Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1–2 days for identification of bacterial agent and an additional 2–3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10 minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and bla_CTX-M subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes.

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).

Antibiotic resistance poses a major threat to public health. More effective ways of the antibiotic prescription are needed to delay the spread of antibiotic resistance. Employment of sequencing technologies coupled with the use of trained neural network algorithms for genotype-to-phenotype prediction will reduce the time needed for antibiotic susceptibility profile identification from days to hours.

Siderophores are small molecules synthesized and secreted by bacteria and fungi to scavenge iron. Extracellular ferri-siderohores are recognized by cognate receptors on the cell surface for transport over membranes. Several siderophore systems from Vibrionaceae representatives are known and well understood, e.g., the molecular structure of the siderophore, the biosynthesis gene cluster and pathway, and the gene expression pattern. Less is known about how these systems are distributed among the ~140 Vibrionaceae species, and which evolutionary processes contributed to the present-day distribution. In this work, we compiled existing knowledge on siderophore biosynthesis systems and siderophore receptors from Vibrionaceae and used phylogenetic analyses to investigate their organization, distribution, origin and evolution. Through literature searches, we identified nine different siderophore biosynthesis systems and thirteen siderophore receptors in Vibrionaceae. Homologs were identified by BLAST searches, and the results were mapped onto a Vibrionaceae phylogeny. We identified 81 biosynthetic systems distributed in 45 Vibrionaceae species and 16 unclassified Vibrionaceae strains, and 409 receptors in 89 Vibrionaceae species and 49 unclassified Vibrionaceae strains. The majority of taxa are associated with at least one type of siderophore biosynthesis system, some (e.g., aerobactin and vibrioferrin) of which are widely distributed in the family, whereas others (i.e., bisucaberin and vibriobactin) are found in one lineage. Cognate receptors are found more widespread. Phylogenetic analysis of three siderophore systems (piscibactin, vibrioferrin and aerobactin) show that their present-day distribution can be explained by an old insertion into Vibrionaceae, followed mainly by stable vertical evolution and extensive loss, and some cases of horizontal gene transfers. The present work provides an up to date overview of the distribution of siderophore-based iron acquisition systems in Vibrionaceae, and presents phylogenetic analysis of these systems. Our results suggest that the present-day distribution is a result of several evolutionary processes, such as old and new gene acquisitions, gene loss, and both vertical and horizontal gene transfers.

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.