Mechanisms of Persistence of the Ammonia-Oxidizing Bacteria Nitrosomonas to the Biocide Free Nitrous Acid.

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

Transcriptomics and functional genomics of ROS-induced cell death regulation by RADICAL-INDUCED CELL DEATH1.

Plant responses to changes in environmental conditions are mediated by a network of signaling events leading to downstream responses, including changes in gene expression and activation of cell death programs. Arabidopsis thaliana RADICAL-INDUCED CELL DEATH1 (RCD1) has been proposed to regulate plant stress responses by protein-protein interactions with transcription factors. Furthermore, the rcd1 mutant has defective control of cell death in response to apoplastic reactive oxygen species (ROS). Combining transcriptomic and functional genomics approaches we first used microarray analysis in a time series to study changes in gene expression after apoplastic ROS treatment in rcd1. To identify a core set of cell death regulated genes, RCD1-regulated genes were clustered together with other array experiments from plants undergoing cell death or treated with various pathogens, plant hormones or other chemicals. Subsequently, selected rcd1 double mutants were constructed to further define the genetic requirements for the execution of apoplastic ROS induced cell death. Through the genetic analysis we identified WRKY70 and SGT1b as cell death regulators functioning downstream of RCD1 and show that quantitative rather than qualitative differences in gene expression related to cell death appeared to better explain the outcome. Allocation of plant energy to defenses diverts resources from growth. Recently, a plant response termed stress-induced morphogenic response (SIMR) was proposed to regulate the balance between defense and growth. Using a rcd1 double mutant collection we show that SIMR is mostly independent of the classical plant defense signaling pathways and that the redox balance is involved in development of SIMR.

A Drug Repurposing Pipeline Based on Bladder Cancer Integrated Proteotranscriptomics Signatures.

Delivering better care for patients with bladder cancer (BC) necessitates the development of novel therapeutic strategies that address both the high disease heterogeneity and the limitations of the current therapeutic modalities, such as drug low efficacy and patient resistance acquisition. Drug repurposing is a cost-effective strategy that targets the reuse of existing drugs for new therapeutic purposes. Such a strategy could open new avenues toward more effective BC treatment. BC patients' multi-omics signatures can be used to guide the investigation of existing drugs that show an effective therapeutic potential through drug repurposing. In this book chapter, we present an integrated multilayer approach that includes cross-omics analyses from publicly available transcriptomics and proteomics data derived from BC tissues and cell lines that were investigated for the development of disease-specific signatures. These signatures are subsequently used as input for a signature-based repurposing approach using the Connectivity Map (CMap) tool. We further explain the steps that may be followed to identify and select existing drugs of increased potential for repurposing in BC patients.

Challenges, Strategies, and Perspectives for Reference-Independent Longitudinal Multi-Omic Microbiome Studies.

In recent years, multi-omic studies have enabled resolving community structure and interrogating community function of microbial communities. Simultaneous generation of metagenomic, metatranscriptomic, metaproteomic, and (meta) metabolomic data is more feasible than ever before, thus enabling in-depth assessment of community structure, function, and phenotype, thus resulting in a multitude of multi-omic microbiome datasets and the development of innovative methods to integrate and interrogate those multi-omic datasets. Specifically, the application of reference-independent approaches provides opportunities in identifying novel organisms and functions. At present, most of these large-scale multi-omic datasets stem from spatial sampling (e.g., water/soil microbiomes at several depths, microbiomes in/on different parts of the human anatomy) or case-control studies (e.g., cohorts of human microbiomes). We believe that longitudinal multi-omic microbiome datasets are the logical next step in microbiome studies due to their characteristic advantages in providing a better understanding of community dynamics, including: observation of trends, inference of causality, and ultimately, prediction of community behavior. Furthermore, the acquisition of complementary host-derived omics, environmental measurements, and suitable metadata will further enhance the aforementioned advantages of longitudinal data, which will serve as the basis to resolve drivers of community structure and function to understand the biotic and abiotic factors governing communities and specific populations. Carefully setup future experiments hold great potential to further unveil ecological mechanisms to evolution, microbe-microbe interactions, or microbe-host interactions. In this article, we discuss the challenges, emerging strategies, and best-practices applicable to longitudinal microbiome studies ranging from sampling, biomolecular extraction, systematic multi-omic measurements, reference-independent data integration, modeling, and validation.

Roles of bacteriophages, plasmids and CRISPR immunity in microbial community dynamics revealed using time-series integrated meta-omics.

Viruses and plasmids (invasive mobile genetic elements (iMGEs)) have important roles in shaping microbial communities, but their dynamic interactions with CRISPR-based immunity remain unresolved. We analysed generation-resolved iMGE-host dynamics spanning one and a half years in a microbial consortium from a biological wastewater treatment plant using integrated meta-omics. We identified 31 bacterial metagenome-assembled genomes encoding complete CRISPR-Cas systems and their corresponding iMGEs. CRISPR-targeted plasmids outnumbered their bacteriophage counterparts by at least fivefold, highlighting the importance of CRISPR-mediated defence against plasmids. Linear modelling of our time-series data revealed that the variation in plasmid abundance over time explained more of the observed community dynamics than phages. Community-scale CRISPR-based plasmid-host and phage-host interaction networks revealed an increase in CRISPR-mediated interactions coinciding with a decrease in the dominant 'Candidatus Microthrix parvicella' population. Protospacers were enriched in sequences targeting genes involved in the transmission of iMGEs. Understanding the factors shaping the fitness of specific populations is necessary to devise control strategies for undesirable species and to predict or explain community-wide phenotypes.

Genomic Sequencing Capacity, Data Retention, and Personal Access to Raw Data in Europe.

Whole genome/exome sequencing (WGS/WES) has become widely adopted in research and, more recently, in clinical settings. Many hope that the information obtained from the interpretation of these data will have medical benefits for patients and-in some cases-also their biological relatives. Because of the manifold possibilities to reuse genomic data, enabling sequenced individuals to access their own raw (uninterpreted) genomic data is a highly debated issue. This paper reports some of the first empirical findings on personal genome access policies and practices. We interviewed 39 respondents, working at 33 institutions in 21 countries across Europe. These sequencing institutions generate massive amounts of WGS/WES data and represent varying organisational structures and operational models. Taken together, in total, these institutions have sequenced ∼317,259 genomes and exomes to date. Most of the sequencing institutions reported that they are able to store raw genomic data in compliance with various national regulations, although there was a lack of standardisation of storage formats. Interviewees from 12 of the 33 institutions included in our study reported that they had received requests for personal access to raw genomic data from sequenced individuals. In the absence of policies on how to process such requests, these were decided on an ad hoc basis; in the end, at least 28 requests were granted, while there were no reports of requests being rejected. Given the rights, interests, and liabilities at stake, it is essential that sequencing institutions adopt clear policies and processes for raw genomic data retention and personal access.

Integration of time-series meta-omics data reveals how microbial ecosystems respond to disturbance.

The development of reliable, mixed-culture biotechnological processes hinges on understanding how microbial ecosystems respond to disturbances. Here we reveal extensive phenotypic plasticity and niche complementarity in oleaginous microbial populations from a biological wastewater treatment plant. We perform meta-omics analyses (metagenomics, metatranscriptomics, metaproteomics and metabolomics) on in situ samples over 14 months at weekly intervals. Based on 1,364 de novo metagenome-assembled genomes, we uncover four distinct fundamental niche types. Throughout the time-series, we observe a major, transient shift in community structure, coinciding with substrate availability changes. Functional omics data reveals extensive variation in gene expression and substrate usage amongst community members. Ex situ bioreactor experiments confirm that responses occur within five hours of a pulse disturbance, demonstrating rapid adaptation by specific populations. Our results show that community resistance and resilience are a function of phenotypic plasticity and niche complementarity, and set the foundation for future ecological engineering efforts.

Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential.

The rate of caesarean section delivery (CSD) is increasing worldwide. It remains unclear whether disruption of mother-to-neonate transmission of microbiota through CSD occurs and whether it affects human physiology. Here we perform metagenomic analysis of earliest gut microbial community structures and functions. We identify differences in encoded functions between microbiomes of vaginally delivered (VD) and CSD neonates. Several functional pathways are over-represented in VD neonates, including lipopolysaccharide (LPS) biosynthesis. We link these enriched functions to individual-specific strains, which are transmitted from mothers to neonates in case of VD. The stimulation of primary human immune cells with LPS isolated from early stool samples of VD neonates results in higher levels of tumour necrosis factor (TNF-α) and interleukin 18 (IL-18). Accordingly, the observed levels of TNF-α and IL-18 in neonatal blood plasma are higher after VD. Taken together, our results support that CSD disrupts mother-to-neonate transmission of specific microbial strains, linked functional repertoires and immune-stimulatory potential during a critical window for neonatal immune system priming.

Colonization and Succession within the Human Gut Microbiome by Archaea, Bacteria, and Microeukaryotes during the First Year of Life.

Perturbations to the colonization process of the human gastrointestinal tract have been suggested to result in adverse health effects later in life. Although much research has been performed on bacterial colonization and succession, much less is known about the other two domains of life, archaea, and eukaryotes. Here we describe colonization and succession by bacteria, archaea and microeukaryotes during the first year of life (samples collected around days 1, 3, 5, 28, 150, and 365) within the gastrointestinal tract of infants delivered either vaginally or by cesarean section and using a combination of quantitative real-time PCR as well as 16S and 18S rRNA gene amplicon sequencing. Sequences from organisms belonging to all three domains of life were detectable in all of the collected meconium samples. The microeukaryotic community composition fluctuated strongly over time and early diversification was delayed in infants receiving formula milk. Cesarean section-delivered (CSD) infants experienced a delay in colonization and succession, which was observed for all three domains of life. Shifts in prokaryotic succession in CSD infants compared to vaginally delivered (VD) infants were apparent as early as days 3 and 5, which were characterized by increased relative abundances of the genera Streptococcus and Staphylococcus, and a decrease in relative abundance for the genera Bifidobacterium and Bacteroides. Generally, a depletion in Bacteroidetes was detected as early as day 5 postpartum in CSD infants, causing a significantly increased Firmicutes/Bacteroidetes ratio between days 5 and 150 when compared to VD infants. Although the delivery mode appeared to have the strongest influence on differences between the infants, other factors such as a younger gestational age or maternal antibiotics intake likely contributed to the observed patterns as well. Our findings complement previous observations of a delay in colonization and succession of CSD infants, which affects not only bacteria but also archaea and microeukaryotes. This further highlights the need for resolving bacterial, archaeal, and microeukaryotic dynamics in future longitudinal studies of microbial colonization and succession within the neonatal gastrointestinal tract.

Integrated meta-omic analyses of the gastrointestinal tract microbiome in patients undergoing allogeneic hematopoietic stem cell transplantation.

In patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), treatment-induced changes to the gastrointestinal tract (GIT) microbiome have been linked to adverse outcomes, most notably graft-versus-host disease (GvHD). However, it is presently unknown whether this relationship is causal or consequential. Here, we performed an integrated meta-omic analysis to probe deeper into the GIT microbiome changes during allo-HSCT and its accompanying treatments. We used 16S and 18S rRNA gene amplicon sequencing to resolve archaea, bacteria, and eukaryotes within the GIT microbiomes of 16 patients undergoing allo-HSCT for the treatment of hematologic malignancies. These results revealed a major shift in the GIT microbiome after allo-HSCT including a marked reduction in bacterial diversity, accompanied by only limited changes in eukaryotes and archaea. An integrated analysis of metagenomic and metatranscriptomic data was performed on samples collected from a patient before and after allo-HSCT for acute myeloid leukemia. This patient developed severe GvHD, leading to death 9 months after allo-HSCT. In addition to drastically decreased bacterial diversity, the post-treatment microbiome showed a higher overall number and higher expression levels of antibiotic resistance genes (ARGs). One specific Escherichia coli strain causing a paravertebral abscess was linked to GIT dysbiosis, suggesting loss of intestinal barrier integrity. The apparent selection for bacteria expressing ARGs suggests that prophylactic antibiotic administration may adversely affect the overall treatment outcome. We therefore assert that such analyses including information about the selection of pathogenic bacteria expressing ARGs may assist clinicians in "personalizing" regimens for individual patients to improve overall outcomes.