The complete genome sequence of probiotic Lactobacillus acidophilus ATCC 9224 isolated from sour milk.

We present the complete genome sequence of the probiotic strain Lactobacillus acidophilus ATCC 9224. The genome sequence provides a valuable resource for investigating the phylogenetic evolution of this lineage and conducting comparative genomics with other Lactobacillus strains and species.

Bridging Place-Based Astrobiology Education with Genomics, Including Descriptions of Three Novel Bacterial Species Isolated from Mars Analog Sites of Cultural Relevance.

Democratizing genomic data science, including bioinformatics, can diversify the STEM workforce and may, in turn, bring new perspectives into the space sciences. In this respect, the development of education and research programs that bridge genome science with "place" and world-views specific to a given region are valuable for Indigenous students and educators. Through a multi-institutional collaboration, we developed an ongoing education program and model that includes Illumina and Oxford Nanopore sequencing, free bioinformatic platforms, and teacher training workshops to address our research and education goals through a place-based science education lens. High school students and researchers cultivated, sequenced, assembled, and annotated the genomes of 13 bacteria from Mars analog sites with cultural relevance, 10 of which were novel species. Students, teachers, and community members assisted with the discovery of new, potentially chemolithotrophic bacteria relevant to astrobiology. This joint education-research program also led to the discovery of species from Mars analog sites capable of producing N-acyl homoserine lactones, which are quorum-sensing molecules used in bacterial communication. Whole genome sequencing was completed in high school classrooms, and connected students to funded space research, increased research output, and provided culturally relevant, place-based science education, with participants naming three novel species described here. Students at St. Andrew's School (Honolulu, Hawai'i) proposed the name Bradyrhizobium prioritasuperba for the type strain, BL16AT, of the new species (DSM 112479T = NCTC 14602T). The nonprofit organization Kauluakalana proposed the name Brenneria ulupoensis for the type strain, K61T, of the new species (DSM 116657T = LMG = 33184T), and Hawai'i Baptist Academy students proposed the name Paraflavitalea speifideiaquila for the type strain, BL16ET, of the new species (DSM 112478T = NCTC 14603T).

A contiguous de novo genome assembly of sugar beet EL10 (Beta vulgaris L.).

A contiguous assembly of the inbred 'EL10' sugar beet (Beta vulgaris ssp. vulgaris) genome was constructed using PacBio long-read sequencing, BioNano optical mapping, Hi-C scaffolding, and Illumina short-read error correction. The EL10.1 assembly was 540 Mb, of which 96.2% was contained in nine chromosome-sized pseudomolecules with lengths from 52 to 65 Mb, and 31 contigs with a median size of 282 kb that remained unassembled. Gene annotation incorporating RNA-seq data and curated sequences via the MAKER annotation pipeline generated 24,255 gene models. Results indicated that the EL10.1 genome assembly is a contiguous genome assembly highly congruent with the published sugar beet reference genome. Gross duplicate gene analyses of EL10.1 revealed little large-scale intra-genome duplication. Reduced gene copy number for well-annotated gene families relative to other core eudicots was observed, especially for transcription factors. Variation in genome size in B. vulgaris was investigated by flow cytometry among 50 individuals producing estimates from 633 to 875 Mb/1C. Read-depth mapping with short-read whole-genome sequences from other sugar beet germplasm suggested that relatively few regions of the sugar beet genome appeared associated with high-copy number variation.

Complete Genome Sequences of the Soil Oxalotrophic Bacterium Cupriavidus oxalaticus Strain Ox1 and Its Derived mCherry-Tagged Strain.

Here, we report the complete genome sequences of the soil oxalotrophic bacterium Cupriavidus oxalaticus Ox1 and a derived mCherry-tagged strain. The genome size is approximately 6.69 Mb, with a GC content of 66.9%. The genome sequence of C. oxalaticus Ox1 contains a complete operon for the degradation and assimilation of oxalate.

EDGE COVID-19: a web platform to generate submission-ready genomes from SARS-CoV-2 sequencing efforts.

SUMMARY: Genomics has become an essential technology for surveilling emerging infectious disease outbreaks. A range of technologies and strategies for pathogen genome enrichment and sequencing are being used by laboratories worldwide, together with different and sometimes ad hoc, analytical procedures for generating genome sequences. A fully integrated analytical process for raw sequence to consensus genome determination, suited to outbreaks such as the ongoing COVID-19 pandemic, is critical to provide a solid genomic basis for epidemiological analyses and well-informed decision making. We have developed a web-based platform and integrated bioinformatic workflows that help to provide consistent high-quality analysis of SARS-CoV-2 sequencing data generated with either the Illumina or Oxford Nanopore Technologies (ONT). Using an intuitive web-based interface, this workflow automates data quality control, SARS-CoV-2 reference-based genome variant and consensus calling, lineage determination and provides the ability to submit the consensus sequence and necessary metadata to GenBank, GISAID and INSDC raw data repositories. We tested workflow usability using real world data and validated the accuracy of variant and lineage analysis using several test datasets, and further performed detailed comparisons with results from the COVID-19 Galaxy Project workflow. Our analyses indicate that EC-19 workflows generate high-quality SARS-CoV-2 genomes. Finally, we share a perspective on patterns and impact observed with Illumina versus ONT technologies on workflow congruence and differences. AVAILABILITY AND IMPLEMENTATION: https://edge-covid19.edgebioinformatics.org, and https://github.com/LANL-Bioinformatics/EDGE/tree/SARS-CoV2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods.

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making-as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as "Candidatus Synoicihabitans palmerolidicus." The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium. IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, "Candidatus Synoicihabitans palmerolidicus," in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer's genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

Regulation of provider networks in response to COVID-19.

In public health insurance programs, federal and state regulators use network adequacy standards to ensure that health plans provide enrollees with adequate access to care. These standards are based on provider availability, anticipated enrollment, and patterns of care delivery. We anticipate that the coronavirus disease 2019 pandemic will have 3 main effects on provider networks and their regulation: enrollment changes, changes to the provider landscape, and changes to care delivery. Regulators will need to ensure that plans adjust their network size should there be increased enrollment or increased utilization caused by forgone care. Regulators will also require updated monitoring data and plan network data that reflect postpandemic provider availability. Telehealth will have a larger role in care delivery than in the prepandemic period, and regulators will need to adapt network standards to accommodate in-person and virtual care delivery.

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya.

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003-2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi.

Bacteria-fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments.

Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian.

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

Challenges in Bioinformatics Workflows for Processing Microbiome Omics Data at Scale.

The nascent field of microbiome science is transitioning from a descriptive approach of cataloging taxa and functions present in an environment to applying multi-omics methods to investigate microbiome dynamics and function. A large number of new tools and algorithms have been designed and used for very specific purposes on samples collected by individual investigators or groups. While these developments have been quite instructive, the ability to compare microbiome data generated by many groups of researchers is impeded by the lack of standardized application of bioinformatics methods. Additionally, there are few examples of broad bioinformatics workflows that can process metagenome, metatranscriptome, metaproteome and metabolomic data at scale, and no central hub that allows processing, or provides varied omics data that are findable, accessible, interoperable and reusable (FAIR). Here, we review some of the challenges that exist in analyzing omics data within the microbiome research sphere, and provide context on how the National Microbiome Data Collaborative has adopted a standardized and open access approach to address such challenges.

A public website for the automated assessment and validation of SARS-CoV-2 diagnostic PCR assays.

SUMMARY: Polymerase chain reaction-based assays are the current gold standard for detecting and diagnosing SARS-CoV-2. However, as SARS-CoV-2 mutates, we need to constantly assess whether existing PCR-based assays will continue to detect all known viral strains. To enable the continuous monitoring of SARS-CoV-2 assays, we have developed a web-based assay validation algorithm that checks existing PCR-based assays against the ever-expanding genome databases for SARS-CoV-2 using both thermodynamic and edit-distance metrics. The assay-screening results are displayed as a heatmap, showing the number of mismatches between each detection and each SARS-CoV-2 genome sequence. Using a mismatch threshold to define detection failure, assay performance is summarized with the true-positive rate (recall) to simplify assay comparisons. AVAILABILITY AND IMPLEMENTATION: The assay evaluation website and supporting software are Open Source and freely available at https://covid19.edgebioinformatics.org/#/assayValidation, https://github.com/jgans/thermonucleotide BLAST and https://github.com/LANL-Bioinformatics/assay_validation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Uncovering the Core Microbiome and Distribution of Palmerolide in Synoicum adareanum Across the Anvers Island Archipelago, Antarctica.

Polar marine ecosystems hold the potential for bioactive compound biodiscovery, based on their untapped macro- and microorganism diversity. Characterization of polar benthic marine invertebrate-associated microbiomes is limited to few studies. This study was motivated by our interest in better understanding the microbiome structure and composition of the ascidian, Synoicum adareanum, in which palmerolide A (PalA), a bioactive macrolide with specificity against melanoma, was isolated. PalA bears structural resemblance to a hybrid nonribosomal peptide-polyketide that has similarities to microbially-produced macrolides. We conducted a spatial survey to assess both PalA levels and microbiome composition in S. adareanum in a region of the Antarctic Peninsula near Anvers Island (64° 46'S, 64° 03'W). PalA was ubiquitous and abundant across a collection of 21 ascidians (3 subsamples each) sampled from seven sites across the Anvers Island Archipelago. The microbiome composition (V3-V4 16S rRNA gene sequence variants) of these 63 samples revealed a core suite of 21 bacterial amplicon sequence variants (ASVs)-20 of which were distinct from regional bacterioplankton. ASV co-occurrence analysis across all 63 samples yielded subgroups of taxa that may be interacting biologically (interacting subsystems) and, although the levels of PalA detected were not found to correlate with specific sequence variants, the core members appeared to occur in a preferred optimum and tolerance range of PalA levels. These results, together with an analysis of the biosynthetic potential of related microbiome taxa, describe a conserved, high-latitude core microbiome with unique composition and substantial promise for natural product biosynthesis that likely influences the ecology of the holobiont.

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life.

There is growing interest in reconstructing phylogenies from the copious amounts of genome sequencing projects that target related viral, bacterial or eukaryotic organisms. To facilitate the construction of standardized and robust phylogenies for disparate types of projects, we have developed a complete bioinformatic workflow, with a web-based component to perform phylogenetic and molecular evolutionary (PhaME) analysis from sequencing reads, draft assemblies or completed genomes of closely related organisms. Furthermore, the ability to incorporate raw data, including some metagenomic samples containing a target organism (e.g. from clinical samples with suspected infectious agents), shows promise for the rapid phylogenetic characterization of organisms within complex samples without the need for prior assembly.

Microbial Diversity in Bushmeat Samples Recovered from the Serengeti Ecosystem in Tanzania.

Bushmeat, the meat and organs derived from wildlife species, is a common source of animal protein in the diets of those living in sub-Saharan Africa and is frequently associated with zoonotic spillover of dangerous pathogens. Given the frequent consumption of bushmeat in this region and the lack of knowledge about the microbial communities associated with this meat, the microbiome of 56 fresh and processed bushmeat samples ascertained from three districts in the Western Serengeti ecosystem in Tanzania was characterized using 16S rRNA metagenomic sequencing. The results show that the most abundant phyla present in bushmeat samples include Firmicutes (67.8%), Proteobacteria (18.4%), Cyanobacteria (8.9%), and Bacteroidetes (3.1%). Regardless of wildlife species, sample condition, season, or region, the microbiome is diverse across all samples, with no significant difference in alpha or beta diversity. The findings also suggest the presence of DNA signatures of potentially dangerous zoonotic pathogens, including those from the genus Bacillus, Brucella, Coxiella, and others, in bushmeat. Together, this investigation provides a better understanding of the microbiome associated with this major food source in samples collected from the Western Serengeti in Tanzania and highlights a need for future investigations on the potential health risks associated with the harvesting, trade, and consumption of bushmeat in Sub-Saharan Africa.

In-Vivo Retention of 5-Fluorouracil Using 19F Magnetic Resonance Chemical Shift Imaging in Colorectal Cancer in a Murine Model.

Colorectal cancer is the third leading cause of cancer death worldwide. 5-Fluorouracil (5-FU) is one of the most commonly used chemotherapies for treatment of solid tumours, including colorectal cancer. The efficacy of treatment is dependent on tumour type and can only be determined six weeks after beginning chemotherapy, with only 40-50% of patients responding positively to the 5-FU therapy. In this paper, we demonstrate the potential of using Magnetic Resonance (MR) Chemical Shift Imaging (CSI) for in-vivo monitoring of 5-FU tumor-retention in two different colorectal tumour types (HT-29 & H-508). Time curves for 5-FU signals from the liver and bladder were also acquired. We observed significant differences (p < 0.01) in 5-FU signal time dependencies for the HT-29 and H-508 tumours. Retention of 5-FU occurred in the H-508 tumour, whereas the HT-29 tumour is not expected to retain 5FU due to the observation of the negative b time constant indicating a decline in 5FU within the tumour. This study successfully demonstrates that CSI may be a useful tool for early identification of 5-FU responsive tumours based on observed tumour retention of the 5-FU.