Broad time-dependent transcriptional activity of metabolic genes of E. coli O104:H4 strain C227/11Φcu in a soil microenvironment at low temperature.

In the current study, metabolic genes and networks that influence the persistence of pathogenic Escherichia coli O104:H4 strain C227/11Φcu in agricultural soil microenvironments at low temperature were investigated. The strain was incubated in alluvial loam (AL) and total RNA was prepared from samples at time point 0, and after 1 and 4 weeks. Differential transcriptomic analysis was performed by RNA sequencing analysis and values obtained at weeks 1 and 4 were compared to those of time point 0. We found differential expression of more than 1500 genes for either time point comparison. The two lists of differentially expressed genes were then subjected to gene set enrichment of Gene Ontology terms. In total, 17 GO gene sets and 3 Pfam domains were found to be enriched after 1 week. After 4 weeks, 17 GO gene sets and 7 Pfam domains were statistically enriched. Especially stress response genes and genes of the primary metabolism were particularly affected at both time points. Genes and gene sets for uptake of carbohydrates, amino acids were strongly upregulated, indicating adjustment to a low nutrient environment. The results of this transcriptome analysis show that persistence of C227/11Φcu in soils is associated with a complex interplay of metabolic networks.

The DNA-Buster: The evaluation of an alternative DNA recovery approach.

Touch DNA recovery techniques can have limitations, as their effectiveness depends on the substrate on which the DNA of a person of interest can be found. In this study, an in-house dry-vacuuming device, the DNA-Buster, was compared to traditional methods for its DNA recovery performance from items typically examined in forensic casework. The aim was to evaluate whether this dry-vacuuming approach can recover DNA efficiently, potentially complementing the well-established recovery strategies. For this, the performances of swabbing, taping, wet- (M-Vac®) and dry-vacuuming (DNA-Buster) were investigated quantitatively and qualitatively for touch DNA deposited on carpet, cotton sweater, stone, tile and wood. For the sweater, both vacuuming methods outperformed the other collection tools quantitatively. While the highest DNA amounts for the carpet were yielded by swabbing and taping, dry-vacuuming was equally good in reaching full DNA profiles, whereas less complete profiles were observed for the M-Vac®. For stone and tile, swabbing was optimal, whereas dry-vacuuming clearly underperformed for these substrates. Taping was the best recovery method for wood. Despite applying single donor DNA after thoroughly cleaning the items, undesired DNA mixtures were detected for all recovery techniques and all substrates. The overall research findings show first that the novel dry-vacuuming method is suited for DNA recovery from textiles. Secondly, they indicate that more attention should be paid to the substrate-collection dependency to ensure best practices in recovering genetic material in a precise, confident and targeted manner from the variety of forensic casework material.

Technical note: Comparison of forensic swabs for intravaginal sampling.

This study compared the currently used swab Prionics ForensiX Evidence Collection Kit with the alternatives Prionics ForensiX Evidence Collection Tube SafeDry and Sarstedt Forensic Swab XL. Volunteers provided intravaginal swabs collected with all swab types at specific time points after unprotected sexual intercourse. Quantifiable DNA, detectability of seminal fluid component (prostate specific antigen, PSA) and spermatozoa were evaluated to find the best-performing swab type. While Sarstedt XL showed significantly higher DNA quantities for sperm cell fractions than ForensiX Kit, the more concise PSA test results clearly favour ForensiX SafeDry. Reassuringly, mostly complete autosomal STR profiles of male components were obtained for sperm cell fractions at all time points and tested swabs. Switching to the higher performing ForensiX SafeDry with improved sampling and processing properties will also benefit victims, medical personnel, and investigators.

Collaborative swab performance comparison and the impact of sampling solution volumes on DNA recovery.

The collection of DNA traces marks the first step determining the success of genetic analysis. This study aimed to identify and validate a suitable alternative to the currently used ForensiX Evidence Collection Kit containing a cardboard box for swab storage. This box has to be folded at the crime scene, which is time-consuming and carries the risk of potential contamination and handling difficulties. A collaboration study involving three police departments and one laboratory for forensic genetics was performed to compare the currently used swab against three challenger swabs: ForensiX SafeDry, Copan 4N6FLOQSwab™ Genetics and Copan 4N6FLOQSwab™ Crime Scene. Mock samples consisted mainly of touch DNA, but also blood, saliva and semen were applied to twelve items with different surfaces. Every organisation contributed with three DNA collectors, whose individual collection efficiencies were investigated. The challenge of preparing homogenous traces, especially touch DNA, was addressed by enhancing hand contact frequency and sampling area. As a further part of the swab comparison study, we describe for the first time the influence of different swabbing solution volumes on the sampling efficiency of the different swabs. The application of touch DNA was also tested for a further swab type, the Sarstedt Forensic Swab, which yielded such low DNA concentrations that it was excluded from the collaboration study. The Copan Genetics and Copan Crime Scene swabs yielded significantly lower DNA concentrations than the currently used ForensiX Evidence Collection Kit and ForensiX SafeDry swab. The inter-individual performance results of the operators revealed significant differences in sampling skills. Comparing different swabbing solution volumes showed higher DNA yields or no significant difference for the ForensiX Evidence Collection Kit and ForensiX SafeDry than the Copan Genetics, depending on the item or trace type swabbed. Our results highlight the importance of validating first-step components that are decisive to the success of DNA typing in the context of specific sampling procedures and laboratory methods. Also, the significance of individuals' securing variations, principally unknown for crime scene investigation and laboratory teams, is emphasised for the first time, offering a practical approach for improving and training DNA collecting activities and ensuring the optimal securing evidence process. These findings increase the knowledge of impacts on DNA collection and, thus, benefit other laboratories and forensic services, particularly when using the same extraction methods.