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July 7, 2019

The genomic sequence of lymphocryptovirus from cynomolgus macaque.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) cause persistent infections in human and non-human primates, and suppression of the immune system can increase the risk of lymphocryptovirus (LCV)-associated tumor development in both human and non-human primates. To enable LCV infection as a non-clinical model to study effects of therapeutics on EBV immunity, we determined the genomic DNA sequence of the LCV from cynomolgus macaque, a species commonly used for non-clinical testing. Comparison to rhesus macaque LCV and human EBV sequences indicates that LCV from the cynomolgus macaque has the same genomic arrangement and a high degree of similarity in most genes, especially with rhesus macaque LCV. Genes showing lower similarity were those encoding proteins involved in latency and/or tumor promotion or immune evasion. The genomic sequence of LCV from cynomolgus macaque should aid the development of non-clinical tools for identifying therapeutics that impact LCV immunity and carry potential lymphoma risk. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 7, 2019

Genome analysis of Staphylococcus agnetis, an agent of lameness in broiler chickens.

Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.

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July 7, 2019

De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping.

It remains a challenge to perform de novo assembly using next-generation sequencing (NGS). Despite the availability of multiple sequencing technologies and tools (e.g., assemblers) it is still difficult to assemble new genomes at chromosome resolution (i.e., one sequence per chromosome). Obtaining high quality draft assemblies is extremely important in the case of yeast genomes to better characterise major events in their evolutionary history. The aim of this work is two-fold: on the one hand we want to show how combining different and somewhat complementary technologies is key to improving assembly quality and correctness, and on the other hand we present a de novo assembly pipeline we believe to be beneficial to core facility bioinformaticians. To demonstrate both the effectiveness of combining technologies and the simplicity of the pipeline, here we present the results obtained using the Dekkera bruxellensis genome.In this work we used short-read Illumina data and long-read PacBio data combined with the extreme long-range information from OpGen optical maps in the task of de novo genome assembly and finishing. Moreover, we developed NouGAT, a semi-automated pipeline for read-preprocessing, de novo assembly and assembly evaluation, which was instrumental for this work.We obtained a high quality draft assembly of a yeast genome, resolved on a chromosomal level. Furthermore, this assembly was corrected for mis-assembly errors as demonstrated by resolving a large collapsed repeat and by receiving higher scores by assembly evaluation tools. With the inclusion of PacBio data we were able to fill about 5 % of the optical mapped genome not covered by the Illumina data.

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July 7, 2019

Genomic epidemiology of an endoscope-associated outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae.

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum ß-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.

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July 7, 2019

Finished annotated genome sequence of Burkholderia pseudomallei strain Bp1651, a multidrug-resistant clinical isolate.

Burkholderia pseudomallei strain Bp1651, a human isolate, is resistant to all clinically relevant antibiotics. We report here on the finished genome sequence assembly and annotation of the two chromosomes of this strain. This genome sequence may assist in understanding the mechanisms of antimicrobial resistance for this pathogenic species. Copyright © 2015 Bugrysheva et al.

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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

Complete genome sequence of Pseudomonas aeruginosa PA1, isolated from a patient with a respiratory tract infection.

We report the 6,498,072-bp complete genome sequence of Pseudomonas aeruginosa PA1, which was isolated from a patient with a respiratory tract infection in Chongqing, People’s Republic of China. Whole-genome sequencing was performed using single-molecule real-time (SMRT) technology, and de novo assembly revealed a single contig with 396-fold sequence coverage. Copyright © 2015 Lu et al.

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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

SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs.

Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5′-G(m)ATC-3′ motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %.Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.

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July 7, 2019

Circlator: automated circularization of genome assemblies using long sequencing reads.

The assembly of DNA sequence data is undergoing a renaissance thanks to emerging technologies capable of producing reads tens of kilobases long. Assembling complete bacterial and small eukaryotic genomes is now possible, but the final step of circularizing sequences remains unsolved. Here we present Circlator, the first tool to automate assembly circularization and produce accurate linear representations of circular sequences. Using Pacific Biosciences and Oxford Nanopore data, Circlator correctly circularized 26 of 27 circularizable sequences, comprising 11 chromosomes and 12 plasmids from bacteria, the apicoplast and mitochondrion of Plasmodium falciparum and a human mitochondrion. Circlator is available at http://sanger-pathogens.github.io/circlator/ .

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July 7, 2019

Genome analysis of Kingella kingae strain KWG1 reveals how a ß-Lactamase gene inserted in the chromosome of this species.

We describe the genome of a penicillinase-producing Kingella kingae strain (KWG1), the first to be isolated in continental Europe, whose blaTEM-1 gene was, for the first time in this species, found to be chromosomally inserted. The blaTEM gene is located in an integrative and conjugative element (ICE) inserted in Met-tRNA and comprising genes that encode resistance to sulfonamides, streptomycin, and tetracycline. This ICE is homologous to resistance-conferring plasmids of K. kingae and other Gram-negative bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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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

Complete sequence of conjugative IncA/C plasmid encoding CMY-2 ß-lactamase and RmtE 16S rRNA methyltransferase.

RmtE is a rare 16S-RMTase which was first reported in an aminoglycoside-resistant Escherichia coli strain of calf origin (1). Subsequently, we reported the first human case of infection caused by RmtE-producing E. coli (2). The rmtE gene is carried on a self-conjugative plasmid (pYDC637) in the latter strain. The present work aimed to elucidate the genetic context of rmtE. The sequencing approach has been described previously (3). In brief, the plasmid was extracted from an E. coli TOP10 transformant carrying pYDC637 and sequenced on a PacBio RS II sequencing instrument (Pacific Biosciences, Menlo Park, CA). Assembly was also conducted using the HGAP pipeline (Pacific Biosciences) as previously described (3).

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