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April 21, 2020

Agricultural Origins of a Highly Persistent Lineage of Vancomycin-Resistant Enterococcus faecalis in New Zealand.

Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms.IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.Copyright © 2019 Rushton-Green et al.

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April 21, 2020

Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3.

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ?ORF 0934, ?ORF 0492, and wild type (WT) reached their highest growth rates after 8-10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ?ORF 0934 and ?ORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ?ORF 0934, ?ORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.

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April 21, 2020

A high-quality genome assembly from a single, field-collected spotted lanternfly (Lycorma delicatula) using the PacBio Sequel II system

Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens to damage economically important crop plants in the region. Results The DNA from 1 individual was used to make 1 standard, size-selected library with an average DNA fragment size of ~20 kb. The library was run on 1 Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing ~36× coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Furthermore, it was possible to segregate more than half of the diploid genome into the 2 separate haplotypes. The assembly also recovered 2 microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. Conclusions We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

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April 21, 2020

The Complete Genome of the Atypical Enteropathogenic Escherichia coli Archetype Isolate E110019 Highlights a Role for Plasmids in Dissemination of the Type III Secreted Effector EspT.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated that the type III secreted effector EspT gene, an autotransporter gene, a hemolysin gene, and putative fimbrial genes are all carried on plasmids. Further investigation of 65 espT-containing E. coli genomes demonstrated that different espT alleles are associated with multiple plasmids that differ in their overall gene content from the E110019 espT-containing plasmid. EspT has been previously described with respect to its role in the ability of E110019 to invade host cells. While other type III secreted effectors of E. coli have been identified on insertion elements and prophages of the chromosome, we demonstrated in the current study that the espT gene is located on multiple unique plasmids. These findings highlight a role of plasmids in dissemination of a unique E. coli type III secreted effector that is involved in host invasion and severe diarrheal illness.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Dual Role of gnaA in Antibiotic Resistance and Virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Complete Nucleotide Sequences of mcr-4.3-Carrying Plasmids in Acinetobacter baumannii Sequence Type 345 of Human and Food Origin from the Czech Republic, the First Case in Europe.

Here, we describe two plasmids carrying mcr-4.3 in two Acinetobacter baumannii strains isolated from imported food and a clinical sample. The comparative analysis of these plasmids, with two other plasmids reported in the NCBI database, highlighted the common origin of the plasmidic structure carrying mcr-4.3 This is the first case of the mcr-4.3 gene in a A. baumannii strain isolated from a clinical case in Europe. We hypothesize that food import is initiating the spread in Czech Republic.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

A Highly Unusual V1 Region of Env in an Elite Controller of HIV Infection.

HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of antiretroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19?years, the last 10 of which were in the absence of antiretroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 strain was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.IMPORTANCE Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities. Copyright © 2019 Silver et al.

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April 21, 2020

Hybrid de novo genome assembly of Chinese chestnut (Castanea mollissima).

The Chinese chestnut (Castanea mollissima) is widely cultivated in China for nut production. This plant also plays an important ecological role in afforestation and ecosystem services. To facilitate and expand the use of C. mollissima for breeding and its genetic improvement, we report here the whole-genome sequence of C. mollissima.We produced a high-quality assembly of the C. mollissima genome using Pacific Biosciences single-molecule sequencing. The final draft genome is ~785.53 Mb long, with a contig N50 size of 944 kb, and we further annotated 36,479 protein-coding genes in the genome. Phylogenetic analysis showed that C. mollissima diverged from Quercus robur, a member of the Fagaceae family, ~13.62 million years ago.The high-quality whole-genome assembly of C. mollissima will be a valuable resource for further genetic improvement and breeding for disease resistance and nut quality. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Genome sequence of the Chinese white wax scale insect Ericerus pela: the first draft genome for the Coccidae family of scale insects.

The Chinese white wax scale insect, Ericerus pela, is best known for producing wax, which has been widely used in candle production, casting, Chinese medicine, and wax printing products for thousands of years. The secretion of wax, and other unusual features of scale insects, is thought to be an adaptation to their change from an ancestral ground-dwelling lifestyle to a sedentary lifestyle on the higher parts of plants. As well as helping to improve its economic value, studies of E. pela might also help to explain the adaptation of scale insects. However, no genomic data are currently available for E. pela.To assemble the E. pela genome, 303.92 Gb of data were generated using Illumina and Pacific Biosciences sequencing, producing 277.22 Gb of clean data for assembly. The assembled genome size was 0.66 Gb, with 1,979 scaffolds and a scaffold N50 of 735 kb. The guanine + cytosine content was 33.80%. A total of 12,022 protein-coding genes were predicted, with a mean coding sequence length of 1,370 bp. Twenty-six fatty acyl-CoA reductase genes and 35 acyltransferase genes were identified. Evolutionary analysis revealed that E. pela and aphids formed a sister group and split ~241.1 million years ago. There were 214 expanded gene families and 2,219 contracted gene families in E. pela.We present the first genome sequence from the Coccidae family. These results will help to increase our understanding of the evolution of unique features in scale insects, and provide important genetic information for further research. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

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April 21, 2020

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81?kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3′ end of the tRNA genes pheV or pheR The length of the target site was 52 or 55?bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Tn6674 Is a Novel Enterococcal optrA-Carrying Multiresistance Transposon of the Tn554 Family.

The novel 12,932-bp nonconjugative multiresistance transposon Tn6674 was identified in the chromosomal DNA of a porcine Enterococcus faecalis strain. Tn6674 belongs to the Tn554 family of transposons. It shares the same arrangement of the transposase genes tnpA, tnpB, and tnpC with Tn554 However, in addition to the Tn554-associated resistance genes spc and erm(A), Tn6674 harbored the resistance genes fexA and optrA Circular forms of Tn6674 were detected and suggest the functional activity of this transposon.Copyright © 2019 American Society for Microbiology.

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April 21, 2020

Mitochondrial DNA Variants in Patients with Liver Injury Due to Anti-Tuberculosis Drugs.

Hepatotoxicity is the most severe adverse effect of anti-tuberculosis therapy. Isoniazid’s metabolite hydrazine is a mitochondrial complex II inhibitor. We hypothesized that mitochondrial DNA variants are risk factors for drug-induced liver injury (DILI) due to isoniazid, rifampicin or pyrazinamide.We obtained peripheral blood from tuberculosis (TB) patients before anti-TB therapy. A total of 38 patients developed DILI due to anti-TB drugs. We selected 38 patients with TB but without DILI as controls. Next-generation sequencing detected point mutations in the mitochondrial DNA genome. DILI was defined as ALT =5 times the upper limit of normal (ULN), or ALT =3 times the ULN with total bilirubin =2 times the ULN.In 38 patients with DILI, the causative drug was isoniazid in eight, rifampicin in 14 and pyrazinamide in 16. Patients with isoniazid-induced liver injury had more variants in complex I’s NADH subunit 5 and 1 genes, more nonsynonymous mutations in NADH subunit 5, and a higher ratio of nonsynonymous to total substitutions. Patients with rifampicin- or pyrazinamide-induced liver injury had no association with mitochondrial DNA variants.Variants in complex I’s subunit 1 and 5 genes might affect respiratory chain function and predispose isoniazid-induced liver injury when exposed to hydrazine, a metabolite of isoniazid and a complex II inhibitor.

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April 21, 2020

A chromosomal-level genome assembly for the insect vector for Chagas disease, Triatoma rubrofasciata.

Triatoma rubrofasciata is a widespread pathogen vector for Chagas disease, an illness that affects approximately 7 million people worldwide. Despite its importance to human health, its evolutionary origin has not been conclusively determined. A reference genome for T. rubrofasciata is not yet available.We have sequenced the genome of a female individual with T. rubrofasciatausing a single molecular DNA sequencing technology (i.e., PacBio Sequel platform) and have successfully reconstructed a whole-genome (680-Mb) assembly that covers 90% of the nuclear genome (757 Mb). Through Hi-C analysis, we have reconstructed full-length chromosomes of this female individual that has 13 unique chromosomes (2n = 24 = 22 + X1 + X2) with a contig N50 of 2.72 Mb and a scaffold N50 of 50.7 Mb. This genome has achieved a high base-level accuracy of 99.99%. This platinum-grade genome assembly has 12,691 annotated protein-coding genes. More than 95.1% of BUSCO genes were single-copy completed, indicating a high level of completeness of the genome.The platinum-grade genome assembly and its annotation provide valuable information for future in-depth comparative genomics studies, including sexual determination analysis in T. rubrofasciata and the pathogenesis of Chagas disease. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A chromosome-level draft genome of the grain aphid Sitobion miscanthi.

Sitobion miscanthi is an ideal model for studying host plant specificity, parthenogenesis-based phenotypic plasticity, and interactions between insects and other species of various trophic levels, such as viruses, bacteria, plants, and natural enemies. However, the genome information for this species has not yet to be sequenced and published. Here, we analyzed the entire genome of a parthenogenetic female aphid colony using Pacific Biosciences long-read sequencing and Hi-C data to generate chromosome-length scaffolds and a highly contiguous genome assembly.The final draft genome assembly from 33.88 Gb of raw data was ~397.90 Mb in size, with a 2.05 Mb contig N50. Nine chromosomes were further assembled based on Hi-C data to a 377.19 Mb final size with a 36.26 Mb scaffold N50. The identified repeat sequences accounted for 26.41% of the genome, and 16,006 protein-coding genes were annotated. According to the phylogenetic analysis, S. miscanthi is closely related to Acyrthosiphon pisum, with S. miscanthi diverging from their common ancestor ~25.0-44.9 million years ago.We generated a high-quality draft of the S. miscanthi genome. This genome assembly should help promote research on the lifestyle and feeding specificity of aphids and their interactions with each other and species at other trophic levels. It can serve as a resource for accelerating genome-assisted improvements in insecticide-resistant management and environmentally safe aphid management. © The Author(s) 2019. Published by Oxford University Press.

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