Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).

Emerging new sequencing technologies have provided researchers with a unique opportunity to study factors related to microbial pathogenicity, such as antimicrobial resistance (AMR) genes and virulence factors. However, the use of whole-genome sequence (WGS) data requires good knowledge of the bioinformatics involved, as well as the necessary techniques. In this study, a total of nine Escherichia coli and Klebsiella pneumoniae isolates from Norwegian clinical samples were sequenced using both MinION and Illumina platforms. Three out of nine samples were sequenced directly from blood culture, and one sample was sequenced from a mixed-blood culture. For genome assembly, several long-read, (Canu, Flye, Unicycler, and Miniasm), short-read (ABySS, Unicycler and SPAdes) and hybrid assemblers (Unicycler, hybridSPAdes, and MaSurCa) were tested. Assembled genomes from the best-performing assemblers (according to quality checks using QUAST and BUSCO) were subjected to downstream analyses. Flye and Unicycler assemblers performed best for the assembly of long and short reads, respectively. For hybrid assembly, Unicycler was the top-performing assembler and produced more circularized and complete genome assemblies. Hybrid assembled genomes performed substantially better in downstream analyses to predict putative plasmids, AMR genes and β-lactamase gene variants, compared to MinION and Illumina assemblies. Thus, hybrid assembly has the potential to reveal factors related to microbial pathogenicity in clinical and mixed samples.

Persistent organic pollutants (POPs) are widespread in the environment and some may be neurotoxic. As we are exposed to complex mixtures of POPs, we aimed to investigate how a POP mixture based on Scandinavian human blood data affects behaviour and neurodevelopment during early life in zebrafish. Embryos/larvae were exposed to a series of sub-lethal doses and behaviour was examined at 96 h post fertilization (hpf). In order to determine the sensitivity window to the POP mixture, exposure models of 6 to 48 and 48 to 96 hpf were used. The expression of genes related to neurological development was also assessed. Results indicate that the POP mixture increases the swimming speed of larval zebrafish following exposure between 48 to 96 hpf. This behavioural effect was associated with the perfluorinated compounds, and more specifically with perfluorooctanesulfonic acid (PFOS). The expression of genes related to the stress response, GABAergic, dopaminergic, histaminergic, serotoninergic, cholinergic systems and neuronal maintenance, were altered. However, there was little overlap in those genes that were significantly altered by the POP mixture and PFOS. Our findings show that the POP mixture and PFOS can have a similar effect on behaviour, yet alter the expression of genes relevant to neurological development differently.

Background: Sperm DNA integrity is considered essential for successful transmission of the paternal genome, fertilization and normal embryo development. DNA fragmentation index (DFI, %) has become a key parameter in the swine artificial insemination industry to assess sperm DNA integrity. Recently, in some elite Norwegian Landrace boars (boars with excellent field fertility records), a higher level of sperm DFI has been observed. In order to obtain a better understanding of this, and to study the complexity of sperm DNA integrity, liquid preserved semen samples from elite boars with contrasting DFI levels were examined for protamine deficiency, thiol profile and disulphide bonds. Additionally, the DNA methylation profiles of the samples were determined by reduced representation bisulphite sequencing (RRBS). Results: In this study, different traits related to sperm DNA integrity were investigated (n = 18 ejaculates). Upon liquid storage, the levels of total thiols and disulphide bonds decreased significantly, while the DFI and protamine deficiency level increased significantly. The RRBS results revealed similar global patterns of low methylation from semen samples with different levels of DFI (low, medium and high). Differential methylation analyses indicated that the number of differentially methylated cytosines (DMCs) increased in the low-high compared to the low-medium and the medium-high DFI groups. Annotating the DMCs with gene and CpG features revealed clear differences between DFI groups. In addition, the number of annotated transcription starting sites (TSS) and associated pathways in the low-high comparison was greater than the other two groups. Pathway analysis showed that genes (based on the closest TSS to DMCs) corresponding to low-high DFI comparison were associated with important processes such as membrane function, metabolic cascade and antioxidant defence system. Conclusion: To our knowledge, this is the first study evaluating DNA methylation in boar sperm cells with different levels of DFI. The present study shows that sperm cells with varying levels of DNA fragmentation exhibit similar global methylation, but different site-specific DNA methylation signatures. Moreover, with increasing DNA fragmentation in spermatozoa, there is an increase in the number of potentially affected downstream genes and their respective regulatory pathways.