Microbial Genomics最新文献

A sudden increase in the number of Mpox virus (MPXV) cases worldwide prompted the WHO to declare a Public Health Emergency of International Concern in 2022 and again in 2024. Public health genomic surveillance of MPXV in impacted areas is ongoing to inform national and international situational awareness, with a growing number of sequences available in public sequence repositories. Critical to genomic surveillance is well-curated and harmonized contextual data - the sample metadata, epidemiological and clinical data, lab results and method information that enables the interpretation of sequence data for public health responses and decision-making. Contextual data, however, is often unstructured or highly variable in formats, granularity and terminology. This variability usually requires a great deal of manual clean-up before it can be integrated and used for analysis, which can be laborious, time-consuming and error-prone. To facilitate harmonization of contextual data for genomic surveillance during the 2022 and 2024 epidemics, an MPXV contextual data specification was developed by the Centre for Infectious Disease Genomics and One Health (Simon Fraser University, Canada) in collaboration with several teams at Canada's National Microbiology Lab [Public Health Agency of Canada (PHAC)] as well as provincial public health laboratories. The MPXV specification provides standardized ontology-based fields and terms for capturing information about MPXV samples and infections and prioritizes geo-temporal, data provenance and sampling strategy information for surveillance. The specification utilizes the same semantic framework used to develop other public health pathogen genomics data standards, thus demonstrating its adaptability for additional infectious diseases. The specification has been implemented as a template within an open-source application known as the DataHarmonizer, which provides curation, validation and data transformation features and functions. The MPXV specification is already being utilized in Canada and is freely available for international use. The MPXV specification adds to a growing library of interoperable, harmonized community consensus contextual data standards for public health pathogen genomics.

Pathogen genomic surveillance is globally recognized as a pillar of public health. This field has expanded rapidly following the onset of the coronavirus disease 2019 (COVID-19) pandemic, and there is an urgent need to ensure the quality, comparability and reliability of the results of genomic analyses across diverse settings and analytical platforms. Currently, no methodology or framework has been universally adopted to mitigate this issue. This study aimed to provide a solution within the Canadian public health landscape by using standardized test datasets for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic analysis. In this context, a test dataset refers to a curated set of genomic sequences designed to evaluate the accuracy, consistency and performance of sequencing workflows, bioinformatics pipelines and analytical tools. These datasets serve as benchmarks, allowing laboratories to validate their methodologies and ensure comparability across different platforms. The test datasets included in this analysis were selected based on the use of well-characterized experimental protocols from the application of specimen selection criteria, through to sequence generation. Datasets generated using Illumina and Nanopore sequencing of samples from COVID-19 patients in Saskatchewan, Canada, were used and included clean controls, variable lineages and spiked-in lower-quality run data. Illumina libraries were sequenced using ARTIC network PCR amplification, while Nanopore libraries underwent similar protocols with some modifications. Public test dataset access on Zenodo further facilitates reproducibility, providing data summary outputs and pipeline environment files. A customized R script was developed to compare Illumina data, generating multiple tables and figures highlighting comparisons between analyses. The significance of this study lies in its contribution to the implementation of bioinformatic pipeline validation tools and protocols, which are essential for reliable genomic surveillance and outbreak response. By establishing a structured framework for computational validation, this study enhances the accuracy, comparability and efficiency of genomic surveillance in an evolving landscape of viral strains and testing strategies.

Co-infection with multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, though rare, may have clinical and public health implications, including facilitating variant recombination. Early detection of co-infections is, therefore, crucial. In this study, we report two probable cases of co-infection identified during routine genomic surveillance. Initially suspected as cross-contamination due to the presence of private mutations and nucleotide mixtures flagged by Nextclade and bammix, the samples were re-extracted and re-sequenced after workspace decontamination, yet the anomalies persisted. To investigate further, we developed a bioinformatics pipeline (Katmon) incorporating various tools such as Freyja, with lineage abundance results that illustrated the presence of multiple variants, and VirStrain, which confirmed inconsistent lineage assignments. We also visualized the alternative allele fractions for each lineage-defining mutation and amplicon, showing evidence of two variants, Delta and Omicron, co-existing within a single amplicon. Amplicon sorting effectively separated reads corresponding to the two variants, and the resulting consensus sequences aligned with their respective lineage assignments. These findings suggest that the first sample, PH-RITM-1395, involved a Delta-Omicron co-infection, while the second sample, PH-RITM-4146, probably contains both a co-infection and a recombinant variant. To further support the second sample's recombinant nature, we employed sc2rf, which identified Delta-Omicron breakpoints. Retrospective analysis of 1,078 samples from July 2021 to July 2022, encompassing the period of co-circulation of different variants in the Philippines, flagged four additional co-infection cases, including Delta-Omicron and Beta-Omicron, suggesting a lower bound co-infection prevalence of 0.27% and 0.19%, respectively. Furthermore, the pipeline was used to test previously identified co-infections of different variants from different countries. Our findings underscore the critical importance of real-time genomic surveillance and advanced bioinformatics pipelines in detecting SARS-CoV-2 co-infections and variant recombination.

Marine sponges are found in all of the world's oceans, from the surface waters to the deepest abyssal zones. The marine sponge holobiont is a rich source of microbial and chemical diversity. Up to 63 bacterial phyla have been observed to be associated with sponges, and thousands of unique natural products have been extracted from sponges or their microbial symbionts. However, sponges from the deep sea and their associated microbial communities are relatively understudied, largely due to sampling-associated difficulties. Secondary metabolism biosynthetic gene clusters are phylogenetically distinct and hold the potential to produce novel chemistry with potential pharmacological or industrial utility. In order to gain further insights into the microbiome of the deep-sea sponge Inflatella pellicula, the metagenome of this sponge, sampled from a depth of 2,900 m, was sequenced. A large fraction of the sequence reads appeared to be 'biological dark matter' and could not be taxonomically classified. Further, unlike similar studies from different marine ecosystems, relatively few metagenome-assembled genomes (MAGs) could be assembled, and relatively few secondary metabolism biosynthetic gene clusters were identified. The identified clusters were, however, very dissimilar to known characterized clusters, but some shared similarities with clusters annotated in MAGs assembled from sponge metagenomes from disparate geographic locations. Therefore, renewed efforts to cultivate the hosts of these gene clusters may yield valuable small-molecule natural products.

The genus Vibrio encompasses globally relevant pathogens, of which Vibrio cholerae is the best known due to its role in cholera. Closely related species within the Cholerae clade - Vibrio paracholerae, Vibrio metoecus and Vibrio tarriae - were long misclassified as non-O1/O139 Vibrio cholerae. The objective of this study was to analyse all 13,000+ available V. cholerae genomes in GenBank to determine the presence of species from the Cholerae clade. Genome-wide analyses using Mash, whole-genome-based Average Nucleotide Identity and digital DNA-DNA hybridization reclassified 190 unique genomes as V. paracholerae, while V. metoecus and V. tarriae were not detected. Phylogenomic analyses revealed that V. paracholerae forms distinct lineages, spanning clinical, environmental and animal sources over a period of more than a century. Virulence profiling revealed the absence of cholera toxin and toxin-coregulated pilus; however, most genomes exhibited other virulence factors, including haemolysins, RTX toxins, cholix toxin and a conserved type VI secretion system. Resistome analysis revealed multiple antibiotic resistance genes, several of which were embedded within superintegron regions, reinforcing the role of V. paracholerae as a reservoir of resistance determinants. Importantly, we identified five putative gene markers with high sensitivity and specificity for discriminating the two species, providing a tool for diagnostic applications and epidemiological surveillance. These findings reveal an unsuspected epidemiological scenario for V. paracholerae, which should be considered in clinical monitoring and public health strategies involving the Cholerae clade.

Vibrio europaeus has emerged as a significant pathogen in shellfish aquaculture, causing mass mortality outbreaks in key bivalve species and leading to severe economic losses for the industry. Studies on the structure and characteristics of the accessory genome in aquaculture pathogens remain scarce, despite its crucial role in evolutionary and ecological adaptation. The accessory genome provides indeed genetic variability that enables rapid responses to environmental challenges, host adaptation and selective pressures such as antibiotics or phage predation. Here, we present the first comprehensive comparative genomic analysis of the V. europaeus pangenome to investigate the structural organization and functional content of its accessory genome. The soft mobilome of V. europaeus comprises 73% of accessory genes and 44% of the total pangenome, including non-chromosomic (plasmids) and chromosomic genetic elements such as prophages, integrative and conjugative/mobilizable elements, phage satellites and other mobile genetic elements (MGEs) designated as unclassified chromosomic regions of genomic plasticity (unclassified chromosomic RGPs). Among accessory elements, unclassified chromosomic RGPs were the primary drivers of evolutionary dynamics in V. europaeus, acting as the main genetic reservoir of anti-phage defence systems and antimicrobial resistance genes. Notably, the identification of abundant insertion hotspots in chromosomic genetic elements facilitates the rapid acquisition of anti-phage defence systems, thereby enabling rapid turnover of these systems and enhancing host fitness. In addition, novel pVE1-like plasmids (>300 kb) - only found in this species and its closest relative Vibrio tubiashii - emerged as the largest and most ubiquitous MGEs in V. europaeus. These plasmids encode the highest number of virulence genes and secondary metabolite biosynthetic genes, as well as a remarkable diversity of anti-phage defence systems among closely related strains. Although the genome dataset analysed here is limited to strains isolated from moribund/dead animals in aquaculture environments, this study provides new insights into the role of accessory genetic elements in the evolution, adaptation and diversification of the shellfish pathogen V. europaeus. The findings reveal the complexity and plasticity of its pangenome and highlight the importance of RGPs and plasmids in bacterial fitness.

Background. The propensity of Streptococcus pyogenes (group A Streptococcus) to invade normally sterile sites and cause invasive group A Streptococcus (iGAS) disease varies across strains, which are classified using the emm gene. Between 2015 and 2017, multistate iGAS surveillance identified an ~150-fold increase of one particular emm type, emm49. This genomic epidemiological analysis aimed to identify bacterial, patient and societal factors associated with this expansion.Methods. We analysed 1322 emm49 iGAS cases and the genome sequences of the clinical isolates acquired through the population-based Active Bacterial Core surveillance during 2015-2022. For each invasive case, we received both a cultured isolate and a standardized case report form that included basic demographic attributes and risk factors of infection. A phylogeographic analysis was performed to reconstruct the divergence times and spatial dispersal history within our emm49 collection.Results. Compared to other emm types, emm49 cases were more common in males (63.5% vs. 58.3%, P=0.0143), in people experiencing homelessness (34.0% vs. 17.5%, P<0.0001) and in people who inject drugs (23.7% vs. 13.1%, P<0.0001). Time-scaled phylogeographic analysis estimated that the most recent common ancestor of the post-2015 expansion isolates occurred around 2004 and that emm49 emerged in the western USA.Conclusion. Our findings suggest that the current nationwide outbreak may have originated from the introduction of emm49 into disadvantaged (homeless and/or injecting drug users) adult subpopulations. This study underscores how social marginalization and broader social determinants of health can shape iGAS strain epidemiology in the USA.

Metagenomic sequencing of clinical samples has significantly enhanced our understanding of microbial communities. However, microbial contamination and host-derived DNA remain a major obstacle to accurate data interpretation. Here, we present a methodology called 'Stop-Check-Go' for detecting and mitigating contaminants in metagenomic datasets obtained from neonatal patient samples (nasal and rectal swabs). This method incorporates laboratory and bioinformatics work combining a prevalence method, coverage estimation and microbiological reports. We compared the 'Stop-Check-Go' decontamination system with other published decontamination tools and commonly found poor performance in decontaminating microbiologically negative patients (false positives). We emphasize that host DNA decreased by an average of 76% per sample using a lysis method and was further reduced during post-sequencing analysis. Microbial species were classified as putative contaminants and assigned to 'Stop' in nearly 60% of the dataset. The 'Stop-Check-Go' system was developed to address the specific need of decontaminating low-biomass samples, where existing tools primarily designed for short-read metagenomic data showed limited performance.

In 2019, an outbreak of acute haemorrhagic diarrhoea (AHD) in dogs in Norway was linked to Providencia alcalifaciens. A marked increase in diarrhoeal cases in dogs during the autumn, along with the frequent identification of P. alcalifaciens in affected dogs, supports its role as a causative agent in seasonal diarrhoea in dogs in Norway. Previous phylogenetic studies have revealed two major genetic lineages of P. alcalifaciens, one of which is associated with diarrhoeal disease and carries a plasmid-borne type 3 transport system (T3SS_1a). The aim of this study was to place Norwegian P. alcalifaciens isolates in a global phylogenetic context and to characterize the distribution of putative virulence-associated genes (VAGs) in isolates from dogs with AHD. Whole-genome sequencing was performed on 273 isolates, and 48 publicly available genomes were included for comparative analyses. Bioinformatic processing and phylogenetic analyses were conducted on the complete dataset of 321 P. alcalifaciens genomes. VAGs were identified using ABRicate and two virulence gene databases. Phylogenetic analyses revealed two main clusters, A and B. Most isolates from sick dogs belonged to cluster A, particularly to subcluster A-1 - the largest of five subclusters - which primarily contained isolates from sick dogs. All isolates in subclusters A-1 and A-4 harboured genes encoding T3SS_1a, a type II secretion system and the effector genes stcE, yopJ and cdtA-C. blast searches using closed reference genomes showed high sequence identity (>90%) to plasmid elements in 196 of 197 isolates from subclusters A-1 and A-4, suggesting plasmid-mediated acquisition of virulence factors. These findings support that specific genetic variants of P. alcalifaciens, particularly those in subclusters A-1 and A-4, exhibit increased virulence potential and are associated with AHD in dogs. The high proportion of isolates from clinically affected dogs, including many from the 2019 outbreak, further underscores the link between subcluster A-1 and disease.

The microbiome is an important part of the complete nutritional and genomic profile of insects. The species-rich insect order Hemiptera (aphids, cicadas and true bugs) is highly diverse for mode of microbiome acquisition, with the conundrum that species in the seed-feeding subfamily Lygaeinae have lost obvious anatomy for housing bacteria, either in bacteriocytes or midgut crypts. Here, we characterize the microbiome of the milkweed bug Oncopeltus fasciatus as a tractable lygaeinid, using 16S rRNA gene sequencing. We assess how bacterial taxa vary between the sexes and across life history stages in a controlled environment, focusing on maternal-to-embryo transmission and distinguishing egg-stage constituents that are superficial or internal (transovarially transmitted). Among a core microbiome of 28 genera, the egg stage shows the greatest diversity, with a particular expansion of the family Comamonadaceae. We also analyse inter-individual variability in nymphs and adults and validate structured, stage-specific detection of seed material. Comparative analysis identifies Rhizobium as a notable microbiome constituent in seed-feeding Hemiptera, which we had previously shown to lack nitrogen metabolism components in the genome. Overall, we provide a nuanced assessment of bacterial abundance dynamics between individuals and across the life cycle and discuss the implications for acquisition and potential relevance as nutritional endosymbionts. This will underpin comparative investigations in seed-feeding bugs and future work in O. fasciatus on tissue-specific and diet-specific microbiome profiles, including in natural populations.