Most Downloaded Articles of 2018

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), which is a commercially important disease of farmed salmonids. Typing by conventional methods provides limited information on the evolution and spread of this pathogen, as there is a low level of standing variation within the R. salmoninarum population. Here, we apply whole-genome sequencing to 42 R. salmoninarum isolates from Chile, primarily from salmon farms, in order to understand the epidemiology of BKD in this country. The patterns of genomic variation are consistent with multiple introductions to Chile, followed by rapid dissemination over a 30 year period. The estimated dates of introduction broadly coincide with major events in the development of the Chilean aquaculture industry. We find evidence for significant barriers to transmission of BKD in the Chilean salmon production chain that may also be explained by previously undescribed signals of host tropism in R. salmoninarum. Understanding the genomic epidemiology of BKD can inform disease intervention and improve sustainability of the economically important salmon industry. This article contains data hosted by Microreact.

Average nucleotide identity analysis is a useful tool to verify taxonomic identities in prokaryotic genomes, for both complete and draft assemblies. Using optimum threshold ranges appropriate for different prokaryotic taxa, we have reviewed all prokaryotic genome assemblies in GenBank with regard to their taxonomic identity. We present the methods used to make such comparisons, the current status of GenBank verifications, and recent developments in confirming species assignments in new genome submissions.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi’s genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a ‘core compartment’ and a ‘disruptive compartment’ which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

Hepatitis E virus (HEV) is an RNA virus that is an important cause of both acute and chronic hepatitis worldwide. To date, there are eight HEV genotypes that can infect mammals. HEV-1 and HEV-2 infect exclusively humans, while HEV-3 and HEV-4 infect humans and various animals, mainly pigs and deer. Additionally, two new genotypes (HEV-5 and HEV-6) infect mainly wild boar. Recently, newly discovered genotypes HEV-7 and HEV-8 were found to infect camels and possibly humans. Nevertheless, the epidemiological distribution of HEV-7 is not well established. HEV-8 is another newly discovered genotype that was identified in 2016 in Chinese Bactrian camels. Although faecal–oral transmission is the most common route of HEV transmission, HEV can be vertically transmitted from infected mothers to their fetuses. HEV may also spread by zoonotic transmission from infected animals to humans and through person-to-person contact. Nowadays, since the number of reported cases linked to blood donations is increasing annually, HEV is recognized as a transfusion-transmitted virus. Laboratory diagnostic techniques vary in their specificity and sensitivity for HEV detection. Direct techniques allow for detection of the viral proteins, antigens and viral nucleic acid, while HEV-specific IgG and IgM antibodies can help establish a diagnosis in acute and chronic infections. In this review, we will discuss recent technologies in the laboratory diagnosis of HEV, including serological and molecular methods to assess the specificity and sensitivity of currently available HEV commercial assays.

Purpose. Speed of bloodstream infection diagnosis is vital to reduce morbidity and mortality. Whole genome sequencing (WGS) performed directly from liquid blood culture could provide single-assay species and antibiotic susceptibility prediction; however, high inhibitor and human cell/DNA concentrations limit pathogen recovery. We develop a method for the preparation of bacterial DNA for WGS-based diagnostics direct from liquid blood culture.

Methodology. We evaluate three commercial DNA extraction kits: BiOstic Bacteraemia, Amplex Hyplex and MolYsis Plus. Differential centrifugation, filtration, selective lysis and solid-phase reversible immobilization bead clean-up are tested to improve human cells/DNA and inhibitor removal. Using WGS (Illumina/MinION), we assess human DNA removal, pathogen recovery, and predict species and antibiotic susceptibility inpositive blood cultures of 44 Gram-negative and 54 Staphylococcus species.

Results/Key findings. BiOstic kit extractions yield the greatest mean DNA concentration, 94–301 ng µl−1, versus 0–2.5 ng µl−1 using Amplex and MolYsis kits. However, we note higher levels of inhibition (260/280 ratio 0.9–2.1) and human DNA (0.0–4.4×106 copies) in BiOstic extracts. Differential centrifugation (2000  g , 1 min) prior to BiOstic extraction reduces human DNA by 63–89 % with selective lysis minimizing by a further 62 %. Post-extraction bead clean-up lowers inhibition. Overall, 67 % of sequenced samples (Illumina MiSeq) contain <10 % human DNA, with >93 % concordance between WGS-based species and susceptibility predictions and clinical diagnosis. If >60 % of sequencing reads are human (7/98 samples) susceptibility prediction becomes compromised. Novel MinION-based WGS (n=9) currently gives rapid species identification but not susceptibility prediction.

Conclusion. Our method for DNA preparation allows WGS-based diagnosis direct from blood culture bottles, providing species and antibiotic susceptibility prediction in a single assay.

The stringent response is a conserved bacterial stress response mechanism that allows bacteria to respond to nutritional challenges. It is mediated by the alarmones pppGpp and ppGpp, nucleotides that are synthesized and hydrolyzed by members of the RSH superfamily. Whilst there are key differences in the binding targets for (p)ppGpp between Gram-negative and Gram-positive bacterial species, the transient accumulation of (p)ppGpp caused by nutritional stresses results in a global change in gene expression in all species. The RSH superfamily of enzymes is ubiquitous throughout the bacterial kingdom, and can be split into three main groups: the long-RSH enzymes; the small alarmone synthetases (SAS); and the small alarmone hydrolases (SAH). Despite the prevalence of these enzymes, there are important differences in the way in which they are regulated on a transcriptional and post-translational level. Here we provide an overview of the diverse regulatory mechanisms that are involved in governing this crucial signalling network. Understanding how the RSH superfamily members are regulated gives insights into the varied important biological roles for this signalling pathway across the bacteria.

The chloroplast of Chlamydomonas reinhardtii and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed in silico, assembled from validated DNA parts and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the C. reinhardtii chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, some of the features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins; and the opportunity to use the whole alga as a low-cost oral vaccine. In this paper we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral ‘pathogens’ cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.