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September 22, 2019

Diversity of hepatitis E virus genotype 3

Summary Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.

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September 22, 2019

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.© 2017 BSPP AND JOHN WILEY & SONS LTD.

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September 22, 2019

Plasmids carrying DHA-1 ß-lactamases.

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. BlaDHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum ß-lactamases (blaCTX-M-, bla SHV -types), oxacillinases (blaOXA-1, blaOXA-30), penicillinases (bla TEM -type), carbapenemases (bla OXA48 , blaKPC-2), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6′-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.

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September 22, 2019

Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.© 2018 The Authors New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019

Investigating the central metabolism of Clostridium thermosuccinogenes.

Clostridium thermosuccinogenes is a thermophilic anaerobic bacterium able to convert various carbohydrates to succinate and acetate as main fermentation products. Genomes of the four publicly available strains have been sequenced, and the genome of the type strain has been closed. The annotated genomes were used to reconstruct the central metabolism, and enzyme assays were used to validate annotations and to determine cofactor specificity. The genes were identified for the pathways to all fermentation products, as well as for the Embden-Meyerhof-Parnas pathway and the pentose phosphate pathway. Notably, a candidate transaldolase was lacking, and transcriptomics during growth on glucose versus that on xylose did not provide any leads to potential transaldolase genes or alternative pathways connecting the C5 with the C3/C6 metabolism. Enzyme assays showed xylulokinase to prefer GTP over ATP, which could be of importance for engineering xylose utilization in related thermophilic species of industrial relevance. Furthermore, the gene responsible for malate dehydrogenase was identified via heterologous expression in Escherichia coli and subsequent assays with the cell extract, which has proven to be a simple and powerful method for the basal characterization of thermophilic enzymes.IMPORTANCE Running industrial fermentation processes at elevated temperatures has several advantages, including reduced cooling requirements, increased reaction rates and solubilities, and a possibility to perform simultaneous saccharification and fermentation of a pretreated biomass. Most studies with thermophiles so far have focused on bioethanol production. Clostridium thermosuccinogenes seems an attractive production organism for organic acids, succinic acid in particular, from lignocellulosic biomass-derived sugars. This study provides valuable insights into its central metabolism and GTP and PPi cofactor utilization. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Clonal emergence of invasive multidrug-resistant Staphylococcus epidermidis deconvoluted via a combination of whole-genome sequencing and microbiome analyses.

Pathobionts, bacteria that are typically human commensals but can cause disease, contribute significantly to antimicrobial resistance. Staphylococcus epidermidis is a prototypical pathobiont as it is a ubiquitous human commensal but also a leading cause of healthcare-associated bacteremia. We sought to determine the etiology of a recent increase in invasive S. epidermidis isolates resistant to linezolid.Whole-genome sequencing (WGS) was performed on 176 S. epidermidis bloodstream isolates collected at the MD Anderson Cancer Center in Houston, Texas, between 2013 and 2016. Molecular relationships were assessed via complementary phylogenomic approaches. Abundance of the linezolid resistance determinant cfr was determined in stool samples via reverse-transcription quantitative polymerase chain reaction.Thirty-nine of the 176 strains were linezolid resistant (22%). Thirty-one of the 39 linezolid-resistant S. epidermidis infections were caused by a particular clone resistant to multiple antimicrobials that spread among leukemia patients and carried cfr on a 49-kb plasmid (herein called pMB151a). The 6 kb of pMB151a surrounding the cfr gene was nearly 100% identical to a cfr-containing plasmid isolated from livestock-associated staphylococci in China. Analysis of serial stool samples from leukemia patients revealed progressive staphylococcal domination of the intestinal microflora and an increase in cfr abundance following linezolid use.The combination of linezolid use plus transmission of a multidrug-resistant clone drove expansion of invasive, linezolid-resistant S. epidermidis. Our results lend support to the notion that a combination of antibiotic stewardship plus infection control measures may help to control the spread of a multidrug-resistant pathobiont.

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September 22, 2019

Biosynthesis of abscisic acid in fungi: identification of a sesquiterpene cyclase as the key enzyme in Botrytis cinerea.

While abscisic acid (ABA) is known as a hormone produced by plants through the carotenoid pathway, a small number of phytopathogenic fungi are also able to produce this sesquiterpene but they use a distinct pathway that starts with the cyclization of farnesyl diphosphate (FPP) into 2Z,4E-a-ionylideneethane which is then subjected to several oxidation steps. To identify the sesquiterpene cyclase (STC) responsible for the biosynthesis of ABA in fungi, we conducted a genomic approach in Botrytis cinerea. The genome of the ABA-overproducing strain ATCC58025 was fully sequenced and five STC-coding genes were identified. Among them, Bcstc5 exhibits an expression profile concomitant with ABA production. Gene inactivation, complementation and chemical analysis demonstrated that BcStc5/BcAba5 is the key enzyme responsible for the key step of ABA biosynthesis in fungi. Unlike what is observed for most of the fungal secondary metabolism genes, the key enzyme-coding gene Bcstc5/Bcaba5 is not clustered with the other biosynthetic genes, i.e., Bcaba1 to Bcaba4 that are responsible for the oxidative transformation of 2Z,4E-a-ionylideneethane. Finally, our study revealed that the presence of the Bcaba genes among Botrytis species is rare and that the majority of them do not possess the ability to produce ABA.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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September 22, 2019

A molecular window into the biology and epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world’s most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line (‘HapOB’) from Rosa chinensis ‘Old Blush’ and generated a rose genome assembly anchored to seven pseudo-chromosomes (512?Mb with N50 of 3.4?Mb and 564 contigs). The length of 512?Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

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September 22, 2019

Diversity among blaKPC-containing plasmids in Escherichia coli and other bacterial species isolated from the same patients.

Carbapenem resistant Enterobacteriaceae are a significant public health concern, and genes encoding the Klebsiella pneumoniae carbapenemase (KPC) have contributed to the global spread of carbapenem resistance. In the current study, we used whole-genome sequencing to investigate the diversity of blaKPC-containing plasmids and antimicrobial resistance mechanisms among 26 blaKPC-containing Escherichia coli, and 13 blaKPC-containing Enterobacter asburiae, Enterobacter hormaechei, K. pneumoniae, Klebsiella variicola, Klebsiella michiganensis, and Serratia marcescens strains, which were isolated from the same patients as the blaKPC-containing E. coli. A blaKPC-containing IncN and/or IncFIIK plasmid was identified in 77% (30/39) of the E. coli and other bacterial species analyzed. Complete genome sequencing and comparative analysis of a blaKPC-containing IncN plasmid from one of the E. coli strains demonstrated that this plasmid is present in the K. pneumoniae and S. marcescens strains from this patient, and is conserved among 13 of the E. coli and other bacterial species analyzed. Interestingly, while both IncFIIK and IncN plasmids were prevalent among the strains analyzed, the IncN plasmids were more often identified in multiple bacterial species from the same patients, demonstrating a contribution of this IncN plasmid to the inter-genera dissemination of the blaKPC genes between the E. coli and other bacterial species analyzed.

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September 22, 2019

Molecular characterization of invasive meningococcal isolates in Burkina Faso as the relative importance of serogroups X and W increases, 2008-2012.

Neisseria meningitidis serogroup A disease in Burkina Faso has greatly decreased following introduction of a meningococcal A conjugate vaccine in 2010, yet other serogroups continue to pose a risk of life-threatening disease. Capsule switching among epidemic-associated serogroup A N. meningitidis strains could allow these lineages to persist despite vaccination. The introduction of new strains at the national or sub-national levels could affect the epidemiology of disease.Isolates collected from invasive meningococcal disease in Burkina Faso between 2008 and 2012 were characterized by serogrouping and molecular typing. Genome sequences from a subset of isolates were used to infer phylogenetic relationships.The ST-5 clonal complex (CC5) was identified only among serogroup A isolates, which were rare after 2010. CC181 and CC11 were the most common clonal complexes after 2010, having serogroup X and W isolates, respectively. Whole-genome phylogenetic analysis showed that the CC181 isolates collected during and after the epidemic of 2010 formed a single clade that was closely related to isolates collected in Niger during 2005 and Burkina Faso during 2007. Geographic population structure was identified among the CC181 isolates, where pairs of isolates collected from the same region of Burkina Faso within a single year had less phylogenetic diversity than the CC181 isolate collection as a whole. However, the reduction of phylogenetic diversity within a region did not extend across multiple years. Instead, CC181 isolates collected during the same year had lower than average diversity, even when collected from different regions, indicating geographic mixing of strains across years. The CC11 isolates were primarily collected during the epidemic of 2012, with sparse sampling during 2011. These isolates belong to a clade that includes previously described isolates collected in Burkina Faso, Mali, and Niger from 2011 to 2015. Similar to CC181, reduced phylogenetic diversity was observed among CC11 isolate pairs collected from the same regions during a single year.The population of disease-associated N. meningitidis strains within Burkina Faso was highly dynamic between 2008 and 2012, reflecting both vaccine-imposed selection against serogroup A strains and potentially complex clonal waves of serogroup X and serogroup W strains.

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September 22, 2019

Genomic comparison of highly virulent, moderately virulent, and avirulent strains from a genetically closely-related MRSA ST239 sub-lineage provides insights into pathogenesis.

The genomic comparison of virulent (TW20), moderately virulent (CMRSA6/CMRSA3), and avirulent (M92) strains from a genetically closely-related MRSA ST239 sub-lineage revealed striking similarities in their genomes and antibiotic resistance profiles, despite differences in virulence and pathogenicity. The main differences were in the spa gene (coding for staphylococcal protein A), lpl genes (coding for lipoprotein-like membrane proteins), cta genes (genes involved in heme synthesis), and the dfrG gene (coding for a trimethoprim-resistant dihydrofolate reductase), as well as variations in the presence or content of some prophages and plasmids, which could explain the virulence differences of these strains. TW20 was positive for all genetic traits tested, compared to CMRSA6, CMRSA3, and M92. The major components differing among these strains included spa and lpl with TW20 carrying both whereas CMRSA6/CMRSA3 carry spa identical to TW20 but have a disrupted lpl. M92 is devoid of both these traits. Considering the role played by these components in innate immunity and virulence, it is predicted that since TW20 has both the components intact and functional, these traits contribute to its pathogenesis. However, CMRSA6/CMRSA3 are missing one of these components, hence their intermediately virulent nature. On the contrary, M92 is completely devoid of both the spa and lpl genes and is avirulent. Mobile genetic elements play a potential role in virulence. TW20 carries three prophages (?Sa6, ?Sa3, and ?SPß-like), a pathogenicity island and two plasmids. CMRSA6, CMRSA3, and M92 contain variations in one or more of these components. The virulence associated genes in these components include staphylokinase, entertoxins, antibiotic/antiseptic/heavy metal resistance and bacterial persistence. Additionally, there are many hypothetical proteins (present with variations among strains) with unknown function in these mobile elements which could be making an important contribution in the virulence of these strains. The above mentioned repertoire of virulence components in TW20 likely contributes to its increased virulence, while the absence and/or modification of one or more of these components in CMRSA6/CMRSA3 and M92 likely affects the virulence of the strains.

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September 22, 2019

Comparative genomic analysis of Bacillus thuringiensis reveals molecular adaptation to copper tolerance

Bacillus thuringiensis is a type of Gram positive and rod shaped bacterium that is found in a wide range of habitats. Despite the intensive studies conducted on this bacterium, most of the information available are related to its pathogenic characteristics, with only a limited number of publications mentioning its ability to survive in extreme environments. Recently, a B. thuringiensis MCMY1 strain was successfully isolated from a copper contaminated site in Mamut Copper Mine, Sabah. This study aimed to conduct a comparative genomic analysis by using the genome sequence of MCMY1 strain published in GenBank (PRJNA374601) as a target genome for comparison with other available B. thuringiensis genomes at the GenBank. Whole genome alignment, Fragment all-against-all comparison analysis, phylogenetic reconstruction and specific copper genes comparison were applied to all forty-five B. thuringiensis genomes to reveal the molecular adaptation to copper tolerance. The comparative results indicated that B. thuringiensis MCMY1 strain is closely related to strain Bt407 and strain IS5056. This strain harbors almost all available copper genes annotated from the forty-five B. thuringiensis genomes, except for the gene for Magnesium and cobalt efflux protein (CorC) which plays an indirect role in reducing the oxidative stress that caused by copper and other metal ions. Furthermore, the findings also showed that the Copper resistance gene family, CopABCDZ and its repressor (CsoR) are conserved in almost all sequenced genomes but the presence of the genes for Cytoplasmic copper homeostasis protein (CutC) and CorC across the sample genomes are highly inconsonant. The variation of these genes across the B. thuringiensis genomes suggests that each strain may have adapted to their specific ecological niche. However, further investigations will be need to support this preliminary hypothesis.

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September 22, 2019

Draft genome sequence of an NDM-1-, OXA-421- and AmpC-producing Acinetobacter pittii ST220 in Anhui Province, China.

Acinetobacter pittii carrying the blaNDM-1 gene is frequently reported in the world recently, however most of the blaNDM-1 genes are located on plasmids. Here we report a multidrug-resistant (MDR) A. pittii isolated in China co-harbouring blaNDM-1, blaOXA-421 and blaAmpC in the genome.Bacterial genomic DNA was extracted using the cetyl trimethylammonium bromide (CTAB) method. Whole-genome sequencing of A. pittii was performed using an Illumina MiSeq system (2×251bp) in combination with PacBio single-molecule real-time (SMRT) sequencing. De novo genome assembly was performed using SPAdes v.3.9.0, A5-miseq v.20150522 and Canu v.1.4, respectively. The genome sequence was analysed by bioinformatics methods.The 4211131-bp genome with 38.99% G+C content displayed several resistance genes, including blaNDM-1, blaOXA-421 and blaAmpC. Meanwhile, 4426 protein-coding sequences were predicted within the genome.The genome sequence reported here can be compared with the already published genomes of NDM-1-producing isolates. These data might facilitate further understanding of the specific genomic feature of MDR A. pittii in China. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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