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September 22, 2019  |  

The complete chloroplast genome sequence of Coix lacryma-jobi L.(Poaceae), a cereal and medicinal crop

Coix lacryma-jobi is a cereal and medicinal crop belonging to the Poaceae family. This study characterized complete chloroplast genome sequence of a Korean cultivar Johyun of C. lacryma-jobi var. ma-yuen through the de novo hybrid assembly with Illumina and PacBio genomic reads. The chloroplast genome is 140,863?bp long and composed of large single copy (82,827?bp), small single copy (12,522?bp), and a pair of inverted repeats (each 22,757?bp). A total of 123 genes including 87 protein-coding genes, 32 tRNA genes, and four rRNA genes were predicted in the genome. Phylogenetic analysis confirmed a close relationship of C. lacryma-jobi with species in the Panicoideae subfamily of the Poaceae family.


September 22, 2019  |  

Complete genomic analysis of a kingdom crossing Klebsiella variicola isolate.

Bacterial isolate X39 was isolated from a community-acquired pneumonia patient in Beijing, China. A phylogenetic tree based on rpoB genes and average nucleotide identity data confirmed that isolate X39 belonged to Klebsiella variicola. The genome of K. variicola X39 contained one circular chromosome and nine plasmids. Comparative genomic analyses with other K. variicola isolates revealed that K. variicola X39 contained the most unique genes. Of these unique genes, many were prophages and transposases. Many virulence factors were shared between K. variicola X39 and Klebsiella pneumoniae F1. The pathogenicity of K. variicola X39 was compared with that of K. pneumoniae F1 in an abdominal infection model. The results indicated that K. variicola X39 was less virulent than typical clinical K. pneumoniae F1. The genome of K. variicola X39 also contained some genes involved in plant colonization, nitrogen fixation, and defense against oxidative stress. GFP-labeled K. variicola X39 could colonize maize as an endophytic bacterium. We concluded that K. variicola X39 was a kingdom-crossing strain.


September 22, 2019  |  

Discovery of mcr-1-mediated colistin resistance in a highly virulent Escherichia coli lineage.

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage. Copyright © 2018 Forde et al.


September 22, 2019  |  

An introduced crop plant is driving diversification of the virulent bacterial pathogen Erwinia tracheiphila.

Erwinia tracheiphila is the causal agent of bacterial wilt of cucurbits, an economically important phytopathogen affecting an economically important phytopathogen affecting few cultivated Cucurbitaceae few cultivated Cucurbitaceae host plant species in temperate eastern North America. However, essentially nothing is known about E. tracheiphila population structure or genetic diversity. To address this shortcoming, a representative collection of 88 E. tracheiphila isolates was gathered from throughout its geographic range, and their genomes were sequenced. Phylogenomic analysis revealed three genetic clusters with distinct hrpT3SS virulence gene repertoires, host plant association patterns, and geographic distributions. Low genetic heterogeneity within each cluster suggests a recent population bottleneck followed by population expansion. We showed that in the field and greenhouse, cucumber (Cucumis sativus), which was introduced to North America by early Spanish conquistadors, is the most susceptible host plant species and the only species susceptible to isolates from all three lineages. The establishment of large agricultural populations of highly susceptible C. sativus in temperate eastern North America may have facilitated the original emergence of E. tracheiphila into cucurbit agroecosystems, and this introduced plant species may now be acting as a highly susceptible reservoir host. Our findings have broad implications for agricultural sustainability by drawing attention to how worldwide crop plant movement, agricultural intensification, and locally unique environments may affect the emergence, evolution, and epidemic persistence of virulent microbial pathogens.IMPORTANCEErwinia tracheiphila is a virulent phytopathogen that infects two genera of cucurbit crop plants, Cucurbita spp. (pumpkin and squash) and Cucumis spp. (muskmelon and cucumber). One of the unusual ecological traits of this pathogen is that it is limited to temperate eastern North America. Here, we complete the first large-scale sequencing of an E. tracheiphila isolate collection. From phylogenomic, comparative genomic, and empirical analyses, we find that introduced Cucumis spp. crop plants are driving the diversification of E. tracheiphila into multiple lineages. Together, the results from this study show that locally unique biotic (plant population) and abiotic (climate) conditions can drive the evolutionary trajectories of locally endemic pathogens in unexpected ways. Copyright © 2018 Shapiro et al.


September 22, 2019  |  

pYR4 from a Norwegian isolate of Yersinia ruckeri is a putative virulence plasmid encoding both a type IV pilus and a type IV secretion system

Enteric redmouth disease caused by the pathogen Yersinia ruckeri is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of Y. ruckeri have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian Y. ruckeri isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic Yersiniae, pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other Y. ruckeri plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the Y. ruckeri NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (tra). In contrast to other Yersinia plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other Y. ruckeri plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the Y. ruckeri 150 and Y. ruckeri ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for de novo genome sequencing. As the tra operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to Y. ruckeri virulence.


September 22, 2019  |  

A continuous genome assembly of the corkwing wrasse (Symphodus melops).

The wrasses (Labridae) are one of the most successful and species-rich families of the Perciformes order of teleost fish. Its members display great morphological diversity, and occupy distinct trophic levels in coastal waters and coral reefs. The cleaning behaviour displayed by some wrasses, such as corkwing wrasse (Symphodus melops), is of particular interest for the salmon aquaculture industry to combat and control sea lice infestation as an alternative to chemicals and pharmaceuticals. There are still few genome assemblies available within this fish family for comparative and functional studies, despite the rapid increase in genome resources generated during the past years. Here, we present a highly continuous genome assembly of the corkwing wrasse using PacBio SMRT sequencing (x28.8) followed by error correction with paired-end Illumina data (x132.9). The present genome assembly consists of 5040 contigs (N50?=?461,652?bp) and a total size of 614 Mbp, of which 8.5% of the genome sequence encode known repeated elements. The genome assembly covers 94.21% of highly conserved genes across ray-finned fish species. We find evidence for increased copy numbers specific for corkwing wrasse possibly highlighting diversification and adaptive processes in gene families including N-linked glycosylation (ST8SIA6) and stress response kinases (HIPK1). By comparative analyses, we discover that de novo repeats, often not properly investigated during genome annotation, encode hundreds of immune-related genes. This new genomic resource, together with the ballan wrasse (Labrus bergylta), will allow for in-depth comparative genomics as well as population genetic analyses for the understudied wrasses. Copyright © 2018 Elsevier Inc. All rights reserved.


September 22, 2019  |  

Genomic analysis of multi-resistant Staphylococcus capitis associated with neonatal sepsis.

Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen. Copyright © 2018 Carter et al.


September 22, 2019  |  

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested.Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378.Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.


September 22, 2019  |  

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component “Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing” we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin. Copyright © 2018 American Society for Histocompatibility and Immunogenetics. All rights reserved.


September 22, 2019  |  

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.


September 22, 2019  |  

Unraveling microbial communities associated with methylmercury production in paddy soils.

Rice consumption is now recognized as an important pathway of human exposure to the neurotoxin methylmercury (MeHg), particularly in countries where rice is a staple food. Although the discovery of a two-gene cluster hgcAB has linked Hg methylation to several phylogenetically diverse groups of anaerobic microorganisms converting inorganic mercury (Hg) to MeHg, the prevalence and diversity of Hg methylators in microbial communities of rice paddy soils remain unclear. We characterized the abundance and distribution of hgcAB genes using third-generation PacBio long-read sequencing and Illumina short-read metagenomic sequencing, in combination with quantitative PCR analyses in several mine-impacted paddy soils from southwest China. Both Illumina and PacBio sequencing analyses revealed that Hg methylating communities were dominated by iron-reducing bacteria (i.e., Geobacter) and methanogens, with a relatively low abundance of hgcA + sulfate-reducing bacteria in the soil. A positive correlation was observed between the MeHg content in soil and the relative abundance of Geobacter carrying the hgcA gene. Phylogenetic analysis also uncovered some hgcAB sequences closely related to three novel Hg methylators, Geobacter anodireducens, Desulfuromonas sp. DDH964, and Desulfovibrio sp. J2, among which G. anodireducens was validated for its ability to methylate Hg. These findings shed new light on microbial community composition and major clades likely driving Hg methylation in rice paddy soils.


September 22, 2019  |  

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased ß-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease.


September 22, 2019  |  

Purge Haplotigs: allelic contig reassignment for third-gen diploid genome assemblies.

Recent developments in third-gen long read sequencing and diploid-aware assemblers have resulted in the rapid release of numerous reference-quality assemblies for diploid genomes. However, assembly of highly heterozygous genomes is still problematic when regional heterogeneity is so high that haplotype homology is not recognised during assembly. This results in regional duplication rather than consolidation into allelic variants and can cause issues with downstream analysis, for example variant discovery, or haplotype reconstruction using the diploid assembly with unpaired allelic contigs.A new pipeline-Purge Haplotigs-was developed specifically for third-gen sequencing-based assemblies to automate the reassignment of allelic contigs, and to assist in the manual curation of genome assemblies. The pipeline uses a draft haplotype-fused assembly or a diploid assembly, read alignments, and repeat annotations to identify allelic variants in the primary assembly. The pipeline was tested on a simulated dataset and on four recent diploid (phased) de novo assemblies from third-generation long-read sequencing, and compared with a similar tool. After processing with Purge Haplotigs, haploid assemblies were less duplicated with minimal impact on genome completeness, and diploid assemblies had more pairings of allelic contigs.Purge Haplotigs improves the haploid and diploid representations of third-gen sequencing based genome assemblies by identifying and reassigning allelic contigs. The implementation is fast and scales well with large genomes, and it is less likely to over-purge repetitive or paralogous elements compared to alignment-only based methods. The software is available at https://bitbucket.org/mroachawri/purge_haplotigs under a permissive MIT licence.


September 22, 2019  |  

Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China.

Florfenicol is a derivative of chloramphenicol that is used only for the treatment of animal diseases. A key resistance gene for florfenicol, floR, can spread among bacteria of the same and different species or genera through horizontal gene transfer. To analyze the potential transmission of resistance genes between animal and human pathogens, we investigated floR in Klebsiella pneumoniae isolates from patient samples. floR in human pathogens may originate from animal pathogens and would reflect the risk to human health of using antimicrobial agents in animals.PCR was used to identify floR-positive strains. The floR genes were cloned, and the minimum inhibitory concentrations (MICs) were determined to assess the relative resistance levels of the genes and strains. Sequencing and comparative genomics methods were used to analyze floR gene-related sequence structure as well as the molecular mechanism of resistance dissemination.Of the strains evaluated, 20.42% (67/328) were resistant to florfenicol, and 86.96% (20/23) of the floR-positive strains demonstrated high resistance to florfenicol with MICs =512 µg/mL. Conjugation experiments showed that transferrable plasmids carried the floR gene in three isolates. Sequencing analysis of a plasmid approximately 125 kb in size (pKP18-125) indicated that the floR gene was flanked by multiple copies of mobile genetic elements. Comparative genomics analysis of a 9-kb transposon-like fragment of pKP18-125 showed that an approximately 2-kb sequence encoding lysR-floR-virD2 was conserved in the majority (79.01%, 83/105) of floR sequences collected from NCBI nucleotide database. Interestingly, the most similar sequence was a 7-kb fragment of plasmid pEC012 from an Escherichia coli strain isolated from a chicken.Identified on a transferable plasmid in the human pathogen K. pneumoniae, the floR gene may be disseminated through horizontal gene transfer from animal pathogens. Studies on the molecular mechanism of resistance gene dissemination in different bacterial species of animal origin could provide useful information for preventing or controlling the spread of resistance between animal and human pathogens.


September 22, 2019  |  

Insights into the microbiota of Asian seabass (Lates calcarifer) with tenacibaculosis symptoms and description of sp. nov. Tenacibaculum singaporense

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the Bacteroidetes genus Tenacibaculum, most notably T. maritimum. The impact of tenacibaculosis on fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissue types of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farm fish. The microbiota of diseased farm fish was dominated by Proteobacteria (relative abundancetextpmstandard deviation, 74.5%textpm22.8%) and Bacteroidetes (18.07%textpm21.7%), the latter mainly comprised by a high abundance of Tenacibaculum species (17.6%textpm20.7%). In healthy seabass Proteobacteria had also highest relative abundance (48.04%textpm0.02%), but Firmicutes (34.2%textpm0.02%) and Fusobacteria (12.0%textpm0.03%) were the next two major constituents. Experimentally infected fish developed lesions characteristic for tenacibaculosis, but the microbiota was primarily dominated by Proteobacteria (90.4%textpm0.2%) and Firmicutes (6.2%textpm0.1%). The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in the commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One strain was isolated and is described here as sp. nov. Tenacibaculum singaporense TLL-A1T (=DSM 106434T, KCTC 62393T). The genome of T. singaporense was sequenced and compared to those of T. maritimum DSM 17995T and the newly sequenced T. mesophilum DSM 13764T.


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