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July 7, 2019

Complete genome sequence of Bacillus velezensis L-1, which has antagonistic activity against pear diseases.

Bacillus velezensis L-1 is an effective biocontrol agent against pear diseases. Here, we report the complete genome sequence of B. velezensis L-1 in which clusters related to the biosynthesis of secondary metabolites were predicted. This genome provides insights into the possible biocontrol mechanisms and furthers application of this specific bacterium. Copyright © 2017 Sun et al.

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July 7, 2019

Phenotypic and genotypic features of a Salmonella Heidelberg strain isolated in broilers in Brazil and their possible association to antibiotics and short-chain organic acids resistance and susceptibility.

Salmonella enterica serovar Heidelberg is a human pathogen also found in broilers. A strain (UFPR1) has been associated with field reports of resistance to short-chain organic acids (SCOA) in broilers in the South of Brazil, but was susceptible to aBacillus subtilis-based probiotic added in feed in a related study. This work aimed to (i) report clinical symptoms caused by SH UFPR1 in broilers, (ii) study its susceptibility to some antibioticsin vitro, and (iii) SCOAin vivo; and (iv) relate these phenotypic observations with its genome characteristics. Twoin vivotrials used 1-day-old chicks housed for 21?days in 8 sterilized isolated negative pressure rooms with 4 battery cages of 12 birds each. Birds were challenged or not with 107?CFU/bird of SH UFPR1 orally and exposed or not to SCOA in a 2?×?2 factorial design. Zootechnical parameters were unaffected (P?>?0.05), no clinical signs were observed, and few cecal and hepatic histologic and immune-related alterations were seen, in birds challenged with SH. Formic and propionic acids added together in drinking water, fumaric and benzoic acid in feed (Trial 1), and coated calcium butyrate in feed (Trial 2) did not reduce the SH isolation frequencies seen in cecum and liver in broilers after SH challenge (P?>?0.05). SH UFPR1 was susceptible to amikacin, amoxicillin?+?clavulanate, ceftiofur, cephalexin, doxycycline and oxytetracycline; and mildly susceptible to ampicillin?+?sulbactam, cephalothin, ciprofloxacin, enrofloxacin, and gentamycin in anin vitrominimum inhibitory concentration model using Mueller-Hinton agar. The whole genome of SH UFPR1 was sequenced and consisted of a circular chromosome, spanning 4,760,321?bp with 52.18% of GC-content encoding 84 tRNA, 22 rRNA, and 4,427 protein-coding genes. The comparison between SH UFPR1 genome and a multidrug-resistant SL476 strain revealed 11 missing genomic fragments and 5 insertions related tobgt, bgr, andrpoSgenes. The deleted genes codify proteins associated with cell cycle regulation, virulence, drug resistance, cellular adhesion, and salt efflux which collectively reveal key aspects of the evolution and adaptation of SH strains such as organic acids resistance and antibiotic sensitivity and provide information relevant to the control of SH in poultry.

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July 7, 2019

Comparative genomics reveals specific genetic architectures in nicotine metabolism of Pseudomonassp. JY-Q.

Microbial degradation of nicotine is an important process to control nicotine residues in the aqueous environment. In this study, a high active nicotine degradation strain namedPseudomonassp. JY-Q was isolated from tobacco waste extract (TWE). This strain could completely degrade 5.0 g l-1nicotine in 24 h under optimal culture conditions, and it showed some tolerance even at higher concentrations (10.0 g l-1) of nicotine. The complete genome of JY-Q was sequenced to understand the mechanism by which JY-Q could degrade nicotine and tolerate such high nicotine concentrations. Comparative genomic analysis indicated that JY-Q degrades nicotine through putative novel mechanisms. Two candidate gene cluster duplications located separately at distant loci were predicted to be responsible for nicotine degradation. These two nicotine (Nic) degradation-related loci (AA098_21325-AA098_21340, AA098_03885-AA098_03900) exhibit nearly completely consistent gene organization and component synteny. The nicotinic acid(NA)degradation gene cluster (AA098_17770-AA098_17790) andNic-like clusters were both predicted to be flanked by mobile genetic elements (MGE). Furthermore, we analyzed the regions of genomic plasticity (RGP) in the JY-Q strain and found a dynamic genome carrying a type VI secretion system (T6SS) that promotes nicotine metabolism and tolerance based on transcriptomics and usedin silicomethods to identify the T6SS effector protein. Thus, a novel nicotine degradation mechanism was elucidated forPseudomonassp. JY-Q, suggesting its potential application in the bioremediation of nicotine-contaminated environments, such as TWEs.

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July 7, 2019

Comparative genomic analysis of two clonally related multidrug resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB underin vivochallenge of anti-TB drugs.Result:Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations atrpoBS531L,inhAC-15T, andembBM306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 ofrv0888in pre-treatment strain, and a missense mutation atrv3303c(lpdA)V44I and a 6-bp inframe deletion at codon 67-68 inrv2071c(cobM)in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function.Conclusion:This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations atrv0888, lpdA, andcobMthat might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

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July 7, 2019

Post genomics era for orchid research.

Among 300,000 species in angiosperms, Orchidaceae containing 30,000 species is one of the largest families. Almost every habitats on earth have orchid plants successfully colonized, and it indicates that orchids are among the plants with significant ecological and evolutionary importance. So far, four orchid genomes have been sequenced, including Phalaenopsis equestris, Dendrobium catenatum, Dendrobium officinale, and Apostaceae shengen. Here, we review the current progress and the direction of orchid research in the post genomics era. These include the orchid genome evolution, genome mapping (genome-wide association analysis, genetic map, physical map), comparative genomics (especially receptor-like kinase and terpene synthase), secondary metabolomics, and genome editing.

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July 7, 2019

Probing genomic aspects of the multi-host pathogen Clostridium perfringens reveals significant pangenome diversity, and a diverse array of virulence factors.

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including a-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.

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July 7, 2019

Safety evaluation of HOWARU®Restore (Lactobacillus acidophilus NCFM, Lactobacillus paracasei Lpc-37, Bifidobacterium animalis subsp. lactis Bl-04 and B. lactis Bi-07) for antibiotic resistance, genomic risk factors, and acute toxicity.

Although probiotic lactobacilli and bifidobacteria are generally considered safe by various regulatory agencies, safety properties, such as absence of transferable antibiotic resistance, must still be determined for each strain prior to market introduction as a probiotic. Safety requirements for probiotics vary regionally and evaluation methods are not standardized, therefore methodologies are often adopted from food ingredients or chemicals to assess microbial safety. Four individual probiotic strains, Lactobacillus acidophilus NCFM®, Lactobacillus paracasei Lpc-37®, Bifidobacterium animalis subsp. lactis strains Bl-04®, and Bi-07®, and their combination (HOWARU®Restore) were examined for antibiotic resistance by broth microdilution culture, toxin genes by PCR and genome mining, and acute oral toxicity in rats. Only B. lactis Bl-04 exhibited antibiotic resistance above a regulated threshold due to a tetW gene previously demonstrated to be non-transferable. Genomic mining did not reveal any bacterial toxin genes known to harm mammalian hosts in any of the strains. The rodent studies did not indicate any evidence of acute toxicity following a dose of 1.7-4.1 × 1012 CFU/kg body weight. Considering a 100-fold safety margin, this corresponds to 1.2-2.8 × 1012 CFU for a 70 kg human. Our findings demonstrate a comprehensive approach of in vitro, in silico, and in vivo safety testing for probiotics. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019

Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species’ lifestyles and taxonomic dilemma.

The Gram-negative bacterium Aeromonas salmonicida contains five subspecies: salmonicida, smithia, achromogenes, masoucida and pectinolytica. Pectinolytica is a mesophilic subspecies with the ability to thrive at a wide range of temperatures, including 37°C, while the four other subspecies are psychrophilic, restricted to lower temperatures. The psychrophilic subspecies are known to infect a wide range of fishes. However, there is no evidence of pathogenicity for the mesophilic subspecies pectinolytica. Study of the differences between the mesophilic and psychrophilic subspecies is hampered by the lack of completely sequenced and closed genomes from the mesophilic subspecies. A previous study reported that insertion sequences, which can induce genomic rearrangements at temperatures around 25°C, could be one of the determinants explaining the differences in lifestyle (mesophilic or psychrophilic) between the subspecies. In this study, the genome of mesophilic strain A527 of A. salmonicida was sequenced, closed and analyzed to investigate the mesophilic-psychrophilic discrepancy. This reference genome supports the hypothesis that insertion sequences are major determinants of the lifestyle differences between the A. salmonicida subspecies. Moreover, the phylogenetic analysis performed to position strain A527 within the taxonomy raises an issue regarding the intraspecies structure of A. salmonicida.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Evolutionary context of non-sorbitol-fermenting Shiga toxin-producing Escherichia coli O55:H7.

In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.

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July 7, 2019

An update on bioinformatics resources for plant genomics research

Next-generation sequencing and traditional Sanger sequencing methods are of great significance in unraveling the complexity of plant genomes. These are constantly generating heaps of sequence data to be analyzed, annotated and stored. This has created a revolutionary demand for bioinformatics tools and software that can perform these functions. A large number of potentially useful bioinformatics tools and plant genome databases are created that have greatly simplified the analysis and storage of vast amounts of sequence data. The information garnered using the available bioinformatics methods have greatly helped in understanding the plant genome structure. Despite the availability of a good number of such tools, the information pouring from single gene-sequencing, and various whole-genome sequencing projects is overwhelming; thus, further innovations and improved methods are needed to sift through this sequence data, and assemble genomes. The current review focuses on diverse bioinformatics approaches and methods developed to systematically analyze and store plant sequence data. Finally, it outlines the bottlenecks in plant genome analysis, and some possible solutions that could be utilized to overcome the problems associated with plant genome analysis.

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July 7, 2019

Ultraaccurate genome sequencing and haplotyping of single human cells.

Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10-8and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

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July 7, 2019

Characterization of Fusobacterium varium Fv113-g1 isolated from a patient with ulcerative colitis based on complete genome sequence and transcriptome analysis.

Fusobacterium spp. present in the oral and gut flora is carcinogenic and is associated with the risk of pancreatic and colorectal cancers. Fusobacterium spp. is also implicated in a broad spectrum of human pathologies, including Crohn’s disease and ulcerative colitis (UC). Here we report the complete genome sequence of Fusobacterium varium Fv113-g1 (genome size, 3.96 Mb) isolated from a patient with UC. Comparative genome analyses totally suggested that Fv113-g1 is basically assigned as F. varium, in particular, it could be reclassified as notable F. varium subsp. similar to F. ulcerans because of partial shared orthologs. Compared with the genome sequences of F. varium ATCC 27725 (genome size, 3.30 Mb) and other strains of Fusobacterium spp., Fv113-g1 possesses many accessary pan-genome sequences with noteworthy multiple virulence factors, including 44 autotransporters (type V secretion system, T5SS) and 13 Fusobacterium adhesion (FadA) paralogs involved in potential mucosal inflammation. Indeed, transcriptome analysis demonstrated that Fv113-g1-specific accessary genes, such as multiple T5SS and fadA paralogs, showed notably increased expression with D-MEM cultivation than with brain heart infusion broth. This implied that growth condition may enhance the expression of such potential virulence factors, leading to remarkable survival against other gut microorganisms and to the pathogenicity to human intestinal epithelium.

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July 7, 2019

Complete genome sequence and comparative genomics of the golden pompano (Trachinotus ovatus) pathogen, Vibrio harveyistrain QT520.

Vibrio harveyi is a Gram-negative, halophilic bacterium that is an opportunistic pathogen of commercially farmed marine vertebrate species. To understand the pathogenicity of this species, the genome of V. harveyi QT520 was analyzed and compared to that of other strains. The results showed the genome of QT520 has two unique circular chromosomes and three endogenous plasmids, totaling 6,070,846 bp with a 45% GC content, 5,701 predicted ORFs, 134 tRNAs and 37 rRNAs. Common virulence factors, including ACF, IlpA, OmpU, Flagellin, Cya, Hemolysin and MARTX, were detected in the genome, which are likely responsible for the virulence of QT520. The results of genomes comparisons with strains ATCC 33843 (392 (MAV)) and ATCC 43516 showed that greater numbers genes associated with types I, II, III, IV and VI secretion systems were detected in QT520 than in other strains, suggesting that QT520 is a highly virulent strain. In addition, three plasmids were only observed in the complete genome sequence of strain QT520. In plasmid p1 of QT520, specific virulence factors (cyaB, hlyB and rtxA) were identified, suggesting that the pathogenicity of this strain is plasmid-associated. Phylogenetic analysis of 12 complete Vibrio sp. genomes using ANI values, core genes and MLST revealed that QT520 was most closely related to ATCC 33843 (392 (MAV)) and ATCC 43516, suggesting that QT520 belongs to the species V. harveyi. This report is the first to describe the complete genome sequence of a V. harveyi strain isolated from an outbreak in a fish species in China. In addition, to the best of our knowledge, this report is the first to compare the V. harveyi genomes of several strains. The results of this study will expand our understanding of the genome, genetic characteristics, and virulence factors of V. harveyi, setting the stage for studies of pathogenesis, diagnostics, and disease prevention.

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July 7, 2019

On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data.

To benchmark algorithms for automated plasmid sequence reconstruction from short-read sequencing data, we selected 42 publicly available complete bacterial genome sequences spanning 12 genera, containing 148 plasmids. We predicted plasmids from short-read data with four programs (PlasmidSPAdes, Recycler, cBar and PlasmidFinder) and compared the outcome to the reference sequences. PlasmidSPAdes reconstructs plasmids based on coverage differences in the assembly graph. It reconstructed most of the reference plasmids (recall=0.82), but approximately a quarter of the predicted plasmid contigs were false positives (precision=0.75). PlasmidSPAdes merged 84?% of the predictions from genomes with multiple plasmids into a single bin. Recycler searches the assembly graph for sub-graphs corresponding to circular sequences and correctly predicted small plasmids, but failed with long plasmids (recall=0.12, precision=0.30). cBar, which applies pentamer frequency analysis to detect plasmid-derived contigs, showed a recall and precision of 0.76 and 0.62, respectively. However, cBar categorizes contigs as plasmid-derived and does not bin the different plasmids. PlasmidFinder, which searches for replicons, had the highest precision (1.0), but was restricted by the contents of its database and the contig length obtained fromde novoassembly (recall=0.36). PlasmidSPAdes and Recycler detected putative small plasmids (<10?kbp), which were also predicted as plasmids by cBar, but were absent in the original assembly. This study shows that it is possible to automatically predict small plasmids. Prediction of large plasmids (>50?kbp) containing repeated sequences remains challenging and limits the high-throughput analysis of plasmids from short-read whole-genome sequencing data.

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July 7, 2019

Genomic characterization of a local epidemic Pseudomonas aeruginosa reveals specific features of the widespread clone ST395.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen with several clones being frequently associated with outbreaks in hospital settings. ST395 is among these so-called ‘international’ clones. We aimed here to define the biological features that could have helped the implantation and spread of the clone ST395 in hospital settings. The complete genome of a multidrug resistant index isolate (DHS01) of a large hospital outbreak was analysed. We identified DHS01-specific genetic elements, among which were identified those shared with a panel of six independent ST395 isolates responsible for outbreaks in other hospitals. DHS01 has the fifth largest chromosome of the species (7.1 Mbp), with most of its 1555 accessory genes borne by either genomic islands (GIs,n=48) or integrative and conjugative elements (ICEs,n=5). DHS01 is multidrug resistant mostly due to chromosomal mutations. It displayed signatures of adaptation to chronic infection in part due to the loss of a 131 kbp chromosomal fragment. Four GIs were specific to the clone ST395 and contained genes involved in metabolism (GI-4), in virulence (GI-6) and in resistance to copper (GI-7). GI-7 harboured an array of six copper transporters and was shared with non-pathogenicPseudomonassp. retrieved from copper-contaminated environments. Copper resistance was confirmed phenotypically in all other ST395 isolates and possibly accounted for the spreading capability of the clone in hospital outbreaks, where water networks have been incriminated. This suggests that genes transferred from copper-polluted environments may have favoured the implantation and spread of the international cloneP. aeruginosaST395 in hospital settings.

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