ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user’s configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem

EXPLORATION OF ECOLOGICAL AND EVOLUTIONARY MECHANISMS UNDERLYING MICROBIAL BIOGEOGRAPHY AND ADAPTATION

256 pagesAs the most abundant and diverse organisms on Earth, microorganisms perform multiple vital ecological functions in our environments. Unveiling the geographic distribution and structure of microbial populations and communities and elucidating the interaction between microorganisms and environments are fundamental goals in the field of microbial ecology and evolution. There has been growing evidence showing that microorganisms including bacteria display biogeographic patterns and have the ability to endure various stresses and adapt to the environments. However, our mechanistic understanding of the distribution patterns and adaptive traits of bacteria, particularly foodborne pathogens, is still limited largely due to the intertwined effects of eco-evolutionary dynamics and a dearth of data from large-scale investigations and from specific environment. In the studies presented here, we used Listeria including the foodborne pathogen L. monocytogenes, Salmonella enterica (a foodborne pathogen), and Escherichia coli (an enteric bacterium) as three model organisms to understand what ecological and evolutionary mechanisms underpin microbial biogeography and adaptation. We approached this question from a variety of angles including by analyzing sequence data of bacteria isolated from different environments (e.g., natural, food-associated, and host) and at different scales (nationwide, regional, and local), and by focusing on different ecological processes (e.g., environmental selection and dispersal) and evolutionary processes (e.g., positive selection and horizontal gene transfer). We found that the niche breadth, host range, and source of closely related bacteria are strongly associated with the genome variations mediated by horizontal gene transfer and transposition. Positive selection mainly targets cell surface proteins (e.g., porins and transporters), and is a key for bacteria to achieve ecological success in nature. Notably, we showed that genes involved in cell motility (e.g., flagella assembly) and metabolism (e.g., amino acid metabolism) under strong positive selection appear to allow motile and non-motile bacteria to occupy a broad niche at a nationwide scale, respectively. In addition, we found evidence that local and regional factors impact the distribution and genetic divergence of bacteria in combination. Regional factors such as landscape appear to shape the distribution of bacteria via influencing the frequency of dispersal and the strength of environmental selection. Overall, this dissertation provides valuable insights on a number of genetic, ecological, and evolutionary bases of microbial biogeography and adaptation. These insights not only shed light on the biological strategies that bacteria may employ to colonize, survive, and persist in the environments with diverse stressors, but also could benefit the control and traceback investigation of disease outbreaks and contamination events caused by foodborne pathogens.2022-08-2

Taxonomy, ecology, and relevance to food safety of the genus Listeria with a particular consideration of new Listeria species described between 2010 and 2022

ABSTRACTSince 2010, the genus Listeria has had the addition of 22 new species that more than tripled the number of species identified until 2010. Sixteen of these 22 new species are distantly related to the type species, Listeria monocytogenes, and several of these present phenotypes that distinguish them from classical Listeria species (L. monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, and Listeria grayi). These 22 newly described species also show that Listeria is more genetically diverse than previously estimated. While future studies and surveys are needed to clarify the distribution of these species, at least some of these species may not be widely spread, while other species may be frequently found spread to human-related settings (e.g., farms and processing facilities), and others may be adapted to specific environmental habitats. Here, we review the taxonomic, phylogenetic, and ecological characteristics of these new Listeria species identified since 2010 and re-iterate the suggestion of re-classification of some species into three new genera: Murraya, Mesolisteria, and Paenilisteria. We also provide a review of current detection issues and the relevance to food safety related to the identification of these new species. For example, several new non-pathogenic species could be misidentified as the pathogen L. monocytogenes, based on methods that do not target L. monocytogenes-specific virulence genes/factors, leading to unnecessary product recalls. Moreover, eight species in the proposed new genus Mesolisteria are not good indicators of environmental conditions that could allow L. monocytogenes to grow since Mesolisteria species are unable to grow at low temperatures

Microdiversity of the vaginal microbiome is associated with preterm birth

Abstract Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. The vaginal microbiome has been associated with PTB, yet the mechanisms underlying this association are not fully understood. Understanding microbial genetic adaptations to selective pressures, especially those related to the host, may yield insights into these associations. Here, we analyze metagenomic data from 705 vaginal samples collected during pregnancy from 40 women who delivered preterm spontaneously and 135 term controls from the Multi-Omic Microbiome Study-Pregnancy Initiative. We find that the vaginal microbiome of pregnancies that ended preterm exhibited unique genetic profiles. It was more genetically diverse at the species level, a result which we validate in an additional cohort, and harbored a higher richness and diversity of antimicrobial resistance genes, likely promoted by transduction. Interestingly, we find that Gardnerella species drove this higher genetic diversity, particularly during the first half of the pregnancy. We further present evidence that Gardnerella spp. underwent more frequent recombination and stronger purifying selection in genes involved in lipid metabolism. Overall, our population genetics analyses reveal associations between the vaginal microbiome and PTB and suggest that evolutionary processes acting on vaginal microbes may play a role in adverse pregnancy outcomes such as PTB

Ångström- and Nano-scale Pore-Based Nucleic Acid Sequencing of Current and Emergent Pathogens

State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (\u3e 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, ångström- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their ångström- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions

Pharmacokinetics and tissue distribution study of herpetotriol in rats by ultrahigh-performance liquid chromatography with tandem mass spectrometry

Herpetotriol, a typical lignan in Herpetospermum pedunculosum Wall’s seeds that has long been used to treat icterhepatitis and indigestion and other related diseases in Tibet, is of potential hepatoprotection. This study aims to study the pharmacokinetics features of herpetotriol, including the blood drug concentration – time curve and tissue distribution. The ultrahigh-performance liquid chromatography with tandem mass spectrometry method was established to detect herpetotriol concentration in plasma and tissues, and the method showed good linearity from 10 to 2000 ng/mL (r ≥ 0.9972) and sensitivity (≥10 ng/mL). Our blood drug concentration – time curve indicated that herpetotriol was distributed quickly in rats with a Tmax value at about 0.083 h and eliminated rapidly with a clearance rate at 98.13 ± 8.05 and 137.04 ± 9.48 L·h–1·kg–1 with doses of 5 and 2.5 mg/kg, respectively. Although herpetotriol was detectable in all tested tissues, it has a higher concentration in liver than in heart, lung, spleen, and kidney, which is in line with its hepatoprotection. The pharmacokinetics features uncovered by the present study could provide more information for future pharmacological and toxicological study of herpetotriol.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Serotype-specific evolutionary patterns of antimicrobial-resistant Salmonella enterica

Abstract Background The emergence of antimicrobial-resistant (AMR) strains of the important human and animal pathogen Salmonella enterica poses a growing threat to public health. Here, we studied the genome-wide evolution of 90 S. enterica AMR isolates, representing one host adapted serotype (S. Dublin) and two broad host range serotypes (S. Newport and S. Typhimurium). Results AMR S. Typhimurium had a large effective population size, a large and diverse genome, AMR profiles with high diversity, and frequent positive selection and homologous recombination. AMR S. Newport showed a relatively low level of diversity and a relatively clonal population structure. AMR S. Dublin showed evidence for a recent population bottleneck, and the genomes were characterized by a larger number of genes and gene ontology terms specifically absent from this serotype and a significantly higher number of pseudogenes as compared to other two serotypes. Approximately 50% of accessory genes, including specific AMR and putative prophage genes, were significantly over- or under-represented in a given serotype. Approximately 65% of the core genes showed phylogenetic clustering by serotype, including the AMR gene aac (6′)-Iaa. While cell surface proteins were shown to be the main target of positive selection, some proteins with possible functions in AMR and virulence also showed evidence for positive selection. Homologous recombination mainly acted on prophage-associated proteins. Conclusions Our data indicates a strong association between genome content of S. enterica and serotype. Evolutionary patterns observed in S. Typhimurium are consistent with multiple emergence events of AMR strains and/or ecological success of this serotype in different hosts or habitats. Evolutionary patterns of S. Newport suggested that antimicrobial resistance emerged in one single lineage, Lineage IIC. A recent population bottleneck and genome decay observed in AMR S. Dublin are congruent with its narrow host range. Finally, our results suggest the potentially important role of positive selection in the evolution of antimicrobial resistance, host adaptation and serotype diversification in S. enterica

Licochalcone A suppresses the proliferation of sarcoma HT-1080 cells, as a selective R132C mutant IDH1 inhibitor

IDH1 mutations are closely related to the development and progression of various human cancers, such as glioblastoma, sarcoma, and acute myeloid leukemia. By screening dozens of reported natural compounds using both wild-type and mutant IDH1 enzymatic assays, we discovered Licochalcone A is a selective inhibitor to the R132C-mutant IDH1 with an IC50 value of 5.176 μM, and inhibits the proliferation of sarcoma HT-1080 cells with an IC50 value of 10.75 μM. Suggested by the molecular docking results, Licochalcone A might occupy the allosteric pocket between the two monomers of IDH1 homodimer, and the R132H mutation was unfavorable for the binding of Licochalcone A with the IDH1 protein, as compared to the R132C mutation. Revealed by the RNA-Seq data analysis, the Cell Cycle pathway was the most over-represented pathway for HT-1080 cells treated with Licochalcone A. Consistent with these results, Licochalcone A induced apoptosis and cell cycle arrest of HT-1080 cells, while it showed minimal effect against the proliferation of normal RCTEC cells. The discovery of Licochalcone A as a mutation-selective IDH1 inhibitor can serve as a promising starting point for the development of mutation-selective anti-tumor lead compounds targeting IDH1

Data_Sheet_3_Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.XLSX

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro’s infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.</p