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July 7, 2019

Metabolomics-driven discovery of a prenylated isatin antibiotic produced by Streptomyces species MBT28.

Actinomycetes are a major source of antimicrobials, anticancer compounds, and other medically important products, and their genomes harbor extensive biosynthetic potential. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known molecules. Here we report the identification of a previously unidentified isatin-type antibiotic produced by Streptomyces sp. MBT28, following a strategy based on NMR-based metabolomics combined with the introduction of streptomycin resistance in the producer strain. NMR-guided isolation by tracking the target proton signal resulted in the characterization of 7-prenylisatin (1) with antimicrobial activity against Bacillus subtilis. The metabolite-guided genome mining of Streptomyces sp. MBT28 combined with proteomics identified a gene cluster with an indole prenyltransferase that catalyzes the conversion of tryptophan into 7-prenylisatin. This study underlines the applicability of NMR-based metabolomics in facilitating the discovery of novel antibiotics.

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July 7, 2019

Contiguity: Contig adjacency graph construction and visualisation

Contiguity is interactive software for the visualization and manipulation of de novo genome assemblies. 14 Contiguity creates and displays information on contig adjacency which is contextualized by the 15 simultaneous display of a comparison between assembled contigs and reference sequence. Where 16 scaffolders allow unambiguous connections between contigs to be resolved into a single scaffold, 17 Contiguity allows the user to create all potential scaffolds in ambiguous regions of the genome. This 18 enables the resolution of novel sequence or structural variants from the assembly. In addition, 19 Contiguity provides a sequencing and assembly agnostic approach for the creation of contig adjacency 20 graphs. To maximize the number of contig adjacencies determined, Contiguity combines information 21 from read pair mappings, sequence overlap and De Bruijn graph exploration. We demonstrate how 22 highly sensitive graphs can be achieved using this method. Contig adjacency graphs allow the user to 23 visualize potential arrangements of contigs in unresolvable areas of the genome. By combining 24 adjacency information with comparative genomics, Contiguity provides an intuitive approach for 25 exploring and improving sequence assemblies. It is also useful in guiding manual closure of long read 26 sequence assemblies. Contiguity is an open source application, implemented using Python and the 27 Tkinter GUI package that can run on any Unix, OSX and Windows operating system. It has been 28 designed and optimized for bacterial assemblies. Contiguity is available at 29 http://mjsull.github.io/Contiguity .

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July 7, 2019

DNA N(6)-methyladenine: a new epigenetic mark in eukaryotes?

DNA N(6)-adenine methylation (N(6)-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

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July 7, 2019

Complete genome sequences of four Escherichia coli ST95 isolates from bloodstream infections.

Finished genome sequences are presented for four Escherichia coli strains isolated from bloodstream infections at San Francisco General Hospital. These strains provide reference sequences for four major fimH-identified sublineages within the multilocus sequence type (MLST) ST95 group, and provide insights into pathogenicity and differential antimicrobial susceptibility within this group. Copyright © 2015 Stephens et al.

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July 7, 2019

Fosfomycin resistance in Escherichia coli, Pennsylvania, USA.

Fosfomycin resistance in Escherichia coli is rare in the United States. An extended-spectrum ß-lactamase-producing E. coli clinical strain identified in Pennsylvania, USA, showed high-level fosfomycin resistance caused by the fosA3 gene. The IncFII plasmid carrying this gene had a structure similar to those found in China, where fosfomycin resistance is commonly described.

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July 7, 2019

Twenty years of bacterial genome sequencing.

Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a ‘sequencing singularity’, where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.

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July 7, 2019

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in a university hospital in Nepal reveals the emergence of a novel epidemic clonal lineage.

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as bla(NDM-1), bla(OXA-23) or bla(OXA-58). MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA). Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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July 7, 2019

Clonal dissemination of Enterobacter cloacae harboring blaKPC-3 in the upper midwestern United States.

Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583-1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The complete genome sequence of the emerging pathogen Mycobacterium haemophilum explains its unique culture requirements.

Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae.Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium’s growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae. Copyright © 2015 Tufariello et al.

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July 7, 2019

Comparative genomics and metabolic profiling of the genus Lysobacter.

Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.

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July 7, 2019

Coupling of mRNA structure rearrangement to ribosome movement during bypassing of non-coding regions.

Nearly half of the ribosomes translating a particular bacteriophage T4 mRNA bypass a region of 50 nt, resuming translation 3′ of this gap. How this large-scale, specific hop occurs and what determines whether a ribosome bypasses remain unclear. We apply single-molecule fluorescence with zero-mode waveguides to track individual Escherichia coli ribosomes during translation of T4’s gene 60 mRNA. Ribosomes that bypass are characterized by a 10- to 20-fold longer pause in a non-canonical rotated state at the take-off codon. During the pause, mRNA secondary structure rearrangements are coupled to ribosome forward movement, facilitated by nascent peptide interactions that disengage the ribosome anticodon-codon interactions for slippage. Close to the landing site, the ribosome then scans mRNA in search of optimal base-pairing interactions. Our results provide a mechanistic and conformational framework for bypassing, highlighting a non-canonical ribosomal state to allow for mRNA structure refolding to drive large-scale ribosome movements. 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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