Natural variation in wild tomato trichomes; selecting metabolites that contribute to insect resistance using a random forest approach.

BACKGROUND: Plant-produced specialised metabolites are a powerful part of a plant's first line of defence against herbivorous insects, bacteria and fungi. Wild ancestors of present-day cultivated tomato produce a plethora of acylsugars in their type-I/IV trichomes and volatiles in their type-VI trichomes that have a potential role in plant resistance against insects. However, metabolic profiles are often complex mixtures making identification of the functionally interesting metabolites challenging. Here, we aimed to identify specialised metabolites from a wide range of wild tomato genotypes that could explain resistance to vector insects whitefly (Bemisia tabaci) and Western flower thrips (Frankliniella occidentalis). We evaluated plant resistance, determined trichome density and obtained metabolite profiles of the glandular trichomes by LC-MS (acylsugars) and GC-MS (volatiles). Using a customised Random Forest learning algorithm, we determined the contribution of specific specialised metabolites to the resistance phenotypes observed. RESULTS: The selected wild tomato accessions showed different levels of resistance to both whiteflies and thrips. Accessions resistant to one insect can be susceptible to another. Glandular trichome density is not necessarily a good predictor for plant resistance although the density of type-I/IV trichomes, related to the production of acylsugars, appears to correlate with whitefly resistance. For type VI-trichomes, however, it seems resistance is determined by the specific content of the glands. There is a strong qualitative and quantitative variation in the metabolite profiles between different accessions, even when they are from the same species. Out of 76 acylsugars found, the random forest algorithm linked two acylsugars (S3:15 and S3:21) to whitefly resistance, but none to thrips resistance. Out of 86 volatiles detected, the sesquiterpene α-humulene was linked to whitefly susceptible accessions instead. The algorithm did not link any specific metabolite to resistance against thrips, but monoterpenes α-phellandrene, α-terpinene and ß-phellandrene/D-limonene were significantly associated with susceptible tomato accessions. CONCLUSIONS: Whiteflies and thrips are distinctly targeted by certain specialised metabolites found in wild tomatoes. The machine learning approach presented helped to identify features with efficacy toward the insect species studied. These acylsugar metabolites can be targets for breeding efforts towards the selection of insect-resistant cultivars.

Targeting the Cutaneous Microbiota in Atopic Dermatitis by Coal Tar via AHR-Dependent Induction of Antimicrobial Peptides.

Skin colonization by Staphylococcus aureus and its relative abundance is associated with atopic dermatitis (AD) disease severity and treatment response. Low levels of antimicrobial peptides in AD skin may be related to the microbial dysbiosis. Therapeutic targeting of the skin microbiome and antimicrobial peptide expression can, therefore, restore skin homeostasis and combat AD. In this study, we analyzed the cutaneous microbiome composition in 7 patients with AD and 10 healthy volunteers upon topical coal tar or vehicle treatment. We implemented and validated a Staphylococcus-specific single-locus sequence typing approach combined with classic 16S ribosomal RNA marker gene sequencing to study the bacterial composition. During coal tar treatment, Staphylococcus abundance decreased, and Propionibacterium abundance increased, suggesting a shift of the microbiota composition toward that of healthy controls. We, furthermore, identified a hitherto unknown therapeutic mode of action of coal tar, namely the induction of keratinocyte-derived antimicrobial peptides via activation of the aryl hydrocarbon receptor. Restoring antimicrobial peptide levels in AD skin via aryl hydrocarbon receptor-dependent transcription regulation can be beneficial by creating a (anti)microbial milieu that is less prone to infection and inflammation. This underscores the importance of coal tar in the therapeutic aryl hydrocarbon receptor armamentarium and highlights the aryl hydrocarbon receptor as a target for drug development.

A generic workflow for Single Locus Sequence Typing (SLST) design and subspecies characterization of microbiota.

We present TaxPhlAn, a new method and bioinformatics pipeline for design and analysis of single-locus sequence typing (SLST) markers to type and profile bacteria beyond the species-level in a complex microbial community background. TaxPhlAn can be applied to any group of phylogenetically-related bacteria, provided reference genomes are available. As TaxPhlAn requires the SLST targets identified to fit the phylogenetic pattern as determined through comprehensive evolutionary reconstruction of input genomes, TaxPhlAn allows for the identification and phylogenetic inference of new biodiversity. Here, we present a clinically relevant case study of high-resolution Staphylococcus profiling on skin of atopic dermatitis (AD) patients. We demonstrate that SLST enables profiling of cutaneous Staphylococcus members at (sub)species level and provides higher resolution than current 16S-based techniques. With the higher discriminative ability provided by our approach, we further show that the presence of Staphylococcus capitis on the skin together with Staphylococcus aureus associates with AD disease.

Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

The intestinal microbiome is perturbed in patients with new-onset and chronic autoimmune inflammatory arthritis. Recent studies in mouse models suggest that development and progression of autoimmune arthritis is highly affected by the intestinal microbiome. This makes modulation of the intestinal microbiota an interesting novel approach to suppress inflammatory arthritis. Prebiotics, defined as non-digestible carbohydrates that selectively stimulate the growth and activity of beneficial microorganisms, provide a relatively non-invasive approach to modulate the intestinal microbiota. The aim of this study was to assess the therapeutic potential of dietary supplementation with a prebiotic mixture of 90% short-chain galacto-oligosaccharides and 10% long-chain fructo-oligosaccharides (scGOS/lcFOS) in experimental arthritis in mice. We here show that dietary supplementation with scGOS/lcFOS has a pronounced effect on the composition of the fecal microbiota. Interestingly, the genera Enterococcus and Clostridium were markedly decreased by scGOS/lcFOS dietary supplementation. In contrast, the family Lachnospiraceae and the genus Lactobacillus, both associated with healthy microbiota, increased in mice receiving scGOS/lcFOS diet. However, the scGOS/lcFOS induced alterations of the intestinal microbiota did not induce significant effects on the intestinal and systemic T helper cell subsets and were not sufficient to reproducibly suppress arthritis in mice. As expected, we did observe a significant increase in the bone mineral density in mice upon dietary supplementation with scGOS/lcFOS for 8 weeks. Altogether, this study suggests that dietary scGOS/lcFOS supplementation is able to promote presumably healthy gut microbiota and improve bone mineral density, but not inflammation, in arthritis-prone mice.

Transcriptional response of Lactococcus lactis during bacterial emulsification.

Microbial surface properties are important for interactions with the environment in which cells reside. Surface properties of lactic acid bacteria significantly vary and some strains can form strong emulsions when mixed with a hydrocarbon. Lactococcus lactis NCDO712 forms oil-in-water emulsions upon mixing of a cell suspension with petroleum. In the emulsion the bacteria locate at the oil-water interphase which is consistent with Pickering stabilization. Cells of strain NCDO712 mixed with sunflower seed oil did not stabilize the oil droplets. This study shows that the addition of either ethanol or ammonium sulfate led to cell aggregation, which subsequently allowed stabilizing oil-in-water emulsions. From this, we conclude that bacterial cell aggregation is important for emulsion droplet stabilization. To determine how bacterial emulsification influences the microbial transcriptome RNAseq analysis was performed on lactococci taken from the oil-water interphase. In comparison to cells in suspension 72 genes were significantly differentially expressed with a more than 4-fold difference. The majority of these genes encode proteins involved in transport processes and the metabolism of amino acids, carbohydrates and ions. Especially the proportion of genes belonging to the CodY regulon was high. Our results also point out that in a complex environment such as food fermentations a heterogeneous response of microbes might be caused by microbe-matrix interactions. In addition, microdroplet technologies are increasingly used in research. The understanding of interactions between bacterial cells and oil-water interphases is of importance for conducting and interpreting such experiments.

Reconstruction and inference of the Lactococcus lactis MG1363 gene co-expression network.

Lactic acid bacteria are Gram-positive bacteria used throughout the world in many industrial applications for their acidification, flavor and texture formation attributes. One of the species, Lactococcus lactis, is employed for the production of fermented milk products like cheese, buttermilk and quark. It ferments lactose to lactic acid and, thus, helps improve the shelf life of the products. Many physiological and transcriptome studies have been performed in L. lactis in order to comprehend and improve its biotechnological assets. Using large amounts of transcriptome data to understand and predict the behavior of biological processes in bacterial or other cell types is a complex task. Gene networks enable predicting gene behavior and function in the context of transcriptionally linked processes. We reconstruct and present the gene co-expression network (GCN) for the most widely studied L. lactis strain, MG1363, using publicly available transcriptome data. Several methods exist to generate and judge the quality of GCNs. Different reconstruction methods lead to networks with varying structural properties, consequently altering gene clusters. We compared the structural properties of the MG1363 GCNs generated by five methods, namely Pearson correlation, Spearman correlation, GeneNet, Weighted Gene Co-expression Network Analysis (WGCNA), and Sparse PArtial Correlation Estimation (SPACE). Using SPACE, we generated an L. lactis MG1363 GCN and assessed its quality using modularity and structural and biological criteria. The L. lactis MG1363 GCN has structural properties similar to those of the gold-standard networks of Escherichia coli K-12 and Bacillus subtilis 168. We showcase that the network can be used to mine for genes with similar expression profiles that are also generally linked to the same biological process.

Comparative Genome Analysis of Lactococcus lactis Indicates Niche Adaptation and Resolves Genotype/Phenotype Disparity.

Lactococcus lactis is one of the most important micro-organisms in the dairy industry for the fermentation of cheese and buttermilk. Besides the conversion of lactose to lactate it is responsible for product properties such as flavor and texture, which are determined by volatile metabolites, proteolytic activity and exopolysaccharide production. While the species Lactococcus lactis consists of the two subspecies lactis and cremoris their taxonomic position is confused by a group of strains that, despite of a cremoris genotype, display a lactis phenotype. Here we compared and analyzed the (draft) genomes of 43 L. lactis strains, of which 19 are of dairy and 24 are of non-dairy origin. Machine-learning algorithms facilitated the identification of orthologous groups of protein sequences (OGs) that are predictors for either the taxonomic position or the source of isolation. This allowed the unambiguous categorization of the genotype/phenotype disparity of ssp. lactis and ssp. cremoris strains. A detailed analysis of phenotypic properties including plasmid-encoded genes indicates evolutionary changes during niche adaptations. The results are consistent with the hypothesis that dairy isolates evolved from plant isolates. The analysis further suggests that genomes of cremoris phenotype strains are so eroded that they are restricted to a dairy environment. Overall the genome comparison of a diverse set of strains allowed the identification of niche and subspecies specific genes. This explains evolutionary relationships and will aid the identification and selection of industrial starter cultures.

TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A3 Receptor-Mediated Signaling.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

The bacterial and fungal microbiome of the skin of healthy dogs and dogs with atopic dermatitis and the impact of topical antimicrobial therapy, an exploratory study.

Canine atopic dermatitis is a genetically predisposed inflammatory and pruritic allergic skin disease that is often complicated by (secondary) bacterial and fungal (yeast) infections. High-throughput DNA sequencing was used to characterize the composition of the microbiome (bacteria and fungi) inhabiting specific sites of skin in healthy dogs and dogs with atopic dermatitis (AD) before and after topical antimicrobial treatment. Skin microbiome samples were collected from six healthy control dogs and three dogs spontaneously affected by AD by swabbing at (non-) predilection sites before, during and after treatment. Bacteria and fungi were profiled by Illumina sequencing of the 16S ribosomal RNA gene of bacteria (16S) and the internally transcribed spacer of the ribosomal gene cassette in fungi (ITS). The total cohort of dogs showed a high diversity of microbes on skin with a strong individual variability of both 16S and ITS profiles. The genera of Staphylococcus and Porphyromonas were dominantly present both on atopic and healthy skin and across all skin sites studied. In addition, bacterial and fungal alpha diversity were similar at the different skin sites. The topical antimicrobial treatment increased the diversity of bacterial and fungal compositions in course of time on both AD and healthy skin.

The Contribution of Genetic Variation of Streptococcus pneumoniae to the Clinical Manifestation of Invasive Pneumococcal Disease.

Background: Different clinical manifestations of invasive pneumococcal disease (IPD) have thus far mainly been explained by patient characteristics. Here we studied the contribution of pneumococcal genetic variation to IPD phenotype. Methods: The index cohort consisted of 349 patients admitted to 2 Dutch hospitals between 2000-2011 with pneumococcal bacteremia. We performed genome-wide association studies to identify pneumococcal lineages, genes, and allelic variants associated with 23 clinical IPD phenotypes. The identified associations were validated in a nationwide (n = 482) and a post-pneumococcal vaccination cohort (n = 121). The contribution of confirmed pneumococcal genotypes to the clinical IPD phenotype, relative to known clinical predictors, was tested by regression analysis. Results: Among IPD patients, the presence of pneumococcal gene slaA was a nationwide confirmed independent predictor of meningitis (odds ratio [OR], 10.5; P = .001), as was sequence cluster 9 (serotype 7F: OR, 3.68; P = .057). A set of 4 pneumococcal genes co-located on a prophage was a confirmed independent predictor of 30-day mortality (OR, 3.4; P = .003). We could detect the pneumococcal variants of concern in these patients' blood samples. Conclusions: In this study, knowledge of pneumococcal genotypic variants improved the clinical risk assessment for detrimental manifestations of IPD. This provides us with novel opportunities to target, anticipate, or avert the pathogenic effects related to particular pneumococcal variants, and indicates that information on pneumococcal genotype is important for the diagnostic and treatment strategy in IPD. Ongoing surveillance is warranted to monitor the clinical value of information on pneumococcal variants in dynamic microbial and susceptible host populations.

Correction: Supplementation of diet with non-digestible oligosaccharides alters the intestinal microbiota, but not arthritis development, in IL-1 receptor antagonist deficient mice.

[This corrects the article DOI: 10.1371/journal.pone.0219366.].

Transcriptome Analysis of a Spray Drying-Resistant Subpopulation Reveals a Zinc-Dependent Mechanism for Robustness in L. lactis SK11.

The viability of starter cultures is essential for an adequate contribution to the fermentation process and end-product. Therefore, robustness during processing and storage is an important characteristic of starter culture strains. For instance, during spray drying cells are exposed to heat and oxidative stress, generally resulting in loss of viability. In this study, we exposed the industrially relevant but stress-sensitive Lactococcus lactis strain SK11 to two cycles of heat stress, with intermediate recovery and cultivation at moderate temperatures. After these two cycles of heat exposure, the abundance of robust derivatives was increased as compared with the original culture, which enabled isolation of heat-resistant subpopulations displaying up to 1,000-fold enhanced heat stress survival. Moreover, this heat-resistant subpopulation demonstrated an increased survival during spray drying. Derivatives from two independent lineages displayed different transcriptome changes as compared with the wild type strain, indicating that the increased robustness within these lineages was established by different adaptive strategies. Nevertheless, an overlap in differential gene expression in all five derivatives tested in both lineages included three genes in an operon involved in zinc transport. The link between zinc homeostasis and heat stress survival in L. lactis was experimentally established by culturing of the wild type strain SK11 in medium with various levels of zinc ions, which resulted in alterations in heat stress survival phenotypes. This study demonstrates that robust derivatives of a relatively sensitive L. lactis strain can be isolated by repeated exposure to heat stress. Moreover, this work demonstrates that transcriptome analysis of these robust derivatives can provide clues for improvement of the robustness of the original strain. This could boost the industrial application of strains with specific desirable traits but inadequate robustness characteristics.

Integrating glycomics and genomics uncovers SLC10A7 as essential factor for bone mineralization by regulating post-Golgi protein transport and glycosylation.

Genomics methodologies have significantly improved elucidation of Mendelian disorders. The combination with high-throughput functional-omics technologies potentiates the identification and confirmation of causative genetic variants, especially in singleton families of recessive inheritance. In a cohort of 99 individuals with abnormal Golgi glycosylation, 47 of which being unsolved, glycomics profiling was performed of total plasma glycoproteins. Combination with whole-exome sequencing in 31 cases revealed a known genetic defect in 15 individuals. To identify additional genetic factors, hierarchical clustering of the plasma glycomics data was done, which indicated a subgroup of four patients that shared a unique glycomics signature of hybrid type N-glycans. In two siblings, compound heterozygous mutations were found in SLC10A7, a gene of unknown function in human. These included a missense mutation that disrupted transmembrane domain 4 and a mutation in a splice acceptor site resulting in skipping of exon 9. The two other individuals showed a complete loss of SLC10A7 mRNA. The patients' phenotype consisted of amelogenesis imperfecta, skeletal dysplasia, and decreased bone mineral density compatible with osteoporosis. The patients' phenotype was mirrored in SLC10A7 deficient zebrafish. Furthermore, alizarin red staining of calcium deposits in zebrafish morphants showed a strong reduction in bone mineralization. Cell biology studies in fibroblasts of affected individuals showed intracellular mislocalization of glycoproteins and a defect in post-Golgi transport of glycoproteins to the cell membrane. In contrast to yeast, human SLC10A7 localized to the Golgi. Our combined data indicate an important role for SLC10A7 in bone mineralization and transport of glycoproteins to the extracellular matrix.

Primary cilia-regulated transcriptome in the renal collecting duct.

Renal tubular cells respond to mechanical stimuli generated by urinary flow to regulate the activity and transcript abundance of important genes for ion handling, cellular homeostasis, and proper renal development. The primary cilium, a mechanosensory organelle, is postulated to regulate this mRNA response. The aim of this study is to reveal the transcriptome changes of tubular epithelia in response to fluid flow and determine the role of primary cilia in this process. Inner-medullary collecting duct (CD) cells were subjected to either static or physiologically relevant fluid flow (∼0.6 dyn/cm2). RNA-sequencing analysis of ciliated cells subjected to fluid flow showed up-regulation of 1379 genes and down-regulation of 1294 genes compared with static control cells. Strikingly, only 54 of these genes were identified as gene candidates sensitive to primary cilia sensing of fluid flow, of which 16 were linked to ion or water transport pathways in the CD. Validation by quantitative real-time PCR revealed that only the expression of transferrin receptor, which is involved in iron transport; and tribbles pseudokinase 3, which is involved in insulin signaling, were unequivocally regulated by primary cilia sensing of fluid flow. This study shows that the involvement of primary cilia in ion transport in the collecting duct is exceptionally specific.-Mohammed, S. G., Arjona, F. J., Verschuren, E. H. J., Bakey, Z., Alkema, W., van Hijum, S., Schmidts, M., Bindels, R. J. M., Hoenderop, J. G. J. Primary cilia-regulated transcriptome in the renal collecting duct.

Haemophilus is overrepresented in the nasopharynx of infants hospitalized with RSV infection and associated with increased viral load and enhanced mucosal CXCL8 responses.

BACKGROUND: While almost all infants are infected with respiratory syncytial virus (RSV) before the age of 2 years, only a small percentage develops severe disease. Previous studies suggest that the nasopharyngeal microbiome affects disease development. We therefore studied the effect of the nasopharyngeal microbiome on viral load and mucosal cytokine responses, two important factors influencing the pathophysiology of RSV disease. To determine the relation between (i) the microbiome of the upper respiratory tract, (ii) viral load, and (iii) host mucosal inflammation during an RSV infection, nasopharyngeal microbiota profiles of RSV infected infants (< 6 months) with different levels of disease severity and age-matched healthy controls were determined by 16S rRNA marker gene sequencing. The viral load was measured using qPCR. Nasopharyngeal CCL5, CXCL10, MMP9, IL6, and CXCL8 levels were determined with ELISA. RESULTS: Viral load in nasopharyngeal aspirates of patients associates significantly to total nasopharyngeal microbiota composition. Healthy infants (n = 21) and RSV patients (n = 54) display very distinct microbial patterns, primarily characterized by a loss in commensals like Veillonella and overrepresentation of opportunistic organisms like Haemophilus and Achromobacter in RSV-infected individuals. Furthermore, nasopharyngeal microbiota profiles are significantly different based on CXCL8 levels. CXCL8 is a chemokine that was previously found to be indicative for disease severity and for which we find Haemophilus abundance as the strongest predictor for CXCL8 levels. CONCLUSIONS: The nasopharyngeal microbiota in young infants with RSV infection is marked by an overrepresentation of the genus Haemophilus. We present that this bacterium is associated with viral load and mucosal CXCL8 responses, both which are involved in RSV disease pathogenesis.

Gut microbiome in ADHD and its relation to neural reward anticipation.

BACKGROUND: Microorganisms in the human intestine (i.e. the gut microbiome) have an increasingly recognized impact on human health, including brain functioning. Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder associated with abnormalities in dopamine neurotransmission and deficits in reward processing and its underlying neuro-circuitry including the ventral striatum. The microbiome might contribute to ADHD etiology via the gut-brain axis. In this pilot study, we investigated potential differences in the microbiome between ADHD cases and undiagnosed controls, as well as its relation to neural reward processing. METHODS: We used 16S rRNA marker gene sequencing (16S) to identify bacterial taxa and their predicted gene functions in 19 ADHD and 77 control participants. Using functional magnetic resonance imaging (fMRI), we interrogated the effect of observed microbiome differences in neural reward responses in a subset of 28 participants, independent of diagnosis. RESULTS: For the first time, we describe gut microbial makeup of adolescents and adults diagnosed with ADHD. We found that the relative abundance of several bacterial taxa differed between cases and controls, albeit marginally significant. A nominal increase in the Bifidobacterium genus was observed in ADHD cases. In a hypothesis-driven approach, we found that the observed increase was linked to significantly enhanced 16S-based predicted bacterial gene functionality encoding cyclohexadienyl dehydratase in cases relative to controls. This enzyme is involved in the synthesis of phenylalanine, a precursor of dopamine. Increased relative abundance of this functionality was significantly associated with decreased ventral striatal fMRI responses during reward anticipation, independent of ADHD diagnosis and age. CONCLUSIONS: Our results show increases in gut microbiome predicted function of dopamine precursor synthesis between ADHD cases and controls. This increase in microbiome function relates to decreased neural responses to reward anticipation. Decreased neural reward anticipation constitutes one of the hallmarks of ADHD.

Genomic and functional analysis of Romboutsia ilealis CRIBT reveals adaptation to the small intestine.

BACKGROUND: The microbiota in the small intestine relies on their capacity to rapidly import and ferment available carbohydrates to survive in a complex and highly competitive ecosystem. Understanding how these communities function requires elucidating the role of its key players, the interactions among them and with their environment/host. METHODS: The genome of the gut bacterium Romboutsia ilealis CRIBT was sequenced with multiple technologies (Illumina paired-end, mate-pair and PacBio). The transcriptome was sequenced (Illumina HiSeq) after growth on three different carbohydrate sources, and short chain fatty acids were measured via HPLC. RESULTS: We present the complete genome of Romboutsia ilealis CRIBT, a natural inhabitant and key player of the small intestine of rats. R. ilealis CRIBT possesses a circular chromosome of 2,581,778 bp and a plasmid of 6,145 bp, carrying 2,351 and eight predicted protein coding sequences, respectively. Analysis of the genome revealed limited capacity to synthesize amino acids and vitamins, whereas multiple and partially redundant pathways for the utilization of different relatively simple carbohydrates are present. Transcriptome analysis allowed identification of the key components in the degradation of glucose, L-fucose and fructo-oligosaccharides. DISCUSSION: This revealed that R. ilealis CRIBT is adapted to a nutrient-rich environment where carbohydrates, amino acids and vitamins are abundantly available.

Whole-Genome Sequencing of Bacterial Pathogens: the Future of Nosocomial Outbreak Analysis.

Outbreaks of multidrug-resistant bacteria present a frequent threat to vulnerable patient populations in hospitals around the world. Intensive care unit (ICU) patients are particularly susceptible to nosocomial infections due to indwelling devices such as intravascular catheters, drains, and intratracheal tubes for mechanical ventilation. The increased vulnerability of infected ICU patients demonstrates the importance of effective outbreak management protocols to be in place. Understanding the transmission of pathogens via genotyping methods is an important tool for outbreak management. Recently, whole-genome sequencing (WGS) of pathogens has become more accessible and affordable as a tool for genotyping. Analysis of the entire pathogen genome via WGS could provide unprecedented resolution in discriminating even highly related lineages of bacteria and revolutionize outbreak analysis in hospitals. Nevertheless, clinicians have long been hesitant to implement WGS in outbreak analyses due to the expensive and cumbersome nature of early sequencing platforms. Recent improvements in sequencing technologies and analysis tools have rapidly increased the output and analysis speed as well as reduced the overall costs of WGS. In this review, we assess the feasibility of WGS technologies and bioinformatics analysis tools for nosocomial outbreak analyses and provide a comparison to conventional outbreak analysis workflows. Moreover, we review advantages and limitations of sequencing technologies and analysis tools and present a real-world example of the implementation of WGS for antimicrobial resistance analysis. We aimed to provide health care professionals with a guide to WGS outbreak analysis that highlights its benefits for hospitals and assists in the transition from conventional to WGS-based outbreak analysis.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis.

BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.