Figure 1 New sequencing technologies in combination with further omics approaches and improved wet-lab and bioinformatics development advance microbial characterization and molecular surveillance of samples taken from various environments.Our journey into the era of new sequencing technologies begins with the monitoring of microbial communities in environmental samples. Studies focus on the detection of unknown microbes, the survey of the global distribution of known taxa, and the causes and implications of the composition of certain microbial communities. As an example, Ríos-Castro et al. use DNA metabarcoding to analyze environmental samples from sediment and marine water samples of a marine ecosystem. They identified pathogens with high relevance for the aquacultural sector and which are of medical and veterinary interest. This study demonstrates the potential of NGS technology for environmental monitoring. Although 16S high-throughput sequencing is widely used worldwide, most protocols do not take into account the variable biomass of microbes, which affects the relative bacterial abundances per sample. Here, Cruz et al. describe a novel equivolumetric protocol that generates library sizes proportional to the total microbial load in 16S rRNA amplicon sequencing. Therefore, the authors introduced 16S amplicon sequencing with short reads in combination with Bayesian cumulative probability models to address this issue and to ultimately achieve higher proportionality between library sizes and total microbial load.Current NGS technology allows for in-depth characterization and molecular surveillance of certain bacterial pathogens. The authors of four studies in the research topic utilized different technologies to characterize outbreaks of these pathogens via bioinformatic approaches comprising pangenome comparisons, Multilocus Sequence Typing (MLST), Genomewide Association Studies (GWAS), and core-genome based phylogenies. All four studies involve at least short-read based Whole-Genome Sequencing (WGS) but also highlight the advantages and challenges of integrating data derived from technologies such as Oxford Nanopore longread sequences (ONT). In the first study, Karthik et al. performed comparative genomics and WGS short-read sequencing of Indian isolates of Brucella melitensis, a zoonotic pathogen prevalent worldwide. Via pangenome analysis and MLST, the authors were able to predict several virulence genes and prophage regions and they were able to phylogenetically resolve different lineages of this species within India, where many isolates have been previously reported. The decreasing costs of NGS, open-source bioinformatics tools, and more easily operated laboratory technology make this approach more globally accessible for monitoring (Khan et al.; Holzer et al.). In another study, Parra-Flores et al. were able to give a detailed profile of virulence and antimicrobial resistance (AMR) proteins of Cronobacter sakazakii strains by adding MALDI-TOF-MS data to WGS data. The strains were isolated from powdered and dairy formulas and showed diverse virulence factors as well as resistance to beta-lactam antibiotics both in silico and in vitro. Their results reinforce a governmental decision to recall all involved powdered and dairy formulas in Chile in 2017 and further highlight the importance of continued molecular surveillance to detect product contamination by this pathogen. A third study deals with a foodborne pathogen, Listeria monocytogenes. Here, Chiaverini et al. performed phylogenetic analysis and GWAS on WGS short-read data to describe an outbreak due to this pathogen in Central Italy. Besides they were not able to identify any genomic differences between the outbreak and a reemergent strain, they found a possible differentiation by transcriptional factors and phage genes. Phages also play an important role in the dissemination of AMR genes of an unusual chimeric methicillin-resistant Staphylococcus aureus (MRSA) strain, as presented by Burgold-Voigt et al. In this study, the authors combined short-read WGS with long-read ONT sequencing to perform hybrid-assembly of an MRSA strain and its bacteriophages. By combining different sequencing technologies, they were able to shed light on several interesting features and the evolution of S. aureus.Due to the novelty and rapid development of molecular technologies, there are still many challenges in data acquisition, standardization, and reproducibility; especially concerning downstream bioinformatics analysis (Hodcroft et al.). This represents a challenge for wet-lab scientists, who are not trained in bioinformatics and need to process a huge amount of samples. Brandt et al. present an option to tackle this problem with an easy-to-use, fast, and robust workflow for the reconstruction of SARS-CoV-2 genomes, namely poreCov. The study highlights the importance of integrating bioinformatics software into reproducible pipelines using state-of-the-art workflow management systems and containers to finally bring new technologies into production and to allow the usage by non-experts (Hodcroft et al.).Within this research topic, we have assembled a diverse portfolio of studies on the molecular surveillance of bacteria, eukaryotic microbes, and viruses; analyzed using a variety of technologies and computational approaches. The results of these studies could impact important areas such as the development of new vaccines, government decisions on molecular surveillance of pathogens in food, control of pathogens in livestock, and methods for tracking outbreaks. In addition, we are particularly pleased to have attracted studies from researchers around the world: India, Chile, Italy, Spain, Germany, and Brazil; and we are grateful to all authors and reviewers for their contribution to this research topic.
