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July 7, 2019  |  

Whole genome DNA sequence analysis of Salmonella subspecies enterica serotype Tennessee obtained from related peanut butter foodborne outbreaks.

Establishing an association between possible food sources and clinical isolates requires discriminating the suspected pathogen from an environmental background, and distinguishing it from other closely-related foodborne pathogens. We used whole genome sequencing (WGS) to Salmonella subspecies enterica serotype Tennessee (S. Tennessee) to describe genomic diversity across the serovar as well as among and within outbreak clades of strains associated with contaminated peanut butter. We analyzed 71 isolates of S. Tennessee from disparate food, environmental, and clinical sources and 2 other closely-related Salmonella serovars as outgroups (S. Kentucky and S. Cubana), which were also shot-gun sequenced. A whole genome single nucleotide polymorphism (SNP) analysis was performed using a maximum likelihood approach to infer phylogenetic relationships. Several monophyletic lineages of S. Tennessee with limited SNP variability were identified that recapitulated several food contamination events. S. Tennessee clades were separated from outgroup salmonellae by more than sixteen thousand SNPs. Intra-serovar diversity of S. Tennessee was small compared to the chosen outgroups (1,153 SNPs), suggesting recent divergence of some S. Tennessee clades. Analysis of all 1,153 SNPs structuring an S. Tennessee peanut butter outbreak cluster revealed that isolates from several food, plant, and clinical isolates were very closely related, as they had only a few SNP differences between them. SNP-based cluster analyses linked specific food sources to several clinical S. Tennessee strains isolated in separate contamination events. Environmental and clinical isolates had very similar whole genome sequences; no markers were found that could be used to discriminate between these sources. Finally, we identified SNPs within variable S. Tennessee genes that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks. Using WGS can delimit contamination sources for foodborne illnesses across multiple outbreaks and reveal otherwise undetected DNA sequence differences essential to the tracing of bacterial pathogens as they emerge.

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July 7, 2019  |  

Structural and functional analysis of the finished genome of the recently isolated toxic Anabaena sp. WA102.

Very few closed genomes of the cyanobacteria that commonly produce toxic blooms in lakes and reservoirs are available, limiting our understanding of the properties of these organisms. A new anatoxin-a-producing member of the Nostocaceae, Anabaena sp. WA102, was isolated from a freshwater lake in Washington State, USA, in 2013 and maintained in non-axenic culture.The Anabaena sp. WA102 5.7 Mbp genome assembly has been closed with long-read, single-molecule sequencing and separately a draft genome assembly has been produced with short-read sequencing technology. The closed and draft genome assemblies are compared, showing a correlation between long repeats in the genome and the many gaps in the short-read assembly. Anabaena sp. WA102 encodes anatoxin-a biosynthetic genes, as does its close relative Anabaena sp. AL93 (also introduced in this study). These strains are distinguished by differences in the genes for light-harvesting phycobilins, with Anabaena sp. AL93 possessing a phycoerythrocyanin operon. Biologically relevant structural variants in the Anabaena sp. WA102 genome were detected only by long-read sequencing: a tandem triplication of the anaBCD promoter region in the anatoxin-a synthase gene cluster (not triplicated in Anabaena sp. AL93) and a 5-kbp deletion variant present in two-thirds of the population. The genome has a large number of mobile elements (160). Strikingly, there was no synteny with the genome of its nearest fully assembled relative, Anabaena sp. 90.Structural and functional genome analyses indicate that Anabaena sp. WA102 has a flexible genome. Genome closure, which can be readily achieved with long-read sequencing, reveals large scale (e.g., gene order) and local structural features that should be considered in understanding genome evolution and function.

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July 7, 2019  |  

A roadmap for gene system development in Clostridium.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019  |  

First report of blaIMP-14 on a plasmid harboring multiple drug resistance genes in Escherichia coli ST131.

The blaIMP-14 carbapenem resistance gene has largely previously been observed in Pseudomonas aeruginosa and Acinetobacter spp. As part of global surveillance and sequencing of carbapenem-resistant E. coli, we identified an ST131 strain harboring blaIMP-14 within a class 1 integron, itself nested within a ~54kb multi-drug resistance region on an epidemic IncA/C2 plasmid. The emergence of blaIMP-14 in this context in the ST131 lineage is of potential clinical concern. Copyright © 2016 Stoesser et al.

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July 7, 2019  |  

Complete genome sequence of Vibrio vulnificus FORC_017 isolated from a patient with a hemorrhagic rash after consuming raw dotted gizzard shad.

Vibrio vulnificus, a resident in the human gut, is frequently found in seafood, causing food-borne illnesses including gastroenteritis and severe septicemia. While V. vulnificus has been known to be one of the major food-borne pathogens, pathogenicity and virulence factors are not fully understood yet. To extend our understanding of the pathogenesis of V. vulnificus at the genomic level, the genome of V. vulnificus FORC_017 isolated from a female patient experiencing a hemorrhagic rash was completely sequenced and analyzed.Three discontinuous contigs were generated from a hybrid assembly using Illumina MiSeq and PacBio platforms, revealing that the genome of the FORC_017 consists of two circular chromosomes and a plasmid. Chromosome I consists of 3,253,417-bp (GC content 46.49 %) containing 2943 predicted open reading frames (ORFs) and chromosome II of 1,905,745-bp (GC content 46.90 %) containing 1638 ORFs. The plasmid pFORC17 consists of 70,069-bp (GC content 43.77 %) containing 84 ORFs. The average nucleotide identity (ANI) value of the FORC_017 and CMCP6 strains was 98.53, suggesting that they are closely related.Pathogenesis-associated genes including vvhA, rtx gene cluster, and various hemolysin genes were present in FORC_017. In addition, three complete secretion systems (Type I, II and VI) as well as iron uptake-related genes for virulence of the FORC_017 were detected, suggesting that this strain is pathogenic. Further comparative genome analysis revealed that FORC_017 and CMCP6 share major toxin genes including vvhA and rtx for pathogenesis activities. The genome information of the FORC_017 provides novel insights into pathogenicity and virulence factors of V. vulnificus.

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July 7, 2019  |  

Complete genome sequences of three outbreak-associated Legionella pneumophila isolates.

We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires’ disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates. Copyright © 2016 Morrison et al.

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July 7, 2019  |  

Homologous recombination within large chromosomal regions facilitates acquisition of beta-lactam and vancomycin resistance in Enterococcus faecium.

The transfer of DNA between Enterococcus faecium strains has been characterized by both the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work we report on the whole genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and vancomycin susceptible D344RRF to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analysed by whole genome sequencing. In all cases but one, the penicillin binding protein 5 gene (pbp5) and the Tn5382-vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 Single nucleotide variants (SNV) analysis suggests that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. Transfer of genomic DNA was also associated with transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with transfer initiated by a cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid/chromosome cointegrate in the donor prior to transfer. Entry into the recipient chromosome occurs most commonly across regions of homology between donor and recipient chromosomes. Copyright © 2016 García-Solache et al.

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July 7, 2019  |  

Representing genetic variation with synthetic DNA standards.

The identification of genetic variation with next-generation sequencing is confounded by the complexity of the human genome sequence and by biases that arise during library preparation, sequencing and analysis. We have developed a set of synthetic DNA standards, termed ‘sequins’, that emulate human genetic features and constitute qualitative and quantitative spike-in controls for genome sequencing. Sequencing reads derived from sequins align exclusively to an artificial in silico reference chromosome, rather than the human reference genome, which allows them them to be partitioned for parallel analysis. Here we use this approach to represent common and clinically relevant genetic variation, ranging from single nucleotide variants to large structural rearrangements and copy-number variation. We validate the design and performance of sequin standards by comparison to examples in the NA12878 reference genome, and we demonstrate their utility during the detection and quantification of variants. We provide sequins as a standardized, quantitative resource against which human genetic variation can be measured and diagnostic performance assessed.

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July 7, 2019  |  

Complete genome sequence of thermophilic Bacillus smithii type strain DSM 4216(T).

Bacillus smithii is a facultatively anaerobic, thermophilic bacterium able to use a variety of sugars that can be derived from lignocellulosic feedstocks. Being genetically accessible, it is a potential new host for biotechnological production of green chemicals from renewable resources. We determined the complete genomic sequence of the B. smithii type strain DSM 4216(T), which consists of a 3,368,778 bp chromosome (GenBank accession number CP012024.1) and a 12,514 bp plasmid (GenBank accession number CP012025.1), together encoding 3880 genes. Genome annotation via RAST was complemented by a protein domain analysis. Some unique features of B. smithii central metabolism in comparison to related organisms included the lack of a standard acetate production pathway with no apparent pyruvate formate lyase, phosphotransacetylase, and acetate kinase genes, while acetate was the second fermentation product.

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July 7, 2019  |  

Comparative genomic analysis of isoproturon-mineralizing sphingomonads reveals the isoproturon catabolic mechanism.

The worldwide use of the phenylurea herbicide, isoproturon (IPU), has resulted in considerable concern about its environmental fate. Although many microbial metabolites of IPU are known and IPU-mineralizing bacteria have been isolated, the molecular mechanism of IPU catabolism has not been elucidated yet. In this study, complete genes that encode the conserved IPU catabolic pathway were revealed, based on comparative analysis of the genomes of three IPU-mineralizing sphingomonads and subsequent experimental validation. The complete genes included a novel hydrolase gene ddhA, which is responsible for the cleavage of the urea side chain of the IPU demethylated products; a distinct aniline dioxygenase gene cluster adoQTA1A2BR, which has a broad substrate range; and an inducible catechol meta-cleavage pathway gene cluster adoXEGKLIJC. Furthermore, the initial mono-N-demethylation genes pdmAB were further confirmed to be involved in the successive N-demethylation of the IPU mono-N-demethylated product. These IPU-catabolic genes were organized into four transcription units and distributed on three plasmids. They were flanked by multiple mobile genetic elements and highly conserved among IPU-mineralizing sphingomonads. The elucidation of the molecular mechanism of IPU catabolism will enhance our understanding of the microbial mineralization of IPU and provide insights into the evolutionary scenario of the conserved IPU-catabolic pathway. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019  |  

Mobile genetic elements: in silico, in vitro, in vivo.

Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function and evolution. However, there is currently a gap between the fast pace of TE discovery in silico, driven by the exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, ‘Mobile Genetic Elements: in silico, in vitro, in vivo’, held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA.© 2016 John Wiley & Sons Ltd.

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July 7, 2019  |  

Adaptive radiation by waves of gene transfer leads to fine-scale resource partitioning in marine microbes.

Adaptive radiations are important drivers of niche filling, since they rapidly adapt a single clade of organisms to ecological opportunities. Although thought to be common for animals and plants, adaptive radiations have remained difficult to document for microbes in the wild. Here we describe a recent adaptive radiation leading to fine-scale ecophysiological differentiation in the degradation of an algal glycan in a clade of closely related marine bacteria. Horizontal gene transfer is the primary driver in the diversification of the pathway leading to several ecophysiologically differentiated Vibrionaceae populations adapted to different physical forms of alginate. Pathway architecture is predictive of function and ecology, underscoring that horizontal gene transfer without extensive regulatory changes can rapidly assemble fully functional pathways in microbes.

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July 7, 2019  |  

Genomic analysis reveals novel diversity among the 1976 Philadelphia Legionnaires’ disease outbreak isolates and additional ST36 strains.

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires’ disease (LD) at a Pennsylvania veterans’ convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, “Philadelphia-1”, has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of “Broad Street pneumonia”, and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.

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July 7, 2019  |  

Complete circular genome sequence of successful ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (OC8) in Russia: one-megabase genomic inversion, IS256’s spread, and evolution of Russia ST8-IV.

ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaß split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256’s strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

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July 7, 2019  |  

Complete genome sequences of multidrug-resistant Campylobacter jejuni strain 14980A (turkey feces) and Campylobacter coli strain 14983A (housefly from a turkey farm), harboring a novel gentamicin resistance mobile element.

Multidrug resistance (MDR) in foodborne pathogens is a major food safety and public health issue. Here we describe whole-genome sequences of two MDR strains of Campylobacter jejuni and Campylobacter coli from turkey feces and a housefly from a turkey farm. Both strains harbor a novel chromosomal gentamicin resistance mobile element. Copyright © 2016 Miller et al.

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