RESULTS:

Obtaining meaningful viral information from large sequencing datasets presents unique challenges distinct from prokaryotic and eukaryotic sequencing efforts. The difficulties surrounding this issue can be ascribed in part to the genomic plasticity of viruses themselves as well as the scarcity of existing information in genomic databases. The open-source software PhagePhisher (http://www.putonti-lab.com/phagephisher) has been designed as a simple pipeline to extract relevant information from complex and mixed datasets and will improve the examination of bacteriophages viruses and virally related sequences in a range of environments. Key aspects of the software include speed and ease of use; PhagePhisher can be used with limited operator knowledge of bioinformatics on a standard workstation. As a proof-of-concept PhagePhisher was successfully implemented with bacteria–virus mixed samples of varying complexity. Furthermore viral signals within microbial metagenomic datasets were easily and quickly identified by PhagePhisher including those from prophages as well as lysogenic phages an important and often neglected aspect of examining phage populations in the environment. PhagePhisher resolves viral-related sequences which may be obscured by or imbedded in bacterial genomes.

Species definitions of viruses are frequently descriptive with assignments often being based on their disease manifestations host range geographical distribution and transmission routes. This method of categorizing viruses has recently been challenged by technology advances such as high-throughput sequencing. These have dramatically increased knowledge of viral diversity in the wider environment that dwarfs the current catalogue of viruses classified by the International Committee for the Taxonomy of Viruses (ICTV). However because such viruses are known only from their sequences without phenotypic information it is unclear how they might be classified consistently with much of the existing taxonomy framework. This difficulty exposes deeper incompatibilities in how species are conceptualized. The original species assignments based on disease or other biological attributes were primarily descriptive similar to principles used elsewhere in biology for species taxonomies. In contrast purely sequence-based classifications rely on genetic metrics such as divergence thresholds that include or exclude viruses in individual species categories. These different approaches bring different preconceptions about the nature of a virus species the former being more easily conceptualized as a category with a part/whole relationship of individuals and species while species defined by divergence thresholds or other genetic metrics are essentially logically defined groups with specific inclusion and exclusion criteria. While descriptive species definitions match our intuitive division of viruses into natural kinds rules-based genetic classifications are required for viruses known from sequence alone whose incorporation into the ICTV taxonomy is essential if it is to represent the true diversity of viruses in nature.

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes Anopheles Culex Mansonia and Psorophora) were sorted into 37 pools homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses designated Uxmal virus and Mayapan virus were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae) with one and two isolates being recovered respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian human monkey hamster murine lepidopteran and mosquito (Aedes Anopheles and Culex) origin revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico and add to the growing plethora of viruses discovered recently through the use of metagenomics.

Two Bifidobacterium strains i.e. 2176BT and 2177BT were isolated from Golden-Headed Lion Tamarin (Leontopithecus chrysomelas) and Goeldi's monkey (Callimico goeldii). Isolates were shown to be Gram-positive non-motile non-sporulating facultative anaerobic and d-fructose 6-phosphate phosphoketolase-positive. Phylogenetic analyses based on 16S rRNA sequences multilocus sequences (including hsp60 rpoB dnaJ dnaG and clpC genes) and the core genome revealed that bifidobacterial strains 2176BT and 2177BT exhibit close phylogenetic relatedness to Bifidobacterium felsineum DSM 103139T and Bifidobacterium bifidum LMG 11041T respectively. Further genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses clearly show that these strains are distinct from each of the type strains of the so far recognized Bifidobacterium species. Thus Bifidobacterium cebidarum sp. nov. (2176BT=LMG 31469T=CCUG 73785T) and Bifidobacterium leontopitheci sp. nov. (2177BT=LMG 31471T=CCUG 73786T are proposed as novel Bifidobacterium species.

Calf diarrhoea has been a major cause of economic losses in the global dairy industry. Many factors including multiple pathogen infections can directly or indirectly cause calf diarrhoea. This study compared the faecal virome between 15 healthy calves and 15 calves with diarrhoea. Significantly lower diversity of viruses was found in samples from animals with diarrhoea than those in the healthy ones and this feature may also be related to the age of the calves. Viruses belonging to the families Astroviridae and Caliciviridae that may cause diarrhoea in dairy calves have been characterized which revealed that reads of caliciviruses and astroviruses in diarrhoea calves were much higher than those in healthy calves. Five complete genomic sequences closely related to Smacoviridae have been identified which may participate in the regulation of the gut virus community ecology of healthy hosts together with bacteriophages. This research provides a theoretical basis for further understanding of known or potential enteric pathogens related to calf diarrhoea.

Introduction. Studying taxonomic and functional signatures of respiratory microbiomes provide a better understanding of airway diseases.

Gap Statement. Several human airway metagenomics studies have identified taxonomic and functional features restricted to a single disease condition and the findings are not comparable across airway diseases due to use of different samples NGS platforms and bioinformatics databases and tools.

Aim. To study the microbial taxonomic and functional components of sputum microbiome across airway diseases and healthy smokers.

Methodology. Here 57 whole metagenome shotgun sequencing (WMSS) runs coming from the sputum of five airway diseases: asthma bronchiectasis chronic obstructive pulmonary diseases (COPD) cystic fibrosis (CF) tuberculosis (TB) and healthy smokers as the control were reanalysed using a common WMSS analysis pipeline.

Results. Shannon’s index (alpha diversity) of the healthy smoker group was the highest among all. The beta diversity showed that the sputum microbiome is distinct in major airway diseases such as asthma COPD and cystic fibrosis. The microbial composition based on differential analysis showed that there are specific markers for each airway disease like Acinetobacter bereziniae as a marker for COPD and Achromobacter xylosoxidans as a marker of cystic fibrosis. Pathways and metabolites identified from the sputum microbiome of these five diseases and healthy smokers also show specific markers. ‘ppGpp biosynthesis’ and ‘purine ribonucleosides degradation’ pathways were identified as differential markers for bronchiectasis and COPD. In this meta-analysis besides bacteria kingdom Aspergillus fumigatus was detected in asthma and COPD and Roseolovirus human betaherpesvirus 7 was detected in COPD. Our analysis showed that the majority of the gene families specific to the drug-resistant associated genes were detected from opportunistic pathogens across all the groups.

Conclusion. In summary the specific species in the sputum of airway diseases along with the microbial features like specific gene families pathways and metabolites were identified which can be explored for better diagnosis and therapy.

More sustainable approaches to agriculture are urgently needed to protect existing resources and optimize crop yields and to provide food for a growing global human population. More sustainable agricultural practices that utilize plant–microbe relationships across cultivation are urgently needed. The main objectives of this study were to track the prokaryotic and fungal microbiomes associated with key growth stages of developing maize to evaluate the relationships among nitrogen cycling bacteria and major fungal genera including those known to contain arbuscular mycorrhizal fungi and other important taxa. Prokaryotic and fungal microbiomes associated with bulk soils rhizosphere soils and tissues of developing maize were characterized using Illumina MiSeq sequencing. Similarities in microbiome diversity and abundance were compared to sample metadata to explore the influence of external factors on microbiome development. Correlations among target fungal taxa bulk bacteria and nitrogen cycling bacteria were determined using non-parametric Spearman correlations. Important maize-associated fungal taxa were detected in all samples across growth stages with Fusarium Penicillium and Aspergillus fungi comprising up to 4.21 4.26 and 0.28% of all fungal genera respectively. Thirteen statistically significant correlations between nitrogen cycling genera and targeted fungal genera were also identified (r S≥0.70 or r S≤−0.70; P<0.05). This study is the first to note a strong positive association among several nitrifying bacteria and Fusarium (R=0.71; P=0.0046) Aspergillus (R=0.71; P=0.0055) and Cladosporium spcies (R=0.74; P=0.0038) suggesting the levels of soil nitrate nitrite or nitrification intermediates may have large roles in the proliferation of important maize-associated fungi.

Benthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into the diversity of polar Cyanobacteria only a handful of genomes have been sequenced to date. Here we applied a genome-resolved metagenomics approach to data obtained from Arctic sub-Antarctic and Antarctic microbial mats. We recovered 37 metagenome-assembled genomes (MAGs) of Cyanobacteria representing 17 distinct species most of which are only distantly related to genomes that have been sequenced so far. These include (i) lineages that are common in polar microbial mats such as the filamentous taxa Pseudanabaena Leptolyngbya Microcoleus / Tychonema and Phormidium; (ii) the less common taxa Crinalium and Chamaesiphon ; (iii) an enigmatic Chroococcales lineage only distantly related to Microcystis ; and (iv) an early branching lineage in the order Gloeobacterales that is distributed across the cold biosphere for which we propose the name Candidatus Sivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria especially in understudied remote and extreme environments.

Wastewater-based epidemiology (WBE) for population-level surveillance of antimicrobial resistance (AMR) is gaining significant traction but the impact of wastewater sampling methods on results is unclear. In this study we characterized taxonomic and resistome differences between single-timepoint-grab and 24 h composites of wastewater influent from a large UK-based wastewater treatment work [WWTW (population equivalent: 223 435)]. We autosampled hourly influent grab samples (n=72) over three consecutive weekdays and prepared additional 24 h composites (n=3) from respective grabs. For taxonomic profiling metagenomic DNA was extracted from all samples and 16S rRNA gene sequencing was performed. One composite and six grabs from day 1 underwent metagenomic sequencing for metagenomic dissimilarity estimation and resistome profiling. Taxonomic abundances of phyla varied significantly across hourly grab samples but followed a repeating diurnal pattern for all 3 days. Hierarchical clustering grouped grab samples into four time periods dissimilar in both 16S rRNA gene-based profiles and metagenomic distances. 24H-composites resembled mean daily phyla abundances and showed low variability of taxonomic profiles. Of the 122 AMR gene families (AGFs) identified across all day 1 samples single grab samples identified a median of six (IQR: 5–8) AGFs not seen in the composite. However 36/36 of these hits were at lateral coverage <0.5 (median: 0.19; interquartile range: 0.16–0.22) and potential false positives. Conversely the 24H-composite identified three AGFs not seen in any grab with higher lateral coverage (0.82; 0.55–0.84). Additionally several clinically significant human AGFs (bla VIM bla IMP bla KPC) were intermittently or completely missed by grab sampling but captured by the 24 h composite. Wastewater influent undergoes significant taxonomic and resistome changes on short timescales potentially affecting interpretation of results based on sampling strategy. Grab samples are more convenient and potentially capture low-prevalence/transient targets but are less comprehensive and temporally variable. Therefore we recommend 24H-composite sampling where feasible. Further validation and optimization of WBE methods is vital for its development into a robust AMR surveillance approach.

Background. The oesophageal microbiome is thought to contribute to the pathogenesis of oesophageal cancer. However investigations using culture and molecular barcodes have provided only a low-resolution view of this important microbial community. We therefore explored the potential of culturomics and metagenomic binning to generate a catalogue of reference genomes from the healthy human oesophageal microbiome alongside a comparison set from saliva.

Results. Twenty-two distinct colonial morphotypes from healthy oesophageal samples were genome-sequenced. These fell into twelve species clusters eleven of which represented previously defined species. Two isolates belonged to a novel species which we have named Rothia gullae. We performed metagenomic binning of reads generated from UK samples from this study alongside reads generated from Australian samples in a recent study. Metagenomic binning generated 136 medium or high-quality metagenome-assembled genomes (MAGs). MAGs were assigned to 56 species clusters eight representing novel Candidatus species which we have named Ca. Granulicatella gullae Ca. Streptococcus gullae Ca. Nanosynbacter quadramensis Ca. Nanosynbacter gullae Ca. Nanosynbacter colneyensis Ca. Nanosynbacter norwichensis Ca. Nanosynococcus oralis and Ca. Haemophilus gullae. Five of these novel species belong to the recently described phylum Patescibacteria . Although members of the Patescibacteria are known to inhabit the oral cavity this is the first report of their presence in the oesophagus. Eighteen of the metagenomic species were until recently identified only by hard-to-remember alphanumeric placeholder designations. Here we illustrate the utility of a set of recently published arbitrary Latinate species names in providing user-friendly taxonomic labels for microbiome analyses.

Our non-redundant species catalogue contained 63 species derived from cultured isolates or MAGs. Mapping revealed that these species account for around half of the sequences in the oesophageal and saliva metagenomes. Although no species was present in all oesophageal samples 60 species occurred in at least one oesophageal metagenome from either study with 50 identified in both cohorts.

Conclusions. Recovery of genomes and discovery of new species represents an important step forward in our understanding of the oesophageal microbiome. The genes and genomes that we have released into the public domain will provide a base line for future comparative mechanistic and intervention studies.