitability of such procedures for microbial profiling and establish the potential limitations of microbial profiling for forensic purposes. This preliminary study investigated the presence and potential transfer of human-associated microbiomes within a forensic laboratory. Swabs of laboratory surfaces, external surfaces of personal protective equipment (PPE) and equipment were taken before and after mock examinations of cotton swatches, which harboured microbiota transferred from direct hand-contact. Microbial profiles obtained from these samples were compared to reference profiles obtained from the participants, cotton swatches and the researcher to detect microbial transfer from the individuals and determine potential source contributions. The results revealed an apparent transfer of microbiota to the examined swatches, laboratory equipment and surfaces from the participants and/or researcher following the mock examinations, highlighting potential contamination issues regarding microbial profiling when using current laboratory SOPs for nuclear DNA recovery, and cleaning.

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RESUMEN

The human microbiome is comprised of the microbes that live on and within an individual, as well as immediately surrounding them. Microbial profiling may have forensic utility in the identification or association of individuals with criminal activities, using microbial signatures derived from a personal microbiome. This review highlights some important aspects of recent studies, many of which have revealed issues involving the effect of contamination of microbial samples from both technical and environmental sources and their impacts on microbiome research and the potential forensic applications of microbial profiling. It is imperative that these challenges be discussed and evaluated within a forensic context to better understand the future directions and potential applications of microbial profiling for human identification. It is necessary that the limitations identified be resolved prior to the adoption of microbial profiling, or, at a minimum, acknowledged by those applying this new approach.

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RESUMEN

The human microbiome encompasses the fungi, bacteria and viruses that live on, within, and immediately surrounding the body. Microbiomes have potential utility in forensic science as an evidentiary tool to link or exclude persons of interest associated with criminal activities. Research has shown the microbiome is individualised, and that personal microbial signatures can be recovered from surfaces such as phones, shoes and fabrics. Before the human microbiome may be used as an investigative tool, further research is required to investigate the utility and potential limitations surrounding microbial profiling. This includes the detectability of microbial transfer between individuals or items, the associated risks (such as contamination events) and the applicability of microbial profiling for forensic purposes. This research aimed to identify whether an individual's distinguishable microbiome could be transferred to another individual and onto substrates, and vice versa. Paper, cotton, and glass surfaces were chosen to represent a range of substrate matrices. The study involved six participants placed into three pairs; participants took part in two modes of transfer. Transfer Mode 1 involved the pair shaking hands, followed by rubbing a substrate in their right hand. Transfer Mode 2 involved individuals rubbing a substrate in their left hand, swapping substrates with their partner and then rubbing the swapped substrate in their left hand. 16S rRNA sequencing was performed on the extracted microbial DNA from participant and substrate samples. Quantitative Insights into Microbial Ecology 2 (QIIME 2) was used for sequence quality control and beta (between-sample) diversity analyses and taxonomic assignment. Principal Coordinate Analysis (PCoA) based on Jaccard distances was visualised through Emperor software to determine the phylogenetic similarity of bacterial communities between participants and among participant pairs. Statistical testing through PERMANOVA revealed significant differences in the Jaccard distances between each participant pair (P < 0.001), highlighting not only the potential distinguishability of skin microbiomes among individuals, but also the clustering effect observed between participant pairs due to the potential transfer of hand-associated microbiomes between individuals. The study demonstrated that transfer of the human skin microbiome had occurred between all participant pairs, regardless of substrate type or mode of transfer.

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