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Interpretation of microbial genome data will be improved by a fully revised bacterial taxonomy. Taxonomy is an organizing principle of biology and is ideally based on evolutionary relationships among organisms. Development of a robust bacterial taxonomy has been hindered by an inability to obtain most bacteria in pure culture and, to a lesser extent, by the historical use of phenotypes to guide classification. Culture-independent sequencing technologies have matured sufficiently that a comprehensive genome-based taxonomy is now possible. We used a concatenated protein phylogeny as the basis for a bacterial taxonomy that conservatively removes polyphyletic groups and normalizes taxonomic ranks on the basis of relative evolutionary divergence. Under this approach, 58% of the 94,759 genomes comprising the Genome Taxonomy Database had changes to their existing taxonomy. This result includes the description of 99 phyla, including six major monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into a single phylum. Our taxonomy should enable improved classification of uncultured bacteria and provide a sound basis for ecological and evolutionary studies.

Ecological theory suggests that habitat disturbance differentially influences distributions of habitat generalist and specialist species. While well-established for macroorganisms, this theory has rarely been explored for microorganisms. Here we tested these principles in permeable (sandy) sediments, ecosystems with much spatiotemporal variation in resource availability and physicochemical conditions. Microbial community composition and function were profiled in intertidal and subtidal sediments using 16S rRNA gene amplicon sequencing and metagenomics, yielding 135 metagenome-assembled genomes. Community composition and metabolic traits modestly varied with sediment depth and sampling date. Several taxa were highly abundant and prevalent in all samples, including within the orders Woeseiales and Flavobacteriales, and classified as habitat generalists; genome reconstructions indicate these taxa are highly metabolically flexible facultative anaerobes and adapt to resource variability by using different electron donors and acceptors. In contrast, obligately anaerobic taxa such as sulfate reducers and candidate lineage MBNT15 were less abundant overall and only thrived in more stable deeper sediments. We substantiated these findings by measuring three metabolic processes in these sediments; whereas the habitat generalist-associated processes of sulfide oxidation and fermentation occurred rapidly at all depths, the specialist-associated process of sulfate reduction was restricted to deeper sediments. A manipulative experiment also confirmed habitat generalists outcompete specialist taxa during simulated habitat disturbance. Together, these findings show metabolically flexible habitat generalists become dominant in highly dynamic environments, whereas metabolically constrained specialists are restricted to narrower niches. Thus, an ecological theory describing distribution patterns for macroorganisms likely extends to microorganisms. Such findings have broad ecological and biogeochemical ramifications.

The accrual of genomic data from both cultured and uncultured microorganisms provides new opportunities to develop systematic taxonomies based on evolutionary relationships. Previously, we established a bacterial taxonomy through the Genome Taxonomy Database. Here, we propose a standardized archaeal taxonomy that is derived from a 122-concatenated-protein phylogeny that resolves polyphyletic groups and normalizes ranks based on relative evolutionary divergence. The resulting archaeal taxonomy, which forms part of the Genome Taxonomy Database, is stable for a range of phylogenetic variables including marker gene selection, inference methods, corrections for rate heterogeneity and compositional bias, tree rooting scenarios and expansion of the genome database. Rank normalization is shown to robustly correct for substitution rates varying up to 30-fold using simulated datasets. Taxonomic curation follows the rules of the International Code of Nomenclature of Prokaryotes while taking into account proposals to formally recognize the rank of phylum and to use genome sequences as type material. This taxonomy is based on 2,392 archaeal genomes, 93.3% of which required one or more changes to their existing taxonomy, mainly owing to incomplete classification. We identify 16 archaeal phyla and reclassify 3 major monophyletic units from the former Euryarchaeota and one phylum that unites the Thaumarchaeota–Aigarchaeota–Crenarchaeota–Korarchaeota (TACK) superphylum into a single phylum. Resolving widespread incomplete and uneven archaeal classifications is achieved by a rank-normalized genome-based taxonomy.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current 'critically endangered' status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈ throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand's borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.

To protect New Zealand’s unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

The role of Staphylococcus aureus in the pathogenesis of the chronic sinonasal disease chronic rhinosinusitis (CRS), has not been definitively established. Comparative analyses of S. aureus isolates from CRS with those from control participants may offer insight into a possible pathogenic link between this organism and CRS. The intra- and inter-subject S. aureus strain-level diversity in the sinuses of patients with and without CRS were compared in this cross-sectional study. In total, 100 patients (CRS = 64, control = 36) were screened for S. aureus carriage. The overall carriage prevalence of S. aureus in this cohort was 24% (CRS n = 13, control n = 11). Cultured S. aureus isolates from 18 participants were strain-typed using spa gene sequencing. The bacterial community composition of the middle meatus was assessed using amplicon sequencing targeting the V3V4 hypervariable region of the bacterial 16S rRNA gene. S. aureus isolates cultured from patients were grown in co-culture with the commensal bacterium Dolosigranulum pigrum and characterised. All participants harboured a single S. aureus strain and no trend in disease-specific strain-level diversity was observed. Bacterial community analyses revealed a significant negative correlation in the relative abundances of S. aureus and D. pigrum sequences, suggesting an antagonistic interaction between these organisms. Co-cultivation experiments with these bacteria, however, did not confirm this interaction in vitro. We saw no significant associations of CRS disease with S. aureus strain types. The functional role that S. aureus occupies in CRS likely depends on other factors such as variations in gene expression and interactions with other members of the sinus bacterial community.

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.