Evaluating The Resistome And Microbial Composition During Food Waste Feeding And Composting On A Vermont Poultry Farm

While commonly thought of as a waste product, food scraps and residuals represent an important opportunity for energy and nutrient recapture within the food system. As demands on production continue to increase, conservation of these valuable resources has become a priority area. In the wake of new legislation in Vermont, Act 148, the Universal Recycling Law, the fate of microbial species in food waste, scraps and residuals is increasingly important. The presence of antimicrobial resistance genes in all types of foods calls for an increased need to estimate risk of antibiotic resistance transfer and maintenance across all segments of food production and distribution systems, from farm to fork. Specifically, the fate of antibiotic resistance genes (ARGs) in these co-mingled food wastes has not been sufficiently characterized; as legislative programs increase in popularity, surveillance of these materials is pressing and should be documented to assess the risk and potential measures for mitigation and management as we approach commercial scales of implementation Previous studies have relied on a combination of targeted techniques, such as 16S rRNA sequencing and qPCR on a specific subset of ARGs; however, these may not cover the full extent of resistance or microorganisms of concern in any given sample. As sequencing technologies improve and costs continue to drop, more comprehensive tools, such as shotgun metagenomic sequencing, can be applied to these problems for both surveillance and novel gene discovery. In this study, we leveraged the increased screening power of the Illumina HiSeq and shotgun metagenomic sequencing to identify and characterize ARGs, microbial communities, and associated virulence factors of food scraps, on-farm composts, and several consumer products. Isolates were also screened for antibiotic resistance to demonstrate the functionality of ARGs identified. The resistome, microbiome, and virulence genes were characterized in all samples. Fifty unique ARGs were identified that spanned 8 major drug classes. Most frequently found were genes related to aminoglycoside, macrolide, and tetracycline resistance. Additionally, 54 distinct virulence factors and 495 bacterial species were identified. Virulence factors were present across the farm setting and mainly included gene transfer mechanisms, while bacteria clustered distinctly into site and farm, as well as separate on farm niches. The relationship between these categories was also assessed by both Pearson correlation and co-inertia analysis, with the most significant relationship being between ARGs and virulence factors (P = 0.05, RV = 0.67). While limited in this study, these patterns reinforce the finding that spread of antibiotic resistance genes may be dependent on the virulence factors present enabling transfer, rather than total microbial community composition

High prevalence of Phasi Charoen-like virus from wild-caught Aedes aegypti in Grenada, W.I. as revealed by metagenomic analysis.

Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity

Metagenomic analysis of Aedes aegypti and Culex quinquefasciatus mosquitoes from Grenada, West Indies.

The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus

Human alveolar hydrogels promote morphological and transcriptional differentiation in iPSC-derived alveolar type 2 epithelial cells

Abstract Alveolar type 2 epithelial cells (AT2s) derived from human induced pluripotent stem cells (iAT2s) have rapidly contributed to our understanding of AT2 function and disease. However, while iAT2s are primarily cultured in three-dimensional (3D) Matrigel, a matrix derived from cancerous mouse tissue, it is unclear how a physiologically relevant matrix will impact iAT2s phenotype. As extracellular matrix (ECM) is recognized as a vital component in directing cellular function and differentiation, we sought to derive hydrogels from decellularized human lung alveolar-enriched ECM (aECM) to provide an ex vivo model to characterize the role of physiologically relevant ECM on iAT2 phenotype. We demonstrate aECM hydrogels retain critical in situ ECM components, including structural and basement membrane proteins. While aECM hydrogels facilitate iAT2 proliferation and alveolosphere formation, a subset of iAT2s rapidly change morphology to thin and elongated ring-like cells. This morphological change correlates with upregulation of recently described iAT2-derived transitional cell state genetic markers. As such, we demonstrate a potentially underappreciated role of physiologically relevant aECM in iAT2 differentiation

Additional file 10 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 10: Tables S1-S36. Genomic and metabolomic raw data and analyses, metadata and primer sets used in this study

Additional file 2 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 2: Figure S2. Expression levels of putative aromatic amino acid aminotransferase (ArAT) loci identified in the L. reuteri genome. (A) Pathway schematic of bacterial and abbreviated mammalian tryptophan metabolism from Fig. 2A. Enzymes with genomic evidence in Lactobacillus isolates are boxed in orange (ArAT), blue (FldH) and yellow (AmiE). (B) Heatmap of bacterial tryptophan specific enzymes with genomic evidence in Lactobacillus isolates. Enzymes are listed along the left in corresponding colors to the pathway in (A) with isolates and representative strains of the same species along the top and warmer colors indicating increasing copy number. (C) Expression level of araT loci in L. reuteri following 4 or 24hrs (D) of monoculture with 0 or 1mM tryptophan supplementation in brain heart infusion (BHI) medium as measured by qRT-PCR. Data is organized top to bottom corresponding to the heatmap in (B). Cultures were performed in triplicate and expression levels are normalized to a pan-Eubacterial primer set against the 16S rRNA gene. Primer sets are available in Table S35

Additional file 8 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 8: Figure S8. L. reuteri metabolites are ligands for the aryl hydrocarbon receptor. Selected metabolites identified in L. reuteri monoculture or serum by UPLC-MS/MS were analyzed in a cell-based luciferase assay for capacity to activate or inhibit the AhR at 100μM, 10μM, 1μM, and 0.1μM with or without 4hr pre-treatment with the AhR antagonist, CH223191 at 10μM. Transfection and DMSO vehicle controls are included for comparison

Additional file 7 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 7: Figure S7. The abundance of p-cresol sulfate and p-cresol glucuronide correlate with disease severity. Serum was collected from mice fed a low or high tryptophan diet colonized with either the B6 or B6+L. reuteri microbiome following a 30-day EAE course and analyzed via UPLC-MS/MS as outlined in (Fig. 4A and B). Data were analyzed for all four experimental groups: B6 or B6+L. reuteri colonized mice randomized to a low 0.02% or 0.8% high tryptophan diet. (A) Top 25 metabolites correlating with disease severity as measured by cumulative disease score (CDS), the sum of all daily scores over a 30-day disease course. (B) X-Y scatter plot of p-cresol sulfate and p-cresol glucuronide abundance and CDS using linear regression, with the P value indicating significant deviation from a non-zero slope (i.e. significant correlation). A zero value was entered for p-cresol glucuronide abundance in low-tryptophan-fed mice when below the limit of detection. Statistical analysis is provided in Table S31

Additional file 1 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 1: Figure S1. Full radial phylogenetic trees of Lactobacillus isolates and nearest subspecies neighbors. Extended taxonomic data comparing duplicate isolate draft genomes for L. reuteri (A-B), L. murinus (C-D) and L. johnsonii (E-F). Nearest phylogenetic neighbors, including subspecies neighbors, of each Lactobacillus draft genome determined by average amino acid identify (AAI) percent shared genomic content are represented as phylogenetic trees. Color gradients denote percent conserved average nucleotide identity (ANI) between each isolate and respective nearest subspecies phylogenetic neighbors

Additional file 5 of Lactobacillus reuteri tryptophan metabolism promotes host susceptibility to CNS autoimmunity

Additional file 5: Figure S5. Abundance dynamics of L. reuteri in experimental breeding pairs and within dietary intervention studies. Founder G0 B6-GF mice were inoculated with cryopreserved donor cecal contents from PWD, B6, or B6 cecal contents supplemented with 109 CFU of L. reuteri and breeding pairs were established for vertical transmission to G1 offspring. Fecal samples were collected at 4-wks post-inoculation and in experimental offspring prior to dietary intervention (pre-diet), following 1-week of randomized diets (naive) and following a full 30-day disease course (post-EAE). Abundance of L. reuteri and L. murinus was determined by qPCR using species specific primers in (A) G0 breeders and G1 offspring and (B) throughout the course of dietary intervention. Relative abundance of L. reuteri at day 30 post EAE induction was correlated with disease severity as measured by cumulative disease score (CDS) using linear regression with the P value indicating significant deviation from a non-zero slop (i.e. significant correlation) for pre-diet (C), naive (D), and post-EAE (E) samples