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April 21, 2020

WGS of 1058 Enterococcus faecium from Copenhagen, Denmark, reveals rapid clonal expansion of vancomycin-resistant clone ST80 combined with widespread dissemination of a vanA-containing plasmid and acquisition of a heterogeneous accessory genome.

From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized.WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems.Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in =99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ~29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes.Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Sequential evolution of virulence and resistance during clonal spread of community-acquired methicillin-resistant Staphylococcus aureus.

The past two decades have witnessed an alarming expansion of staphylococcal disease caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The factors underlying the epidemic expansion of CA-MRSA lineages such as USA300, the predominant CA-MRSA clone in the United States, are largely unknown. Previously described virulence and antimicrobial resistance genes that promote the dissemination of CA-MRSA are carried by mobile genetic elements, including phages and plasmids. Here, we used high-resolution genomics and experimental infections to characterize the evolution of a USA300 variant plaguing a patient population at increased risk of infection to understand the mechanisms underlying the emergence of genetic elements that facilitate clonal spread of the pathogen. Genetic analyses provided conclusive evidence that fitness (manifest as emergence of a dominant clone) changed coincidently with the stepwise emergence of (i) a unique prophage and mutation of the regulator of the pyrimidine nucleotide biosynthetic operon that promoted abscess formation and colonization, respectively, thereby priming the clone for success; and (ii) a unique plasmid that conferred resistance to two topical microbiocides, mupirocin and chlorhexidine, frequently used for decolonization and infection prevention. The resistance plasmid evolved through successive incorporation of DNA elements from non-S. aureus spp. into an indigenous cryptic plasmid, suggesting a mechanism for interspecies genetic exchange that promotes antimicrobial resistance. Collectively, the data suggest that clonal spread in a vulnerable population resulted from extensive clinical intervention and intense selection pressure toward a pathogen lifestyle that involved the evolution of consequential mutations and mobile genetic elements.

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April 21, 2020

The complete mitochondrial genome of the tree frog, Polypedates braueri (Anura, Rhacophoridae)

We determined the complete mitochondrial genome of the tree frog, Polypedates braueri using next generation sequencing (NGS) and Sanger sequencing. The mitogenome of P. braueri was 19,904?bp in length, which contained 12 protein-coding genes, 22 tRNAs, two rRNAs, and two control regions (D-Loop). A noncoding sequence (NC) was discovered between tRNALys and ATP6 gene, as well as replaced the original position of ATP8 gene. The ND5 gene was found between the two control regions. More mitochondrial genomic information will contribute to revealing the phylogenetic relationships among species of the genus Polypedates.

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April 21, 2020

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone taxa was the highest under organic farming where agricultural intensification was the lowest. We also found a strong negative association (R2?=?0.366; P?

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April 21, 2020

High-Resolution Evolutionary Analysis of Within-Host Hepatitis C Virus Infection.

Despite recent breakthroughs in treatment of hepatitis C virus (HCV) infection, we have limited understanding of how virus diversity generated within individuals impacts the evolution and spread of HCV variants at the population scale. Addressing this gap is important for identifying the main sources of disease transmission and evaluating the risk of drug-resistance mutations emerging and disseminating in a population.We have undertaken a high-resolution analysis of HCV within-host evolution from 4 individuals coinfected with human immunodeficiency virus 1 (HIV-1). We used long-read, deep-sequenced data of full-length HCV envelope glycoprotein, longitudinally sampled from acute to chronic HCV infection to investigate the underlying viral population and evolutionary dynamics.We found statistical support for population structure maintaining the within-host HCV genetic diversity in 3 out of 4 individuals. We also report the first population genetic estimate of the within-host recombination rate for HCV (0.28 × 10-7 recombination/site/year), which is considerably lower than that estimated for HIV-1 and the overall nucleotide substitution rate estimated during HCV infection.Our findings indicate that population structure and strong genetic linkage shapes within-host HCV evolutionary dynamics. These results will guide the future investigation of potential HCV drug resistance adaptation during infection, and at the population scale. © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.

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April 21, 2020

Genome and transcriptome sequencing of the astaxanthin-producing green microalga, Haematococcus pluvialis.

Haematococcus pluvialis is a freshwater species of Chlorophyta, family Haematococcaceae. It is well known for its capacity to synthesize high amounts of astaxanthin, which is a strong antioxidant that has been utilized in aquaculture and cosmetics. To improve astaxanthin yield and to establish genetic resources for H. pluvialis, we performed whole-genome sequencing, assembly, and annotation of this green microalga. A total of 83.1 Gb of raw reads were sequenced. After filtering the raw reads, we subsequently generated a draft assembly with a genome size of 669.0?Mb, a scaffold N50 of 288.6?kb, and predicted 18,545 genes. We also established a robust phylogenetic tree from 14 representative algae species. With additional transcriptome data, we revealed some novel potential genes that are involved in the synthesis, accumulation, and regulation of astaxanthin production. In addition, we generated an isoform-level reference transcriptome set of 18,483 transcripts with high confidence. Alternative splicing analysis demonstrated that intron retention is the most frequent mode. In summary, we report the first draft genome of H. pluvialis. These genomic resources along with transcriptomic data provide a solid foundation for the discovery of the genetic basis for theoretical and commercial astaxanthin enrichment.

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April 21, 2020

The complete mitogenome of clam Corbicula fluminea determined using next-generation and PacBio sequencing

Corbicula fluminea is an important aquatic commercial species in China. In this study, we present the complete mitogenome and a phylogenetic analysis of C. fluminea, determined using next-generation and PacBio long read sequencing. The mitogenome of C. fluminea is 17,423bp in size, including 13 protein-coding genes, two ribosomal RNA genes, 22 tRNA genes, and a putative control region, all located on the same strand. The base composition of the entire mitogenome showed a conspicuous AþT bias of 70.5 %. The entire mitogenome data produced in this study provides the genomic resour- ces available for future evolutionary studies.

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April 21, 2020

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.

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April 21, 2020

Molecular Epidemiology of Candida auris in Colombia Reveals a Highly Related, Countrywide Colonization With Regional Patterns in Amphotericin B Resistance.

Candida auris is a multidrug-resistant yeast associated with hospital outbreaks worldwide. During 2015-2016, multiple outbreaks were reported in Colombia. We aimed to understand the extent of contamination in healthcare settings and to characterize the molecular epidemiology of C. auris in Colombia.We sampled patients, patient contacts, healthcare workers, and the environment in 4 hospitals with recent C. auris outbreaks. Using standardized protocols, people were swabbed at different body sites. Patient and procedure rooms were sectioned into 4 zones and surfaces were swabbed. We performed whole-genome sequencing (WGS) and antifungal susceptibility testing (AFST) on all isolates.Seven of the 17 (41%) people swabbed were found to be colonized. Candida auris was isolated from 37 of 322 (11%) environmental samples. These were collected from a variety of items in all 4 zones. WGS and AFST revealed that although isolates were similar throughout the country, isolates from the northern region were genetically distinct and more resistant to amphotericin B (AmB) than the isolates from central Colombia. Four novel nonsynonymous mutations were found to be significantly associated with AmB resistance.Our results show that extensive C. auris contamination can occur and highlight the importance of adherence to appropriate infection control practices and disinfection strategies. Observed genetic diversity supports healthcare transmission and a recent expansion of C. auris within Colombia with divergent AmB susceptibility.

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April 21, 2020

Development of Tigecycline Resistance in Carbapenemase-Producing Klebsiella pneumoniae Sequence Type 147 via AcrAB Overproduction Mediated by Replacement of the ramA Promoter.

Carbapenem-resistant K. pneumoniae 2297, isolated from a patient treated with tigecycline for pneumonia, developed tigecycline resistance, in contrast to carbapenem-resistant isolate 1215, which was collected four months prior to the 2297 isolate. Mechanisms underlying tigecycline resistance were elucidated for the clinical isolates.The tigecycline minimum inhibitory concentration (MIC) was determined using the broth microdilution method, with or without phenylalanine-arginine ß-naphthylamide (PABN), and whole-genome sequencing was carried out by single-molecule real-time sequencing. The expression levels of the genes acrA, oqxA, ramA, rarA, and rpoB were determined by reverse-transcription quantitative PCR.Both isolates presented identical antibiograms, except for tigecycline, which showed an MIC of 0.5 mg/L in 1215 and 2 mg/L in 2297. The addition of PABN to tigecycline-resistant 2297 caused a four-fold decrease in the tigecycline MIC to 0.5 mg/L, although acrA expression (encoding the AcrAB efflux pump) was upregulated by 2.5 fold and ramA expression (encoding the pump activator RamA) was upregulated by 1.4 fold. We identified a 6,096-bp fragment insertion flanking direct TATAT repeats that disrupted the romA gene located upstream of ramA in the chromosome of K. pneumoniae 2297; the insertion led the ramA gene promoter replacement resulting in stronger activation of the gene.The K. pneumoniae isolate developed tigecycline resistance during tigecycline treatment. It was related to the overexpression of the AcrAB resistance-nodulation-cell division efflux system due to promoter replacement. © The Korean Society for Laboratory Medicine.

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April 21, 2020

Characterization of Mauritian cynomolgus macaque Fc?R alleles using long-read sequencing.

The Fc?Rs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. Fc?Rs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize Fc?R diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM Fc?R genes. We also developed a high-throughput Fc?R genotyping assay, which we used to determine allele frequencies and identify Fc?R haplotypes in more than 500 additional MCMs. We found three alleles for Fc?R1A, seven each for Fc?R2A and Fc?R2B, and four for Fc?R3A; these segregate into eight haplotypes. We also assessed whether different Fc?R alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of Fc?R responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies. Copyright © 2018 by The American Association of Immunologists, Inc.

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April 21, 2020

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected.To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms.The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing.A novel IncZ-7 plasmid pEC013 (~118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013.The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid’s heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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April 21, 2020

Genomic Plasticity Mediated by Transposable Elements in the Plant Pathogenic Fungus Colletotrichum higginsianum.

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020

Full-length transcriptome sequences obtained by a combination of sequencing platforms applied to heat shock proteins and polyunsaturated fatty acids biosynthesis in Pyropia haitanensis

Pyropia haitanensis is a high-yield commercial seaweed in China. Pyropia haitanensis farms often suffer from problems such as severe germplasm degeneration, while the mechanisms underlying resistance to abiotic stresses remain unknown because of lacking genomic information. Although many previous studies focused on using next-generation sequencing (NGS) technologies, the short-read sequences generated by NGS generally prevent the assembly of full-length transcripts, and then limit screening functional genes. In the present study, which was based on hybrid sequencing (NGS and single-molecular real-time sequencing) of the P. haitanensis thallus transcriptome, we obtained high-quality full-length transcripts with a mean length of 2998 bp and an N50 value of 3366 bp. A total of 14,773 unigenes (93.52%) were annotated in at least one database, while approximately 60% of all unigenes were assembled by short Illumina reads. Moreover, we herein suggested that the genes involved in the biosynthesis of polyunsaturated fatty acids and heat shock proteins play an important role in the process of development and resistance to abiotic stresses in P. haitanensis. The present study, together with previously published ones, may facilitate seaweed transcriptome research.

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April 21, 2020

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety “Finola.” The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ~40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant. © 2019 Laverty et al.; Published by Cold Spring Harbor Laboratory Press.

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