The genomic epidemiology of shigellosis in South Africa.

Shigellosis, a leading cause of diarrhoeal mortality and morbidity globally, predominantly affects children under five years of age living in low- and middle-income countries. While whole genome sequence analysis (WGSA) has been effectively used to further our understanding of shigellosis epidemiology, antimicrobial resistance, and transmission, it has been under-utilised in sub-Saharan Africa. In this study, we applied WGSA to large sub-sample of surveillance isolates from South Africa, collected from 2011 to 2015, focussing on Shigella flexneri 2a and Shigella sonnei. We find each serotype is epidemiologically distinct. The four identified S. flexneri 2a clusters having distinct geographical distributions, and antimicrobial resistance (AMR) and virulence profiles, while the four sub-Clades of S. sonnei varied in virulence plasmid retention. Our results support serotype specific lifestyles as a driver for epidemiological differences, show AMR is not required for epidemiological success in S. flexneri, and that the HIV epidemic may have promoted Shigella population expansion.

Evolutionary conservation of Trichomonas-mycoplasma symbiosis across the host species barrier.

Introduction: The protozoan parasite Trichomonas vaginalis is the most common cellular sexually transmitted disease in humans, and the closely related species Trichomonas gallinae is an avian parasite of ecological and economic importance. Phylogenetic evidence suggests T. vaginalis arose during bird to human transmission of a T. gallinae-like ancestor. Trichomonas vaginalis shares a strong clinical association with the independent sexually transmitted pathogen Metamycoplasma (formerly Mycoplasma) hominis, and the uncultured bacterium "Candidatus Malacoplasma (formerly Mycoplasma) girerdii," with the latter association being an order of magnitude stronger. Both bacterial species have been shown to profoundly influence T. vaginalis growth, energy production and virulence-associated mechanisms. Methods: Evidence for a novel Malacoplasma sp. was discovered by in vivo Illumina metatranscriptomics sequencing of the T. gallinae-infected pigeon mouth. We leveraged published 16S rDNA profiling data from digestive tract of 12 healthy and 24 T. gallinae-infected pigeons to investigate association between the novel Malacoplasma sp. and T. gallinae. We utilised Illumina metagenomics sequencing targeted to pigeon oral and crop samples infected with the novel Malacoplasma sp. to generate its full-length genome sequence. Sequence similarity network analysis was used to compare annotated proteins from the novel Malacoplasma sp. with a range of other related species. Results: Here we present evidence for a novel Malacoplasma species, related to "Ca. M. girerdii," that is strongly associated with T. gallinae in the upper digestive tract of domestic pigeons. Analysis of the genome sequence revealed gene features apparently specific to a Trichomonas-symbiotic Malacoplasma lineage. Discussion: These data support a model of long-term association between Trichomonas and Malacoplasma spp. that has been conserved across diversification of the Trichomonas lineage and the host species barrier from birds to human.

Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

Mental Health First Aid Training with the Nepalese Community in Australia: An Evaluation of Effects on Knowledge, Confidence, Intentions, Willingness to have Contact and Stigmatizing Attitudes.

The aim of this study was to evaluate the effectiveness of Mental Health First Aid (MHFA) training amongst the Nepalese community in Australia by exploring the impact on knowledge about mental health first aid, confidence and intentions to help, willingness to have contact and stigmatizing attitudes towards people with mental illness. We hypothesized that since MHFA has been extensively evaluated with other communities and has been found to be effective, it would therefore be effective with this community as well. MHFA training was conducted by an accredited Nepalese-Australian MHFA Instructor with 162 participants from the Nepalese community in four states of Australia. Participants completed an evaluation questionnaire prior to the training (pre-test) and at the end of the training (post-test). The evaluation questionnaire assessed participants? knowledge about what was taught in the course, ability to recognize depression as described in a vignette, confidence in providing help, intentions to provide help, and willingness to have contact and stigmatizing attitudes towards people with mental illness. There were large improvements from pre-test to post-test in knowledge, confidence and intentions to help, medium improvements in willingness to have contact, small-to-medium improvements in stigmatizing attitudes and small improvements in recognition of depression. Participants gave high ratings of the course and the instructor. MHFA training produced improvements in knowledge, confidence, intentions, willingness to have contact and stigmatizing attitudes. The training was also well received. Further research is needed to assess persistence of these effects following the course and any changes in mental health first aid provided to the community.

Mitochondrial RNA editing in Trypanoplasma borreli: New tools, new revelations.

The kinetoplastids are unicellular flagellates that derive their name from the 'kinetoplast', a region within their single mitochondrion harboring its organellar genome of high DNA content, called kinetoplast (k) DNA. Some protein products of this mitochondrial genome are encoded as cryptogenes; their transcripts require editing to generate an open reading frame. This happens through RNA editing, whereby small regulatory guide (g)RNAs direct the proper insertion and deletion of one or more uridines at each editing site within specific transcript regions. An accurate perspective of the kDNA expansion and evolution of their unique uridine insertion/deletion editing across kinetoplastids has been difficult to achieve. Here, we resolved the kDNA structure and editing patterns in the early-branching kinetoplastid Trypanoplasma borreli and compare them with those of the well-studied trypanosomatids. We find that its kDNA consists of circular molecules of about 42 kb that harbor the rRNA and protein-coding genes, and 17 different contigs of approximately 70 kb carrying an average of 23 putative gRNA loci per contig. These contigs may be linear molecules, as they contain repetitive termini. Our analysis uncovered a putative gRNA population with unique length and sequence parameters that is massive relative to the editing needs of this parasite. We validated or determined the sequence identity of four edited mRNAs, including one coding for ATP synthase 6 that was previously thought to be missing. We utilized computational methods to show that the T. borreli transcriptome includes a substantial number of transcripts with inconsistent editing patterns, apparently products of non-canonical editing. This species utilizes the most extensive uridine deletion compared to other studied kinetoplastids to enforce amino acid conservation of cryptogene products, although insertions still remain more frequent. Finally, in three tested mitochondrial transcriptomes of kinetoplastids, uridine deletions are more common in the raw mitochondrial reads than aligned to the fully edited, translationally competent mRNAs. We conclude that the organization of kDNA across known kinetoplastids represents variations on partitioned coding and repetitive regions of circular molecules encoding mRNAs and rRNAs, while gRNA loci are positioned on a highly unstable population of molecules that differ in relative abundance across strains. Likewise, while all kinetoplastids possess conserved machinery performing RNA editing of the uridine insertion/deletion type, its output parameters are species-specific.

Euglena International Network (EIN): Driving euglenoid biotechnology for the benefit of a challenged world.

Euglenoids (Euglenida) are unicellular flagellates possessing exceptionally wide geographical and ecological distribution. Euglenoids combine a biotechnological potential with a unique position in the eukaryotic tree of life. In large part these microbes owe this success to diverse genetics including secondary endosymbiosis and likely additional sources of genes. Multiple euglenoid species have translational applications and show great promise in production of biofuels, nutraceuticals, bioremediation, cancer treatments and more exotically as robotics design simulators. An absence of reference genomes currently limits these applications, including development of efficient tools for identification of critical factors in regulation, growth or optimization of metabolic pathways. The Euglena International Network (EIN) seeks to provide a forum to overcome these challenges. EIN has agreed specific goals, mobilized scientists, established a clear roadmap (Grand Challenges), connected academic and industry stakeholders and is currently formulating policy and partnership principles to propel these efforts in a coordinated and efficient manner.

Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella.

Single-cell DNA sequencing has the potential to reveal detailed hierarchical structures in evolving populations of cells. Single cell approaches are increasingly used to study clonal evolution in human ageing and cancer but have not yet been deployed to study evolving clonal microbial populations. Here, we present an approach for single bacterial genomic analysis for in vitro evolution experiments using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to the experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from populations we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. We were also able to identify that the population exposed to antibiotic stress was able to develop resistance whilst maintaining diversity. This population structure could not be resolved from bulk sequence data, and our results show how high-throughput single-cell sequencing can enhance experimental studies of bacterial evolution.

Flaviviruses hijack the host microbiota to facilitate their transmission.

Flaviviruses, such as Dengue and Zika viruses, infect millions of people worldwide using mosquitos as vectors. In this issue of Cell, Zhang et al. reveal how these viruses manipulate the skin microbiome of infected hosts in a way that increases vector recruitment and viral spread. They propose vitamin A as a way to counteract the virus and decrease transmission.

Pathogenomic analyses of Shigella isolates inform factors limiting shigellosis prevention and control across LMICs.

Shigella spp. are the leading bacterial cause of severe childhood diarrhoea in low- and middle-income countries (LMICs), are increasingly antimicrobial resistant and have no widely available licenced vaccine. We performed genomic analyses of 1,246 systematically collected shigellae sampled from seven countries in sub-Saharan Africa and South Asia as part of the Global Enteric Multicenter Study (GEMS) between 2007 and 2011, to inform control and identify factors that could limit the effectiveness of current approaches. Through contemporaneous comparison among major subgroups, we found that S. sonnei contributes ≥6-fold more disease than other Shigella species relative to its genomic diversity, and highlight existing diversity and adaptative capacity among S. flexneri that may generate vaccine escape variants in <6 months. Furthermore, we show convergent evolution of resistance against ciprofloxacin, the current WHO-recommended antimicrobial for the treatment of shigellosis, among Shigella isolates. This demonstrates the urgent need to integrate existing genomic diversity into vaccine and treatment plans for Shigella, providing a framework for the focused application of comparative genomics to guide vaccine development, and the optimization of control and prevention strategies for other pathogens relevant to public health policy considerations.

Why sequence all eukaryotes?

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine.

Standards recommendations for the Earth BioGenome Project.

A global international initiative, such as the Earth BioGenome Project (EBP), requires both agreement and coordination on standards to ensure that the collective effort generates rapid progress toward its goals. To this end, the EBP initiated five technical standards committees comprising volunteer members from the global genomics scientific community: Sample Collection and Processing, Sequencing and Assembly, Annotation, Analysis, and IT and Informatics. The current versions of the resulting standards documents are available on the EBP website, with the recognition that opportunities, technologies, and challenges may improve or change in the future, requiring flexibility for the EBP to meet its goals. Here, we describe some highlights from the proposed standards, and areas where additional challenges will need to be met.

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes.

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations.

A Bioinformatics Approach to Identifying Potential Biomarkers for Cryptosporidium parvum: A Coccidian Parasite Associated with Fetal Diarrhea.

Cryptosporidium parvum (C. parvum) is a protozoan parasite known for cryptosporidiosis in pre-weaned calves. Animals and patients with immunosuppression are at risk of developing the disease, which can cause potentially fatal diarrhoea. The present study aimed to construct a network biology framework based on the differentially expressed genes (DEGs) of C. parvum infected subjects. In this way, the gene expression profiling analysis of C. parvum infected individuals can give us a snapshot of actively expressed genes and transcripts under infection conditions. In the present study, we have analyzed microarray data sets and compared the gene expression profiles of the patients with the different data sets of the healthy control. Using a network medicine approach to identify the most influential genes in the gene interaction network, we uncovered essential genes and pathways related to C. parvum infection. We identified 164 differentially expressed genes (109 up- and 54 down-regulated DEGs) and allocated them to pathway and gene set enrichment analysis. The results underpin the identification of seven significant hub genes with high centrality values: ISG15, MX1, IFI44L, STAT1, IFIT1, OAS1, IFIT3, RSAD2, IFITM1, and IFI44. These genes are associated with diverse biological processes not limited to host interaction, type 1 interferon production, or response to IL-gamma. Furthermore, four genes (IFI44, IFIT3, IFITM1, and MX1) were also discovered to be involved in innate immunity, inflammation, apoptosis, phosphorylation, cell proliferation, and cell signaling. In conclusion, these results reinforce the development and implementation of tools based on gene profiles to identify and treat Cryptosporidium parvum-related diseases at an early stage.

Inter-kingdom relationships in Crohn's disease explored using a multi-omics approach.

The etiology of Crohn's disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD.Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome.A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated.We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

Subtelomeric assembly of a multi-gene pathway for antimicrobial defense compounds in cereals.

Non-random gene organization in eukaryotes plays a significant role in genome evolution. Here, we investigate the origin of a biosynthetic gene cluster for production of defence compounds in oat-the avenacin cluster. We elucidate the structure and organisation of this 12-gene cluster, characterise the last two missing pathway steps, and reconstitute the entire pathway in tobacco by transient expression. We show that the cluster has formed de novo since the divergence of oats in a subtelomeric region of the genome that lacks homology with other grasses, and that gene order is approximately colinear with the biosynthetic pathway. We speculate that the positioning of the late pathway genes furthest away from the telomere may mitigate against a 'self-poisoning' scenario in which toxic intermediates accumulate as a result of telomeric gene deletions. Our investigations reveal a striking example of adaptive evolution underpinned by remarkable genome plasticity.

Taxon-Specific Proteins of the Pathogenic Entamoeba Species E. histolytica and E. nuttalli.

The human protozoan parasite Entamoeba histolytica can live in the human intestine for months or years without generating any symptoms in the host. For unknown reasons, amoebae can suddenly destroy the intestinal mucosa and become invasive. This can lead to amoebic colitis or extraintestinal amoebiasis whereby the amoebae spread to other organs via the blood vessels, most commonly the liver where abscesses develop. Entamoeba nuttalli is the closest genetic relative of E. histolytica and is found in wild macaques. Another close relative is E. dispar, which asyptomatically infects the human intestine. Although all three species are closely related, only E. histolytica and E. nuttalli are able to penetrate their host's intestinal epithelium. Lineage-specific genes and gene families may hold the key to understanding differences in virulence among species. Here we discuss those genes found in E. histolytica that have relatives in only one or neither of its sister species, with particular focus on the peptidase, AIG, Ariel, and BspA families.

Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture.

BACKGROUND: The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. RESULTS: We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. CONCLUSIONS: Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome.

Ecological niche adaptation of Salmonella Typhimurium U288 is associated with altered pathogenicity and reduced zoonotic potential.

The emergence of new bacterial pathogens is a continuing challenge for agriculture and food safety. Salmonella Typhimurium is a major cause of foodborne illness worldwide, with pigs a major zoonotic reservoir. Two phylogenetically distinct variants, U288 and ST34, emerged in UK pigs around the same time but present different risk to food safety. Here we show using genomic epidemiology that ST34 accounts for over half of all S. Typhimurium infections in people while U288 less than 2%. That the U288 clade evolved in the recent past by acquiring AMR genes, indels in the virulence plasmid pU288-1, and accumulation of loss-of-function polymorphisms in coding sequences. U288 replicates more slowly and is more sensitive to desiccation than ST34 isolates and exhibited distinct pathogenicity in the murine model of colitis and in pigs. U288 infection was more disseminated in the lymph nodes while ST34 were recovered in greater numbers in the intestinal contents. These data are consistent with the evolution of S. Typhimurium U288 adaptation to pigs that may determine their reduced zoonotic potential.