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July 7, 2019

IncI1 plasmids encoding various blaCTX-Ms contributed to ceftriaxone resistance in Salmonella Enteritidis in China.

Resistance to extended spectrum ß-lactams in Salmonella, in particular serotypes such as S. Enteritidis that are frequently associated with clinical infections, is a serious public health concern. In this study, phenotypic characterization of 433 clinical S. Enteritidis strains obtained from a nationwide collection of China CDC during the period of 2005~2010 depicted an increasing trend of resistance to ceftriaxone from 2008 onwards. Seventeen (4%) of the strains were found to be resistant to ceftriaxone, 7% to ciprofloxacin and 0.7% to both ciprofloxacin and ceftriaxone. Most of the ceftriaxone-resistant S. Enteritidis strains (15/17) were genetically unrelated, and originated from Henan province. The complete sequence of an IncI1 plasmid pSE115 which belonged to a novel Sequence Type was obtained. This 87,255bp IncI1 plasmid was found to harbour a blaCTX-M-14 gene located in a novel Multidrug Resistance Region (MRR) within the tra locus. Although the majority of strains were also found to contain conjugative IncI1 plasmids of similar size to pSE115(~90kb) and harbor a variety of blaCTX-MGroup 1 and Group 9 elements, the novel MRR site at the tra locus in pSE115 was not detectable in the other IncI1 plasmids. Findings in this study show that cephalosporin resistance in S. Enteritidis strains collected in China was mainly due to dissemination of blaCTX-M-encoding IncI1 plasmids, resembling the situation in which IncI1 plasmids serve as major vectors of blaCTX-M variants in other members of Enterobacteriaceae. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Molecular characterization using next generation sequencing of plasmids containing blaNDM-7 in Enterobacteriaceae from Calgary, Canada.

Enterobacteriaceae with blaNDM-7 is relatively uncommon and had previously been described in Europe, India, USA and Japan. This study describes the characteristics of Enterobacteriaceae [Klebsiella pneumoniae (n=2), Escherichia coli (n=2), Serratia marcescens (n=1), Enterobacter hormaechei (n=1)] with blaNDM-7 obtained in 4 patients from Calgary, Canada during 2013-4. The 46,161 bp IncX3 plasmids with blaNDM-7 are highly similar to other blaNDM-harboring IncX3 plasmids and interestingly, showed identical structures within the different isolates. This finding may indicate horizontal transmission within our health region or may indicate contact with individuals from endemic areas within the hospital setting. Patients infected or colonized with bacteria containing blaNDM-7 IncX3 plasmids will generate infection control challenges. Epidemiological and molecular studies are required to better understand the dynamics of transmission, risk factors and reservoirs for bacteria harboring blaNDM-7. To the best of our knowledge, this is the first report of S. marcescens, and E. hormaechei with blaNDM-7. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Next-generation sequencing and comparative analysis of sequential outbreaks caused by multidrug-resistant Acinetobacter baumannii at a large academic burn center.

Next-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistant Acinetobacter baumannii (MDRAB) at a large academic burn center. A reference genome from the index case was generated using de novo assembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed a blaOXA-23-like group, while the third-outbreak strains harbored a blaOXA-40-like group. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Global insights into acetic acid resistance mechanisms and genetic stability of Acetobacter pasteurianus strains by comparative genomics.

Acetobacter pasteurianus (Ap) CICC 20001 and CGMCC 1.41 are two acetic acid bacteria strains that, because of their strong abilities to produce and tolerate high concentrations of acetic acid, have been widely used to brew vinegar in China. To globally understand the fermentation characteristics, acid-tolerant mechanisms and genetic stabilities, their genomes were sequenced. Genomic comparisons with 9 other sequenced Ap strains revealed that their chromosomes were evolutionarily conserved, whereas the plasmids were unique compared with other Ap strains. Analysis of the acid-tolerant metabolic pathway at the genomic level indicated that the metabolism of some amino acids and the known mechanisms of acetic acid tolerance, might collaboratively contribute to acetic acid resistance in Ap strains. The balance of instability factors and stability factors in the genomes of Ap CICC 20001 and CGMCC 1.41 strains might be the basis for their genetic stability, consistent with their stable industrial performances. These observations provide important insights into the acid resistance mechanism and the genetic stability of Ap strains and lay a foundation for future genetic manipulation and engineering of these two strains.

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July 7, 2019

Bovine NK-lysin: Copy number variation and functional diversification.

NK-lysin is an antimicrobial peptide and effector protein in the host innate immune system. It is coded by a single gene in humans and most other mammalian species. In this study, we provide evidence for the existence of four NK-lysin genes in a repetitive region on cattle chromosome 11. The NK2A, NK2B, and NK2C genes are tandemly arrayed as three copies in ~30-35-kb segments, located 41.8 kb upstream of NK1. All four genes are functional, albeit with differential tissue expression. NK1, NK2A, and NK2B exhibited the highest expression in intestine Peyer’s patch, whereas NK2C was expressed almost exclusively in lung. The four peptide products were synthesized ex vivo, and their antimicrobial effects against both Gram-positive and Gram-negative bacteria were confirmed with a bacteria-killing assay. Transmission electron microcopy indicated that bovine NK-lysins exhibited their antimicrobial activities by lytic action in the cell membranes. In summary, the single NK-lysin gene in other mammals has expanded to a four-member gene family by tandem duplications in cattle; all four genes are transcribed, and the synthetic peptides corresponding to the core regions are biologically active and likely contribute to innate immunity in ruminants.

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July 7, 2019

Botrytis, the good, the bad and the ugly

Botrytis spp. are efficient pathogens, causing devastating diseases and significant crop losses in a wide variety of plant species. Here we outline our review of these pathogens, as well as highlight the major advances of the past 10 years in studying Botrytis in interaction with its hosts. Progress in molecular genetics and the development of relevant phylogenetic markers in particular, has resulted in the characterisation of approximately 30 species. The host range of Botrytis spp. includes plant species that are members of 170 families of cultivated plants.

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July 7, 2019

Synergistic effect of ATP for RuvA-RuvB-Holliday junction DNA complex formation.

The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday junction DNA complex in the following order: no nucleotide, ADP, ATP?S, and mixture of ADP and ATP?S. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday junction DNA complex formation.

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July 7, 2019

Single molecule sequencing of THCA synthase reveals copy number variation in modern drug-type Cannabis sativa L.

Cannabinoid expression is an important genetically determined feature of cannabis that presents clinical and legal implications for patients seeking cannabinoid specific therapies like Cannabidiol (CBD). Cannabinoid, terpenoid, and flavonoid marker assisted selection can accelerate breeding efforts by offering genetic tools to select for desired traits at an early stage in growth. To this end, multiple models for chemotype inheritance have been described suggesting a complex picture for chemical phenotype determination. Here we explore the potential role of copy number variation of THCA Synthase using phased single molecule sequencing and demonstrate that copy number and sequence variation of this gene is common and suggests a more nuanced view of chemotype prediction.

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July 7, 2019

Leafy spurge genomics: A model perennial weed to investigate development, stress responses, and invasiveness

Leafy spurge is wild flower native to Europe that has become an invasive perennial weed in the northern great plains of the USA and Canada. Leafy spurge primarily infests range and recreation lands and costs US land managers millions dollars annually. In its invaded range, leafy spurge can form vast monocultures that significantly impact native flora and fauna and has been attributed to reduced populations of endangered species such as the prairie fringed orchid. Leafy spurge has remarkable plasticity and can persist under environmental extremes—primarily due to the formation of hundreds of underground adventitious buds that can form on its extensive and deep root system. We have developed genomics-based tools to assist our investigations related to vegetative production from these underground buds, as well as its responses to stress, and the potential mechanisms leading to the invasiveness of leafy spurge. Towards these ends, we have utilized Sanger-based sequencing to develop EST-databases from leafy spurge and cassava (a related species) transcriptomes, and developed textasciitilde23,000 element cDNA microarrays representing all of the unigenes identified in these databases. Additionally, numerous cDNA libraries and genomic libraries have been developed including bacterial artificial chromosome libraries useful for identifying and characterizing promoters of differentially expressed genes. Finally, to enhance our ability to identify promoter sequences and transcription factors involved in vegetative production, stress responses, and invasiveness, we have incorporated next generation sequencing approaches to fully sequence the leafy spurge genome. Using global transcriptome profiles, next generation sequencing, bioinformatics programs has provided insights into molecular mechanisms and regulatory pathways that make leafy spurge a particularly invasive and difficult weed to control.

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July 7, 2019

Lesions from patients with sporadic cerebral cavernous malformations harbor somatic mutations in the CCM genes: evidence for a common biochemical pathway for CCM pathogenesis.

Cerebral cavernous malformations (CCMs) are vascular lesions affecting the central nervous system. CCM occurs either sporadically or in an inherited, autosomal dominant manner. Constitutional (germline) mutations in any of three genes, KRIT1, CCM2 and PDCD10, can cause the inherited form. Analysis of CCM lesions from inherited cases revealed biallelic somatic mutations, indicating that CCM follows a Knudsonian two-hit mutation mechanism. It is still unknown, however, if the sporadic cases of CCM also follow this genetic mechanism. We extracted DNA from 11 surgically excised lesions from sporadic CCM patients, and sequenced the three CCM genes in each specimen using a next-generation sequencing approach. Four sporadic CCM lesion samples (36%) were found to contain novel somatic mutations. Three of the lesions contained a single somatic mutation, and one lesion contained two biallelic somatic mutations. Herein, we also describe evidence of somatic mosaicism in a patient presenting with over 130 CCM lesions localized to one hemisphere of the brain. Finally, in a lesion regrowth sample, we found that the regrown CCM lesion contained the same somatic mutation as the original lesion. Together, these data bolster the idea that all forms of CCM have a genetic underpinning of the two-hit mutation mechanism in the known CCM genes. Recent studies have found aberrant Rho kinase activation in inherited CCM pathogenesis, and we present evidence that this pathway is activated in sporadic CCM patients. These results suggest that all CCM patients, including those with the more common sporadic form, are potentially amenable to the same therapy. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019

Stenotrophomonas comparative genomics reveals genes and functions that differentiate beneficial and pathogenic bacteria.

In recent years, the number of human infections caused by opportunistic pathogens has increased dramatically. Plant rhizospheres are one of the most typical natural reservoirs for these pathogens but they also represent a great source for beneficial microbes with potential for biotechnological applications. However, understanding the natural variation and possible differences between pathogens and beneficials is the main challenge in furthering these possibilities. The genus Stenotrophomonas contains representatives found to be associated with human and plant host.We used comparative genomics as well as transcriptomic and physiological approaches to detect significant borders between the Stenotrophomonas strains: the multi-drug resistant pathogenic S. maltophilia and the plant-associated strains S. maltophilia R551-3 and S. rhizophila DSM14405T (both are biocontrol agents). We found an overall high degree of sequence similarity between the genomes of all three strains. Despite the notable similarity in potential factors responsible for host invasion and antibiotic resistance, other factors including several crucial virulence factors and heat shock proteins were absent in the plant-associated DSM14405T. Instead, S. rhizophila DSM14405T possessed unique genes for the synthesis and transport of the plant-protective spermidine, plant cell-wall degrading enzymes, and high salinity tolerance. Moreover, the presence or absence of bacterial growth at 37°C was identified as a very simple method in differentiating between pathogenic and non-pathogenic isolates. DSM14405T is not able to grow at this human-relevant temperature, most likely in great part due to the absence of heat shock genes and perhaps also because of the up-regulation at increased temperatures of several genes involved in a suicide mechanism.While this study is important for understanding the mechanisms behind the emerging pattern of infectious diseases, it is, to our knowledge, the first of its kind to assess the risk of beneficial strains for biotechnological applications. We identified certain traits typical of pathogens such as growth at the human body temperature together with the production of heat shock proteins as opposed to a temperature-regulated suicide system that is harnessed by beneficials.

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July 7, 2019

Site-specific genetic engineering of the Anopheles gambiae Y chromosome.

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line-specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.

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July 7, 2019

The effects of read length, quality and quantity on microsatellite discovery and primer development: from Illumina to PacBio.

The advent of next-generation sequencing (NGS) technologies has transformed the way microsatellites are isolated for ecological and evolutionary investigations. Recent attempts to employ NGS for microsatellite discovery have used the 454, Illumina, and Ion Torrent platforms, but other methods including single-molecule real-time DNA sequencing (Pacific Biosciences or PacBio) remain viable alternatives. We outline a workflow from sequence quality control to microsatellite marker validation in three plant species using PacBio circular consensus sequencing (CCS). We then evaluate the performance of PacBio CCS in comparison with other NGS platforms for microsatellite isolation, through simulations that focus on variations in read length, read quantity and sequencing error rate. Although quality control of CCS reads reduced microsatellite yield by around 50%, hundreds of microsatellite loci that are expected to have improved conversion efficiency to functional markers were retrieved for each species. The simulations quantitatively validate the advantages of long reads and emphasize the detrimental effects of sequencing errors on NGS-enabled microsatellite development. In view of the continuing improvement in read length on NGS platforms, sequence quality and the corresponding strategies of quality control will become the primary factors to consider for effective microsatellite isolation. Among current options, PacBio CCS may be optimal for rapid, small-scale microsatellite development due to its flexibility in scaling sequencing effort, while platforms such as Illumina MiSeq will provide cost-efficient solutions for multispecies microsatellite projects. © 2014 John Wiley & Sons Ltd.

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July 7, 2019

A fault-tolerant method for HLA typing with PacBio data.

Human leukocyte antigen (HLA) genes are critical genes involved in important biomedical aspects, including organ transplantation, autoimmune diseases and infectious diseases. The gene family contains the most polymorphic genes in humans and the difference between two alleles is only a single base pair substitution in many cases. The next generation sequencing (NGS) technologies could be used for high throughput HLA typing but in silico methods are still needed to correctly assign the alleles of a sample. Computer scientists have developed such methods for various NGS platforms, such as Illumina, Roche 454 and Ion Torrent, based on the characteristics of the reads they generate. However, the method for PacBio reads was less addressed, probably owing to its high error rates. The PacBio system has the longest read length among available NGS platforms, and therefore is the only platform capable of having exon 2 and exon 3 of HLA genes on the same read to unequivocally solve the ambiguity problem caused by the “phasing” issue.We proposed a new method BayesTyping1 to assign HLA alleles for PacBio circular consensus sequencing reads using Bayes’ theorem. The method was applied to simulated data of the three loci HLA-A, HLA-B and HLA-DRB1. The experimental results showed its capability to tolerate the disturbance of sequencing errors and external noise reads.The BayesTyping1 method could overcome the problems of HLA typing using PacBio reads, which mostly arise from sequencing errors of PacBio reads and the divergence of HLA genes, to some extent.

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