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July 7, 2019  |  

Highlights of the 11th International Bordetella Symposium: from basic biology to vaccine development.

Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Genomic analyses of multidrug resistant Pseudomonas aeruginosa PA1 resequenced by single-molecule real-time sequencing.

As a third-generation sequencing (TGS) method, single-molecule real-time (SMRT) technology provides long read length, and it is well suited for resequencing projects and de novo assembly. In the present study, Pseudomonas aeruginosa PA1 was characterized and resequenced using SMRT technology. PA1 was also subjected to genomic, comparative and pan-genomic analyses. The multidrug resistant strain PA1 possesses a 6,498,072 bp genome and a sequence type of ST-782. The genome of PA1 was also visualized, and the results revealed the details of general genome annotations, virulence factors, regulatory proteins (RPs), secretion system proteins, type II toxin-antitoxin (T-A) pairs and genomic islands. Whole genome comparison analysis suggested that PA1 exhibits similarity to other P. aeruginosa strains but differs in terms of horizontal gene transfer (HGT) regions, such as prophages and genomic islands. Phylogenetic analyses based on 16S rRNA sequences demonstrated that PA1 is closely related to PAO1, and P. aeruginosa strains can be divided into two main groups. The pan-genome of P. aeruginosa consists of a core genome of approximately 4,000 genes and an accessory genome of at least 6,600 genes. The present study presented a detailed, visualized and comparative analysis of the PA1 genome, to enhance our understanding of this notorious pathogen. © 2016 The Author(s).

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July 7, 2019  |  

An ethnically relevant consensus Korean reference genome is a step towards personal reference genomes.

Human genomes are routinely compared against a universal reference. However, this strategy could miss population-specific and personal genomic variations, which may be detected more efficiently using an ethnically relevant or personal reference. Here we report a hybrid assembly of a Korean reference genome (KOREF) for constructing personal and ethnic references by combining sequencing and mapping methods. We also build its consensus variome reference, providing information on millions of variants from 40 additional ethnically homogeneous genomes from the Korean Personal Genome Project. We find that the ethnically relevant consensus reference can be beneficial for efficient variant detection. Systematic comparison of human assemblies shows the importance of assembly quality, suggesting the necessity of new technologies to comprehensively map ethnic and personal genomic structure variations. In the era of large-scale population genome projects, the leveraging of ethnicity-specific genome assemblies as well as the human reference genome will accelerate mapping all human genome diversity.

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July 7, 2019  |  

Emerging infectious disease implications of invasive mammalian species: the greater white-toothed shrew (Crocidura russula) is associated with a novel serovar of pathogenic Leptospira in Ireland.

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.

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July 7, 2019  |  

Whole genome analysis of Yersinia ruckeri isolated over 27 years in Australia and New Zealand reveals geographical endemism over multiple lineages and recent evolution under host selection.

Yersinia ruckeri is a salmonid pathogen with widespread distribution in cool-temperate waters including Australia and New Zealand, two isolated environments with recently developed salmonid farming industries. Phylogenetic comparison of 58 isolates from Australia, New Zealand, USA, Chile, Finland and China based on non-recombinant core genome SNPs revealed multiple deep-branching lineages, with a most recent common ancestor estimated at 18?500 years BP (12?355-24?757 95% HPD) and evidence of Australasian endemism. Evolution within the Tasmanian Atlantic salmon serotype O1b lineage has been slow, with 63 SNPs describing the variance over 27 years. Isolates from the prevailing lineage are poorly/non-motile compared to a lineage pre-vaccination, introduced in 1997, which is highly motile but has not been isolated since from epizootics. A non-motile phenotype has arisen independently in Tasmania compared to Europe and USA through a frameshift in fliI, encoding the ATPase of the flagella cluster. We report for the first time lipopolysaccharide O-antigen serotype O2 isolates in Tasmania. This phenotype results from deletion of the O-antigen cluster and consequent loss of high-molecular-weight O-antigen. This phenomenon has occurred independently on three occasions on three continents (Australasia, North America and Asia) as O2 isolates from the USA, China and Tasmania share the O-antigen deletion but occupy distant lineages. Despite the European and North American origins of the Australasian salmonid stocks, the lineages of Y. ruckeri in Australia and New Zealand are distinct from those of the northern hemisphere, suggesting they are pre-existing ancient strains that have emerged and evolved with the introduction of susceptible hosts following European colonization.

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July 7, 2019  |  

Investigation of and response to 2 plague cases, Yosemite National Park, California, USA, 2015.

In August 2015, plague was diagnosed for 2 persons who had visited Yosemite National Park in California, USA. One case was septicemic and the other bubonic. Subsequent environmental investigation identified probable locations of exposure for each patient and evidence of epizootic plague in other areas of the park. Transmission of Yersinia pestis was detected by testing rodent serum, fleas, and rodent carcasses. The environmental investigation and whole-genome multilocus sequence typing of Y. pestis isolates from the patients and environmental samples indicated that the patients had been exposed in different locations and that at least 2 distinct strains of Y. pestis were circulating among vector-host populations in the area. Public education efforts and insecticide applications in select areas to control rodent fleas probably reduced the risk for plague transmission to park visitors and staff.

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July 7, 2019  |  

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under ‘priming’ conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30?kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences – but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event – with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

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July 7, 2019  |  

Complete genome sequences of the Neethling-like lumpy skin disease virus strains obtained directly from three commercial live attenuated vaccines.

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. Here, we report the complete genome sequences of three LSDV strains obtained directly from the live attenuated vaccines: Lumpyvax (MSD Animal Health), Herbivac LS (Deltamune) and Lumpy Skin Disease Vaccine (Onderstepoort Biological Products). Copyright © 2016 Mathijs et al.

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July 7, 2019  |  

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence.A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented.Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.

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July 7, 2019  |  

Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078.

How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome).Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM)  and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for (m4)C methylation, but has one additional spacer  when compared to the clinical isolate M120.These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile.

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July 7, 2019  |  

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance.

The whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is among the 100 worst invasive species in the world. As one of the most important crop pests and virus vectors, B. tabaci causes substantial crop losses and poses a serious threat to global food security. We report the 615-Mb high-quality genome sequence of B. tabaci Middle East-Asia Minor 1 (MEAM1), the first genome sequence in the Aleyrodidae family, which contains 15,664 protein-coding genes. The B. tabaci genome is highly divergent from other sequenced hemipteran genomes, sharing no detectable synteny. A number of known detoxification gene families, including cytochrome P450s and UDP-glucuronosyltransferases, are significantly expanded in B. tabaci. Other expanded gene families, including cathepsins, large clusters of tandemly duplicated B. tabaci-specific genes, and phosphatidylethanolamine-binding proteins (PEBPs), were found to be associated with virus acquisition and transmission and/or insecticide resistance, likely contributing to the global invasiveness and efficient virus transmission capacity of B. tabaci. The presence of 142 horizontally transferred genes from bacteria or fungi in the B. tabaci genome, including genes encoding hopanoid/sterol synthesis and xenobiotic detoxification enzymes that are not present in other insects, offers novel insights into the unique biological adaptations of this insect such as polyphagy and insecticide resistance. Interestingly, two adjacent bacterial pantothenate biosynthesis genes, panB and panC, have been co-transferred into B. tabaci and fused into a single gene that has acquired introns during its evolution.The B. tabaci genome contains numerous genetic novelties, including expansions in gene families associated with insecticide resistance, detoxification and virus transmission, as well as numerous horizontally transferred genes from bacteria and fungi. We believe these novelties likely have shaped B. tabaci as a highly invasive polyphagous crop pest and efficient vector of plant viruses. The genome serves as a reference for resolving the B. tabaci cryptic species complex, understanding fundamental biological novelties, and providing valuable genetic information to assist the development of novel strategies for controlling whiteflies and the viruses they transmit.

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July 7, 2019  |  

Identification of the fluvirucin B2 (Sch 38518) biosynthetic gene cluster from Actinomadura fulva subsp. indica ATCC 53714: substrate specificity of the ß-amino acid selective adenylating enzyme FlvN.

Fluvirucins are 14-membered macrolactam polyketides that show antifungal and antivirus activities. Fluvirucins have the ß-alanine starter unit at their polyketide skeletons. To understand the construction mechanism of the ß-alanine moiety in fluvirucin biosyntheses, we have identified the biosynthetic cluster of fluvirucin B2 produced from Actinomadura fulva subsp. indica ATCC 53714. The identified gene cluster contains three polyketide synthases, four characteristic ß-amino acid-carrying enzymes, one decarboxylase, and one amidohydrolase. We next investigated the activity of the adenylation enzyme FlvN, which is a key enzyme for the selective incorporation of a ß-amino acid substrate. FlvN showed strong preference for l-aspartate over other amino acids such as ß-alanine. Based on these results, we propose a biosynthetic pathway for fluvirucin B2.

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July 7, 2019  |  

Microbial metagenomics mock scenario-based sample simulation (M3S3).

Shotgun sequencing in increasingly applied in clinical microbiology for unbiased culture-independent diagnosis. While software solutions for metagenomics proliferate, integration of metagenomics in clinical care, requires method standardisation and validation. Virtual metagenomics samples could underpin validation by substituting real samples and thus we sought to develop a novel solution for simulation of metagenomics samples based on user-defined clinical scenarios.We designed the Microbial Metagenomics Mock Scenario-based Sample Simulation (M3S3) workflow, which allows users to generate virtual samples from raw reads or assemblies. The M3S3 output is a mock sample in FASTQ or FASTA format. M3S3 was tested by generating virtual samples for ten challenging infectious disease scenarios, involving a background matrix ‘spiked’ in silico with pathogens including mixtures. Replicate samples (seven per scenario) were used to represent different compositional ratios. Virtual samples were analysed using Taxonomer and Kraken db.The ten challenge scenarios were successfully applied, generating 80 samples. For all tested scenarios, the virtual samples showed sequence compositions as predicted from the user input. Spiked pathogen sequences were identified with the majority of the replicates and most exhibited acceptable abundance (deviation between expected and observed abundance of spiked pathogens), with slight differences observed between software tools.Despite demonstrated proof-of-concept, integration of clinical metagenomics in routine microbiology remains a substantial challenge. M3S3 is capable of producing virtual samples on-demand, simulating a spectrum of clinical diagnostic scenarios of varying complexity. The M3S3 tool can therefore support the development and validation of standardised metagenomics applications. Copyright © 2017. Published by Elsevier Ltd.

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