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July 7, 2019

Atypical Salmonella enterica serovars in murine and human infection models: Is it time to reassess our approach to the study of salmonellosis?

Nontyphoidal Salmonella species are globally disseminated pathogens and the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using in vivo murine models and cell lines typically challenged with Salmonella Typhimurium. Although serovars Enteritidis and Typhimurium are responsible for the most of human infections reported to the CDC, several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical S. enterica serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage model of infection is unsuitable for inferring human relevant differences in nontyphoidal Salmonella infections whereas differentiated human THP-1 macrophages allowed these isolates to be further characterised in a more relevant, human context.

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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

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

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July 7, 2019

Horizontal transfer of carbapenemase-encoding plasmids and comparison with hospital epidemiology data.

Carbapenemase-producing organisms have spread worldwide, and infections with these bacteria cause significant morbidity. Horizontal transfer of plasmids that encode carbapenemases plays an important role in the spread of multidrug resistant Gram-negative bacteria. Here we investigate parameters regulating conjugation using an E. coli laboratory strain that lacks plasmids or restriction-enzyme modification systems as a recipient and also using patient isolates as donors and recipients. Because conjugation is tightly regulated, we performed a systematic analysis of the transfer of Klebsiella pneumoniae carbapenemase (blaKPC)-encoding plasmids into multiple strains under different environmental conditions to investigate critical variables. We used four blaKPC-plasmids isolated from patient strains obtained from two hospitals: pKpQIL and pKPC-47e from the National Institutes of Health, and pKPC_UVA01 and pKPC_UVA02 from the University of Virginia. Plasmid transfer frequency differed substantially between different donor and recipient pairs, and was influenced by plasmid content, temperature, and substrate, in addition to donor and recipient strain. pKPC-47e was attenuated in conjugation efficiency across all conditions tested. Despite its presence in multiple clinical species, pKPC_UVA01 had lower conjugation efficiencies than pKpQIL into recipient strains. The conjugation frequency of these plasmids into K. pneumoniae and E. coli patient isolates ranged widely without a clear correlation with clinical epidemiological data. Our results highlight the importance of each variable examined in these controlled experiments. The in vitro models did not reliably predict plasmid mobilization observed in a patient population, indicating that further studies are needed to understand the most important variables affecting horizontal transfer in vivo. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Glutathione-S-transferase FosA6 of Klebsiella pneumoniae origin conferring fosfomycin resistance in ESBL-producing Escherichia coli.

The objectives of this study were to elucidate the genetic context of a novel plasmid-mediated fosA variant, fosA6, conferring fosfomycin resistance and to characterize the kinetic properties of FosA6.The genome of fosfomycin-resistant Escherichia coli strain YD786 was sequenced. Homologues of FosA6 were identified through BLAST searches. FosA6 and FosA(ST258) were purified and characterized using a steady-state kinetic approach. Inhibition of FosA activity was examined with sodium phosphonoformate.Plasmid-encoded glutathione-S-transferase (GST) FosA6 conferring high-level fosfomycin resistance was identified in a CTX-M-2-producing E. coli clinical strain at a US hospital. fosA6 was carried on a self-conjugative, 69 kb IncFII plasmid. The ?lysR-fosA6-?yjiR_1 fragment, located between IS10R and ?IS26, was nearly identical to those on the chromosomes of some Klebsiella pneumoniae strains (MGH78578, PMK1 and KPPR1). FosA6 shared >99% identity with chromosomally encoded FosA(PMK1) in K. pneumoniae of various STs and 98% identity with FosA(ST258), which is commonly found in K. pneumoniae clonal complex (CC) 258 including ST258. FosA6 and FosA(ST258) demonstrated robust GST activities that were comparable to each other. Sodium phosphonoformate, a GST inhibitor, reduced the fosfomycin MICs by 6- to 24-fold for K. pneumoniae and E. coli strains carrying fosA genes on the chromosomes and plasmids, respectively.fosA6, probably captured from the chromosome of K. pneumoniae, conferred high-level fosfomycin resistance in E. coli. FosA6 functioned as a GST and inactivated fosfomycin efficiently. K. pneumoniae may serve as a reservoir of fosfomycin resistance for E. coli.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Structural variation detection using next-generation sequencing data: A comparative technical review.

Structural variations (SVs) are mutations in the genome of size at least fifty nucleotides. They contribute to the phenotypic differences among healthy individuals, cause severe diseases and even cancers by breaking or linking genes. Thus, it is crucial to systematically profile SVs in the genome. In the past decade, many next-generation sequencing (NGS)-based SV detection methods have been proposed due to the significant cost reduction of NGS experiments and their ability to unbiasedly detect SVs to the base-pair resolution. These SV detection methods vary in both sensitivity and specificity, since they use different SV-property-dependent and library-property-dependent features. As a result, predictions from different SV callers are often inconsistent. Besides, the noises in the data (both platform-specific sequencing error and artificial chimeric reads) impede the specificity of SV detection. Poorly characterized regions in the human genome (e.g., repeat regions) greatly impact the reads mapping and in turn affect the SV calling accuracy. Calling of complex SVs requires specialized SV callers. Apart from accuracy, processing speed of SV caller is another factor deciding its usability. Knowing the pros and cons of different SV calling techniques and the objectives of the biological study are essential for biologists and bioinformaticians to make informed decisions. This paper describes different components in the SV calling pipeline and reviews the techniques used by existing SV callers. Through simulation study, we also demonstrate that library properties, especially insert size, greatly impact the sensitivity of different SV callers. We hope the community can benefit from this work both in designing new SV calling methods and in selecting the appropriate SV caller for specific biological studies. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019

The rubber tree genome shows expansion of gene family associated with rubber biosynthesis.

Hevea brasiliensis Muell. Arg, a member of the family Euphorbiaceae, is the sole natural resource exploited for commercial production of high-quality natural rubber. The properties of natural rubber latex are almost irreplaceable by synthetic counterparts for many industrial applications. A paucity of knowledge on the molecular mechanisms of rubber biosynthesis in high yield traits still persists. Here we report the comprehensive genome-wide analysis of the widely planted H. brasiliensis clone, RRIM 600. The genome was assembled based on ~155-fold combined coverage with Illumina and PacBio sequence data and has a total length of 1.55?Gb with 72.5% comprising repetitive DNA sequences. A total of 84,440 high-confidence protein-coding genes were predicted. Comparative genomic analysis revealed strong synteny between H. brasiliensis and other Euphorbiaceae genomes. Our data suggest that H. brasiliensis’s capacity to produce high levels of latex can be attributed to the expansion of rubber biosynthesis-related genes in its genome and the high expression of these genes in latex. Using cap analysis gene expression data, we illustrate the tissue-specific transcription profiles of rubber biosynthesis-related genes, revealing alternative means of transcriptional regulation. Our study adds to the understanding of H. brasiliensis biology and provides valuable genomic resources for future agronomic-related improvement of the rubber tree.

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July 7, 2019

Complete genome sequence of the first KPC-type carbapenemase-positive Proteus mirabilis strain from a bloodstream infectio

Sequencing of the blaKPC-positive strain Proteus mirabilis AOUC-001 was performed using both the MiSeq and PacBio RS II platforms and yielded a single molecule of 4,272,433 bp, representing the complete chromosome. Genome analysis showed the presence of several acquired resistance determinants, including two copies of blaKPC-2 carried on a fragment of a KPC-producing plasmid previously described in Klebsiella pneumoniae. Copyright © 2016 Di Pilato et al.

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July 7, 2019

A commensal bacterium promotes virulence of an opportunistic pathogen via cross-respiration.

Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii Our results show that S. gordonii extensively alters A. actinomycetemcomitans requirements for virulence factors and biosynthetic pathways during infection. In addition, we discovered that the presence of S. gordonii enhances the bioavailability of oxygen during infection, allowing A. actinomycetemcomitans to shift from a primarily fermentative to a respiratory metabolism that enhances its growth yields and persistence. Mechanistically, respiratory metabolism enhances the fitness of A. actinomycetemcomitans in vivo by increasing ATP yields via central metabolism and creating a proton motive force. Our results reveal that, similar to cross-feeding, where one species provides another species with a nutrient, commensal bacteria can also provide electron acceptors that promote the respiratory growth and fitness of pathogens in vivo, an interaction that we term cross-respiration.Commensal bacteria can enhance the virulence of pathogens in mixed-species infections. However, knowledge of the mechanisms underlying this clinically relevant phenomenon is lacking. To bridge this gap, we comprehensively determined the genes a pathogen needs to establish coinfection with a commensal. Our findings show that the metabolism of the pathogen is low-energy-yielding in monoinfection, but in coinfection, the commensal improves the fitness of the pathogen by increasing the bioavailability of oxygen, thereby shifting the pathogen toward a high-energy-yielding metabolism. Similar to cross-feeding, this interaction, which we term cross-respiration, illustrates that commensal bacteria can provide electron acceptors that enhance the virulence of pathogens during infection. Copyright © 2016 Stacy 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

Comparative genomic analysis of Klebsiella pneumoniae subsp. pneumoniae KP617 and PittNDM01, NUHL24835, and ATCC BAA-2146 reveals unique evolutionary history of this strain.

Klebsiella pneumoniae subsp. pneumoniae KP617 is a pathogenic strain that coproduces OXA-232 and NDM-1 carbapenemases. We sequenced the genome of KP617, which was isolated from the wound of a Korean burn patient, and performed a comparative genomic analysis with three additional strains: PittNDM01, NUHL24835 and ATCC BAA-2146.The complete genome of KP617 was obtained via multi-platform whole-genome sequencing. Phylogenetic analysis along with whole genome and multi-locus sequence typing of genes of the Klebsiella pneumoniae species showed that KP617 belongs to the WGLW2 group, which includes PittNDM01 and NUHL24835. Comparison of annotated genes showed that KP617 shares 98.3 % of its genes with PittNDM01. Nineteen antibiotic resistance genes were identified in the KP617 genome: bla OXA-1 and bla SHV-28 in the chromosome, bla NDM-1 in plasmid 1, and bla OXA-232 in plasmid 2 conferred resistance to beta-lactams; however, colistin- and tetracycline-resistance genes were not found. We identified 117 virulence factors in the KP617 genome, and discovered that the genes encoding these factors were also harbored by the reference strains; eight genes were lipopolysaccharide-related and four were capsular polysaccharide-related. A comparative analysis of phage-associated regions indicated that two phage regions are specific to the KP617 genome and that prophages did not act as a vehicle for transfer of antimicrobial resistance genes in this strain.Whole-genome sequencing and bioinformatics analysis revealed similarity in the genome sequences and content, and differences in phage-related genes, plasmids and antimicrobial resistance genes between KP617 and the references. In order to elucidate the precise role of these factors in the pathogenicity of KP617, further studies are required.

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July 7, 2019

The genomic sequence of the oral pathobiont strain NI1060 reveals unique strategies for bacterial competition and pathogenicity.

Strain NI1060 is an oral bacterium responsible for periodontitis in a murine ligature-induced disease model. To better understand its pathogenicity, we have determined the complete sequence of its 2,553,982 bp genome. Although closely related to Pasteurella pneumotropica, a pneumonia-associated rodent commensal based on its 16S rRNA, the NI1060 genomic content suggests that they are different species thriving on different energy sources via alternative metabolic pathways. Genomic and phylogenetic analyses showed that strain NI1060 is distinct from the genera currently described in the family Pasteurellaceae, and is likely to represent a novel species. In addition, we found putative virulence genes involved in lipooligosaccharide synthesis, adhesins and bacteriotoxic proteins. These genes are potentially important for host adaption and for the induction of dysbiosis through bacterial competition and pathogenicity. Importantly, strain NI1060 strongly stimulates Nod1, an innate immune receptor, but is defective in two peptidoglycan recycling genes due to a frameshift mutation. The in-depth analysis of its genome thus provides critical insights for the development of NI1060 as a prime model system for infectious disease.

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July 7, 2019

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen.

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in “arms races” with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence. © 2016 Faino et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Distinct Salmonella enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.

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