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Abstract Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) is the leading cause of childhood chronic kidney failure and a significant cause of chronic kidney disease in adults. Genetic and environmental factors are known to influence CAKUT development, but the currently known disease mechanism remains incomplete. Our goal is to identify affected pathways and networks in CAKUT, and thereby aid in getting a better understanding of its pathophysiology. With this goal, the miRNome, peptidome, and proteome of over 30 amniotic fluid samples of patients with non-severe CAKUT was compared to patients with severe CAKUT. These omics data sets were made findable, accessible, interoperable, and reusable (FAIR) to facilitate their integration with external data resources. Furthermore, we analysed and integrated the omics data sets using three different bioinformatics strategies: integrative analysis with mixOmics, joint dimensionality reduction and pathway analysis. The three bioinformatics analyses provided complementary features, but all pointed towards an important role for collagen in CAKUT development and the PI3K-AKT signalling pathway. Additionally, several key genes (CSF1, IGF2, ITGB1, and RAC1) and microRNAs were identified. We published the three analysis strategies as containerized workflows. These workflows can be applied to other FAIR data sets and help gaining knowledge on other rare diseases.

Abstract Background The potential pathogenic role of Stenotrophomonas maltophilia in lung disease and in particular in cystic fibrosis is unclear. To develop further understanding of the biology of this taxa, the taxonomic position, antibiotic resistance and virulence factors of S. maltophilia isolates from patients with chronic lung disease were studied. Results A total of 111 isolates recovered between 2003 and 2016 from respiratory samples from patients in five different countries were included. Based on a cut-off of 95%, analysis of average nucleotide identity by BLAST (ANIb) showed that the 111 isolates identified as S. maltophilia by Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) belonged to S. maltophilia (n = 65), S. pavanii (n = 6) and 13 putative novel species (n = 40), which each included 1–5 isolates; these groupings coincided with the results of the 16S rDNA analysis, and the L1 and L2 ß-lactamase Neighbor-Joining phylogeny. Chromosomally encoded aminoglycoside resistance was identified in all S. maltophilia and S. pavani isolates, while acquired antibiotic resistance genes were present in only a few isolates. Nevertheless, phenotypic resistance levels against commonly used antibiotics, determined by standard broth microbroth dilution, were high. Although putative virulence genes were present in all isolates, the percentage of positive isolates varied. The Xps II secretion system responsible for the secretion of the StmPr1–3 proteases was mainly limited to isolates identified as S. maltophilia based on ANIb, but no correlation with phenotypic expression of protease activity was found. The RPF two-component quorum sensing system involved in virulence and antibiotic resistance expression has two main variants with one variant lacking 190 amino acids in the sensing region. Conclusions The putative novel Stenotrophomonas species recovered from patient samples and identified by MALDI-TOF/MS as S. maltophilia, differed from S. maltophilia in resistance and virulence genes, and therefore possibly in pathogenicity. Revision of the Stenotrophomonas taxonomy is needed in order to reliably identify strains within the genus and elucidate the role of the different species in disease.

Advances in omics technologies allow for holistic studies into biological systems. These studies rely on integrative data analysis techniques to obtain a comprehensive view of the dynamics of cellular processes, and molecular mechanisms. Network-based integrative approaches have revolutionized multi-omics analysis by providing the framework to represent interactions between multiple different omics-layers in a graph, which may faithfully reflect the molecular wiring in a cell. Here we review network-based multi-omics/multi-modal integrative analytical approaches. We classify these approaches according to the type of omics data supported, the methods and/or algorithms implemented, their node and/or edge weighting components, and their ability to identify key nodes and subnetworks. We show how these approaches can be used to identify biomarkers, disease subtypes, crosstalk, causality, and molecular drivers of physiological and pathological mechanisms. We provide insight into the most appropriate methods and tools for research questions as showcased around the aetiology and treatment of COVID-19 that can be informed by multi-omics data integration. We conclude with an overview of challenges associated with multi-omics network-based analysis, such as reproducibility, heterogeneity, (biological) interpretability of the results, and we highlight some future directions for network-based integration.

Abstract Objective The Pseudomonas koreensis group bacteria are usually found in soil and are associated with plants. Currently they are poorly described. Here we report on the whole genome sequence of a bacterial isolate from a patient with bronchiectasis that was first identified as P. koreensis, and on its position in the P. koreensis group. Results Strain 16-537536 was isolated from a patient with bronchiectasis from Spain and initially identified by MALDI-TOF as P. koreensis, a member of the Pseudomonas fluorescens complex. However, the average nucleotide identity analysis (ANIb) and whole genome alignments of the draft genome sequence of this strain showed it to be a member of the P. koreensis group of the P. fluorescens complex, but belonging to an undescribed species. In addition, based on ANIb analysis, the P. koreensis group contains several other unnamed species. Several genes for putative virulence factors were identified. The only antibiotic resistance gene present in strain 16-537536 was a class C β-lactamase. The correct identification of bacterial species from patients is of utmost importance in order to understand their pathogenesis and to track the potential spread of pathogens between patients. Whole genome sequence data should be included for the description of new species.

The role of Achromobacter species in lung disease remains unclear. The aim of this study was to characterize Achromobacter isolated from persons with cystic fibrosis and from other clinical samples. Whole genome sequences from 101 Achromobacter isolates were determined (81 from patients with cystic fibrosis and 20 from other patients) and analysed. Taxonomic analysis showed nine species including two putative novel species. Thirty-five novel sequence types were present. The most active agent was co-trimoxazole followed by imipenem, but Minimal Inhibitory Concentrations (MICs) were high. Acquired antibiotic resistance genes were rare. Their presence did not correlate with minimal inhibitory concentrations suggesting that other mechanisms are involved. Genes for proposed virulence factors were present in only some isolates. Two putative novel species were identified. The putative virulence properties of Achromobacter involved in infections are variable. Despite the high MICs, acquired resistance genes are uncommon.

Abstract Background The Gram-positive bacterium Enterococcus faecium is a commensal of the human gastrointestinal tract and a frequent cause of bloodstream infections in hospitalized patients. The mechanisms by which E. faecium can survive and grow in blood during an infection have not yet been characterized. Here, we identify genes that contribute to growth of E. faecium in human serum through transcriptome profiling (RNA-seq) and a high-throughput transposon mutant library sequencing approach (Tn-seq). Results We first sequenced the genome of E. faecium E745, a vancomycin-resistant clinical isolate, using a combination of short- and long read sequencing, revealing a 2,765,010 nt chromosome and 6 plasmids, with sizes ranging between 9.3 kbp and 223.7 kbp. We then compared the transcriptome of E. faecium E745 during exponential growth in rich medium and in human serum by RNA-seq. This analysis revealed that 27.8% of genes on the E. faecium E745 genome were differentially expressed in these two conditions. A gene cluster with a role in purine biosynthesis was among the most upregulated genes in E. faecium E745 upon growth in serum. The E. faecium E745 transposon mutant library was then used to identify genes that were specifically required for growth of E. faecium in serum. Genes involved in de novo nucleotide biosynthesis (including pyrK_2, pyrF, purD, purH) and a gene encoding a phosphotransferase system subunit (manY_2) were thus identified to be contributing to E. faecium growth in human serum. Transposon mutants in pyrK_2, pyrF, purD, purH and manY_2 were isolated from the library and their impaired growth in human serum was confirmed. In addition, the pyrK_2 and manY_2 mutants were tested for their virulence in an intravenous zebrafish infection model and exhibited significantly attenuated virulence compared to E. faecium E745. Conclusions Genes involved in carbohydrate metabolism and nucleotide biosynthesis of E. faecium are essential for growth in human serum and contribute to the pathogenesis of this organism. These genes may serve as targets for the development of novel anti-infectives for the treatment of E. faecium bloodstream infections.

Abstract Background The gut microbiota is a reservoir of opportunistic pathogens that can cause life-threatening infections in critically ill patients during their stay in an intensive care unit (ICU). To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed “selective decontamination of the digestive tract” (SDD), is used in some countries where it improves clinical outcome in ICU patients. Yet, the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (“the resistome”) of ICU patients during SDD and of healthy subjects. Results From ten patients that were acutely admitted to the ICU, 30 fecal samples were collected during ICU stay. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. Feces from ten healthy subjects were collected twice, with a 1-year interval. Gut microbiota and resistome composition were determined using 16S rRNA gene phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by lower microbial diversity, decreased levels of Escherichia coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes (aac(6′)-Ii, ermC, qacA, tetQ), providing resistance to aminoglycosides, macrolides, disinfectants, and tetracyclines, respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene (catA) and a tetracycline resistance gene (tetW) were more abundant in healthy subjects. Conclusions The gut microbiota of SDD-treated ICU patients deviated strongly from the gut microbiota of healthy subjects. The negative effects on the resistome were limited to selection for four resistance genes. While it was not possible to disentangle the effects of SDD from confounding variables in the patient cohort, our data suggest that the risks associated with ICU hospitalization and SDD on selection for antibiotic resistance are limited. However, we found evidence indicating that recolonization of the gut by antibiotic-resistant bacteria may occur upon ICU discharge and cessation of SDD.

ABSTRACT The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca2+ levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks. IMPORTANCE Infections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the antibiotics used in hospitals. During antibiotic treatment, the normal bacteria are replaced by resistant enterococci within the gut, from which they can spread and cause infections. We studied antibiotic-mediated intestinal proliferation of multidrug-resistant Enterococcus faecium and the effects on intestinal architecture. We demonstrated that antibiotics allow proliferation of E. faecium in the gut, alter the mucus-associated gut bacterial layer, and reduce the colon wall, mucus thickness, and amount of Muc-2 protein. E. faecium is agglutinated in the intestine in a matrix consisting of host molecules. We hypothesize that this matrix maintains a segregation of E. faecium from the epithelium. Understanding the processes that occur in the gut during antibiotic treatment may provide clues for future mucosal vaccination strategies to control E. faecium or other multidrug-resistant opportunistic pathogens, thereby preventing infections, hospital transmission, and outbreaks.