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September 22, 2019

Sequencing of Panax notoginseng genome reveals genes involved in disease resistance and ginsenoside biosynthesis

Background: Panax notoginseng is a traditional Chinese herb with high medicinal and economic value. There has been considerable research on the pharmacological activities of ginsenosides contained in Panax spp.; however, very little is known about the ginsenoside biosynthetic pathway. Results: We reported the first de novo genome of 2.36 Gb of sequences from P. notoginseng with 35,451 protein-encoding genes. Compared to other plants, we found notable gene family contraction of disease-resistance genes in P. notoginseng, but notable expansion for several ATP-binding cassette (ABC) transporter subfamilies, such as the Gpdr subfamily, indicating that ABCs might be an additional mechanism for the plant to cope with biotic stress. Combining eight transcriptomes of roots and aerial parts, we identified several key genes, their transcription factor binding sites and all their family members involved in the synthesis pathway of ginsenosides in P. notoginseng, including dammarenediol synthase, CYP716 and UGT71. Conclusions: The complete genome analysis of P. notoginseng, the first in genus Panax, will serve as an important reference sequence for improving breeding and cultivation of this important nutraceutical and medicinal but vulnerable plant species.


September 22, 2019

The draft genomes of Elizabethkingia anophelis of equine origin are genetically similar to three isolates from human clinical specimens.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of ß-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.


September 22, 2019

Integrating long-range connectivity information into de Bruijn graphs.

The de Bruijn graph is a simple and efficient data structure that is used in many areas of sequence analysis including genome assembly, read error correction and variant calling. The data structure has a single parameter k, is straightforward to implement and is tractable for large genomes with high sequencing depth. It also enables representation of multiple samples simultaneously to facilitate comparison. However, unlike the string graph, a de Bruijn graph does not retain long range information that is inherent in the read data. For this reason, applications that rely on de Bruijn graphs can produce sub-optimal results given their input data.We present a novel assembly graph data structure: the Linked de Bruijn Graph (LdBG). Constructed by adding annotations on top of a de Bruijn graph, it stores long range connectivity information through the graph. We show that with error-free data it is possible to losslessly store and recover sequence from a Linked de Bruijn graph. With assembly simulations we demonstrate that the LdBG data structure outperforms both our de Bruijn graph and the String Graph Assembler (SGA). Finally we apply the LdBG to Klebsiella pneumoniae short read data to make large (12 kbp) variant calls, which we validate using PacBio sequencing data, and to characterize the genomic context of drug-resistance genes.Linked de Bruijn Graphs and associated algorithms are implemented as part of McCortex, which is available under the MIT license at https://github.com/mcveanlab/mccortex.Supplementary data are available at Bioinformatics online.


September 22, 2019

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids.

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1a, IncX1ß and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (ß-lactam resistance), blaKPC-2 (carbapenem resistance) and ?blaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.


September 22, 2019

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.

Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan.KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features.A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain.We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.


September 22, 2019

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.


September 22, 2019

Novel enterobacter lineage as leading cause of nosocomial outbreak involving carbapenemase-producing strains.

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a blaVIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring blaCTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.


September 22, 2019

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening.

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019

The complete methylome of an entomopathogenic bacterium reveals the existence of loci with unmethylated adenines.

DNA methylation can serve to control diverse phenomena in eukaryotes and prokaryotes, including gene regulation leading to cell differentiation. In bacteria, DNA methylomes (i.e., methylation state of each base of the whole genome) have been described for several species, but methylome profile variation during the lifecycle has rarely been studied, and only in a few model organisms. Moreover, major phenotypic changes have been reported in several bacterial strains with a deregulated methyltransferase, but the corresponding methylome has rarely been described. Here we report the first methylome description of an entomopathogenic bacterium, Photorhabdus luminescens. Eight motifs displaying a high rate of methylation (>94%) were identified. The methylome was strikingly stable over course of growth, but also in a subpopulation responsible for a critical step in the bacterium’s lifecycle: successful survival and proliferation in insects. The rare unmethylated GATC motifs were preferentially located in putative promoter regions, and most of them were methylated after Dam methyltransferase overexpression, suggesting that DNA methylation is involved in gene regulation. Our findings bring key insight into bacterial methylomes and encourage further research to decipher the role of loci protected from DNA methylation in gene regulation.


September 22, 2019

Characterization of a novel blaKLUC variant with reduced ß-lactam resistance from an IncA/C group plasmid in a clinical Klebsiella pneumoniae isolate.

Similar to other CTX-M family enzymes, KLUC is a recently identified and emerging determinant of cefotaxime resistance that has been recovered from at least three Enterobacteriaceae species, including Kluyvera cryocrescens, Escherichia coli, and Enterobacter cloacae. Whether this extended-spectrum ß-lactamase (ESBL) has been disseminated among commonly isolated Enterobacteriaceae is worthy of further investigation. In this study, we screened 739 nosocomial Enterobacteriaceae isolates (240 Klebsiella pneumoniae and 499 E. coli strains) and found that one K. pneumoniae and four E. coli isolates harbored the blaKLUC gene. Three blaKLUC determinants isolated from E. coli were entirely identical to a blaKLUC-3 gene previously recovered in the same hospital. PFGE of four blaKLUC-harboring E. coli strains showed that prevalence of these determinants was most likely mediated by horizontal gene transfer but not clonal dissemination. However, the variant isolated from K. pneumoniae belonged to a novel member of the KLUC enzyme group. This newly identified enzyme (KLUC-5) has an amino acid substitution compared with previously identified KLUC-1 (G18S) and KLUC-3 (G240D). Antimicrobial susceptibility tests showed that KLUC-5 significantly reduced resistance activity to almost all the selected antimicrobials compared to previously identified KLUC-3. Site-directed mutagenesis showed that blaKLUC-5-D240G and blaKLUC-5-S18G significantly enhanced the MIC against its best substrate. Conjugation and S1-PFGE indicated that blaKLUC-5 was located on a transferable plasmid, which was further decoded by single-molecule, real-time sequencing. Comparative genome analysis showed that its backbone exhibited genetic homology to the IncA/C incompatibility group plasmids. A transposable element, ISEcp1, was detected 256-bp upstream of the blaKLUC-5 gene; this location was inconsistent with the previously identified blaKLUC-1 but congruent with the variants recovered from E. coli in the same hospital. These data provide evidence of the increasingly emerging KLUC group of ESBLs in China.


September 22, 2019

In vivo evolution of drug-resistant Mycobacterium tuberculosis in patients during long-term treatment.

In the current scenario, the drug-resistant tuberculosis is a significant challenge in the control of tuberculosis worldwide. In order to investigate the in vivo evolution of drug-resistant M. tuberculosis, the present study envisaged sequencing of the draft genomes of 18 serial isolates from four pre-extensively drug-resistant (pre-XDR) tuberculosis patients for continuous genetic alterations.All of the isolates harbored single nucleotide polymorphisms (SNPs) ranging from 1303 to 1309 with M. tuberculosis H37Rv as the reference. SNPs ranged from 0 to 12 within patients. The evolution rates were higher than the reported SNPs of 0.5 in the four patients. All the isolates exhibited mutations at sites of known drug targets, while some contained mutations in uncertain drug targets including folC, proZ, and pyrG. The compensatory substitutions for rescuing these deleterious mutations during evolution were only found in RpoC I491T in one patient. Many loci with microheterogeneity showed transient mutations in different isolates. Ninety three SNPs exhibited significant association with refractory pre-XDR TB isolates.Our results showed evolutionary changes in the serial genetic characteristics of the pre-XDR TB patients due to accumulation of the fixed drug-resistant related mutations, and the transient mutations under continuous antibiotics pressure over several years.


September 22, 2019

Co-location of the blaKPC-2, blaCTX-M-65, rmtB and virulence relevant factors in an IncFII plasmid from a hypermucoviscous Klebsiella pneumoniae isolate.

Hypervirulent variants of klebsiella pneumoniae (hvKP), which cause serious infections not only healthy individuals, but also the immunocompromised patients, have been increasingly reported recently. One conjugation of a hypermucoviscous strian SWU01 co-carried the resistance gene blaKPC-2 and virulence gene iroN by the PCR detection from three carbapenem-resistance hvKP. To know the genetic context of this plasmid. The whole genome of this strain was sequenced. We got a 162,552-bp plasmid (pSWU01) which co-carried the resistance gene blaKPC-2 and virulence gene iroN. It is composed of a typical IncFII-type backbone, five resistance genes including blaCTX-M-65, blaKPC-2, blaSHV-12, blaTEM-1 and rmtB, and several virulence relevant factors including iroN, traT and toxin-antitoxin systems. The plasmid pSWU01 co-carrying the multidrug resistance determinants and virulence relevant factors from the hypermucoviscous K. pneumoniae represents a novel therapeutic challenge. Copyright © 2018 Elsevier Ltd. All rights reserved.


September 22, 2019

Comparative genomics reveals diverse capsular polysaccharide synthesis gene clusters in emerging Raoultella planticola.

Raoultella planticola is an emerging zoonotic pathogen that is associated with rare but life-threatening cases of bacteremia, biliary tract infections, and urinary tract infections. Moreover, increasing antimicrobial resistance in the organism poses a potential threat to public health. In spite of its importance as a human pathogen, the genome of R. planticola remains largely unexplored and little is known about its virulence factors. Although lipopolysaccharides has been detected in R. planticola and implicated in the virulence in earlier studies, the genetic background is unknown. Here, we report the complete genome and comparative analysis of the multidrug-resistant clinical isolate R. planticola GODA. The complete genome sequence of R. planticola GODA was sequenced using single-molecule real-time DNA sequencing. Comparative genomic analysis reveals distinct capsular polysaccharide synthesis gene clusters in R. planticola GODA. In addition, we found bla TEM-57 and multiple transporters related to multidrug resistance. The availability of genomic data in open databases of this emerging zoonotic pathogen, in tandem with our comparative study, provides better understanding of R. planticola and the basis for future work.


September 22, 2019

Genome-wide SNP and InDel mutations in Mycobacterium tuberculosis associated with rifampicin and isoniazid resistance

Objective: Multiple resistances to isoniazid and rifampicin lead to the majority of death associated with M. tuberculosis infection. This study aimed to characterize the single nucleotide polymorphisms (SNPs) and insertion and deletion (InDel) mutations associated with isoniazid and rifampicin resistance. Methods: The M. tuberculosis strain H37Rv was cultured and treated with isoniazid or rifampicin for generations. Total DNA samples from different generations were extracted for construction of DNA library, and the SNP and InDel mutation in different samples were detected by whole genome sequencing. Bioinformatics analysis such as phylogenetic tree and heap map were also performed. Results: Totally 58 nonsynonymous SNP mutations, 64 synonymous SNP mutations, and 99 SNP mutations in intergenic regions were detected in M. tuberculosis strains treated with rifampicin or isoniazid. Seven InDel mutations were found in the intergenic regions, and also six frameshift InDel mutation and three non- frameshift InDel mutations were also characterized. The phylogenetic tree showed clustering of all samples into three main subgroups. A great number of known and newly identified genes associated with drug resistance were detected in M. tuberculosis, showing distinct mutation patterns. Conclusion: By whole genome sequencing, many genetic mutations in both known and new genes associated with isoniazid and rifampicin resistance were charac- terized in M. tuberculosis.


September 22, 2019

Detection and characterization of a clinical Escherichia coli ST3204 strain coproducing NDM-16 and MCR-1.

A plasmid-mediated colistin resistance gene, mcr-1, has been reported worldwide and has caused concern regarding a major therapeutic challenge. Alarmingly, mcr-1 has spread into clinical carbapenem-resistant Enterobacteriaceae isolates, resulting in extensively drug-resistant and even pan drug-resistant isolates that can cause untreatable infections. In this study, we report isolation of an extensively drug-resistant Escherichia coli strain EC1188 that coproduces NDM-16 and MCR-1 from a urine sample taken from a patient with craniocerebral injury.E. coli strain EC1188 was identified and subjected to genotyping, susceptibility testing and conjugation experiments. The genetic locations of blaNDM-16 and mcr-1 were established with southern blot hybridization. The complete genome sequence of this strain was obtained and the genetic characteristics of the mcr-1- and blaNDM-16-harboring plasmids were analyzed. In addition, comparative genetic analyses of mcr-1 and blaNDM-16 with closely related plasmids were also carried out.Whole-genome sequencing revealed that strain EC1188 possess various resistance genes and virulence genes. S1-pulsed-field gel electrophoresis and southern blot suggested that the blaNDM-16 and mcr-1 genes were located on an ~65 kb plasmid and an ~80 kb plasmid, respectively. Moreover, the two genes could successfully transfer their resistance phenotype to E. coli strain C600. Sequence analysis showed that these two plasmids possessed high sequence similarity to previously reported blaNDM-5-harboring and mcr-1-harboring plasmids in China.To the best of our knowledge, this is the first report to isolate an E. coli strain that coproduces NDM-16 and MCR-1. In addition, we characterized the blaNDM-16-harboring plasmid for the first time. Our study further emphasizes that the co-occurrence of the two prevalent transferrable resistance plasmids in a single isolate is highly significant because infections caused by MCR-1-producing carbapenem-resistant Enterobacteriaceae isolates are increasing each year. It is imperative to perform active surveillance to prevent further dissemination of MCR-1-producing CRE isolates.


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